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Pitt, Geoffrey Stuart

Overview:

Our research focuses on how intracellular Ca2+, the ultimate signal of membrane excitability, regulates membrane excitability and the consequent function of excitable cells. We have discovered and described multiple mechanisms that control Ca2+ influx through voltage-gated Ca2+ channels and feedback mechanisms by which internal calcium regulates other channels that influence Ca2+ channel function. The work has entailed structure-function analyses of ion channels and their regulatory subunits and studies of mutations that lead to inherited channelopathies such as cardiac arrhythmias and epilepsy.

Positions:

Adjunct Professor in the Department of Medicine

Medicine, Cardiology
School of Medicine

Faculty Network Member of the Duke Institute for Brain Sciences

Duke Institute for Brain Sciences
Institutes and Provost's Academic Units

Member of the Duke Cancer Institute

Duke Cancer Institute
School of Medicine

Education:

M.D. 1993

M.D. — Johns Hopkins University

Medical Resident, Medicine

Stanford University

Fellow In Cardiovascular Medicine, Medicine

Stanford University

Grants:

Integrated Training in Anesthesiology Research

Administered By
Anesthesiology
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
July 01, 1996
End Date
June 30, 2021

Multidisciplinary Heart and Vascular Diseases

Administered By
Medicine, Cardiology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
July 01, 1975
End Date
March 31, 2021

Pharmacological Sciences Training Program

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Participating Faculty Member
Start Date
July 01, 1975
End Date
June 30, 2020

Identifying the physical stimuli for activation and sensitization of Piezo mechanosensitive ion channels

Administered By
Neurobiology
AwardedBy
National Institutes of Health
Role
Co-Sponsor
Start Date
June 01, 2016
End Date
May 31, 2019

Calmodulin regulation of Na+ channels in neurons and cardiomyocytes

Administered By
Medicine, Cardiology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
November 15, 2014
End Date
October 31, 2018

Identifying the mechanosensitive domains of the Piezo1 ion channel by application of localized force

Administered By
Neurobiology
AwardedBy
National Institutes of Health
Role
Co-Sponsor
Start Date
August 01, 2015
End Date
July 31, 2018

Fibroblast Growth Factor Homologous Factor Modulation and Effect on Cardiac Ion Channel Trafficking

Administered By
Medicine, Cardiology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
March 01, 2016
End Date
February 28, 2018

Calcium regulation and dysregulation of cardiac ion channels

Administered By
Medicine, Cardiology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
March 15, 2008
End Date
January 31, 2018

Medical Scientist Training Program

Administered By
School of Medicine
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
July 01, 1997
End Date
June 30, 2017

Duke Training Grant in Nephrology

Administered By
Medicine, Nephrology
AwardedBy
National Institutes of Health
Role
Preceptor
Start Date
September 20, 1995
End Date
June 30, 2017

Structural studies of NaV1.5 and functional implications

Administered By
Medicine, Cardiology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
July 01, 2013
End Date
May 31, 2017

Investigation of calcium channel modulation in cardiomyocytes by novel methods

Administered By
Medicine, Cardiology
AwardedBy
Columbia University
Role
Principal Investigator
Start Date
June 01, 2013
End Date
May 31, 2017

Bone-targeted calcium channel agonists for osteoporosis

Administered By
Medicine, Cardiology
AwardedBy
UH Harrington Discovery Institute
Role
Principal Investigator
Start Date
January 01, 2015
End Date
March 31, 2017

The role of STIM1 in cardiac and skeletal muscle function

Administered By
Medicine, Cardiology
AwardedBy
National Institutes of Health
Role
Co Investigator
Start Date
July 01, 2009
End Date
June 30, 2014
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Awards:

Harrington Discovery Institute Scholar-Innovator . Harrington Discovery Institute .

Type
National
Awarded By
Harrington Discovery Institute
Date
January 01, 2015

Elected member. The Association of American Physicians.

Type
National
Awarded By
The Association of American Physicians
Date
January 01, 2013

Elected member. The American Society for Clinical Investigation.

Type
National
Awarded By
The American Society for Clinical Investigation
Date
January 01, 2007

Established Investigator . American Heart Association.

Type
National
Awarded By
American Heart Association
Date
January 01, 2007

Distinguished Young Scholars in Medical Research Program. W.M. Keck Foundation.

Type
National
Awarded By
W.M. Keck Foundation
Date
January 01, 2005

Publications:

Current view on regulation of voltage-gated sodium channels by calcium and auxiliary proteins.

In cardiac and skeletal myocytes, and in most neurons, the opening of voltage-gated Na(+) channels (NaV channels) triggers action potentials, a process that is regulated via the interactions of the channels' intercellular C-termini with auxiliary proteins and/or Ca(2+) . The molecular and structural details for how Ca(2+) and/or auxiliary proteins modulate NaV channel function, however, have eluded a concise mechanistic explanation and details have been shrouded for the last decade behind controversy about whether Ca(2+) acts directly upon the NaV channel or through interacting proteins, such as the Ca(2+) binding protein calmodulin (CaM). Here, we review recent advances in defining the structure of NaV intracellular C-termini and associated proteins such as CaM or fibroblast growth factor homologous factors (FHFs) to reveal new insights into how Ca(2+) affects NaV function, and how altered Ca(2+) -dependent or FHF-mediated regulation of NaV channels is perturbed in various disease states through mutations that disrupt CaM or FHF interaction.

Authors
Pitt, GS; Lee, SY
MLA Citation
Pitt, GS, and Lee, SY. "Current view on regulation of voltage-gated sodium channels by calcium and auxiliary proteins." Protein science : a publication of the Protein Society 25.9 (September 2016): 1573-1584. (Review)
PMID
27262167
Source
epmc
Published In
Protein Science
Volume
25
Issue
9
Publish Date
2016
Start Page
1573
End Page
1584
DOI
10.1002/pro.2960

FGF13 modulates the gating properties of the cardiac sodium channel Nav1.5 in an isoform-specific manner.

FGF13 (FHF2), the major fibroblast growth factor homologous factor (FHF) in rodent heart, directly binds to the C-terminus of the main cardiac sodium channel, NaV1.5. Knockdown of FGF13 in cardiomyocytes induces slowed ventricular conduction by altering NaV1.5 function. FGF13 has five splice variants, each of which possess the same core region and C terminus but differing in their respective N termini. Whether and how these alternatively spliced N termini impart isoform-specific regulation of NaV1.5, however, has not been reported. Here, we exploited a heterologous expression to explore the specific modulatory effects of FGF13 splice variants FGF13S, FGF13U and FGF13YV on NaV1.5 function. We found these three splice variants differentially modulated NaV1.5 current density. Although steady-state activation was unaltered by any of the FGF13 isoforms (compared to control cells expressing Nav1.5 but not expressing FGF13), open-state fast inactivation and closed-state fast inactivation were markedly slowed, steady-state availability was significantly shifted toward the depolarizing direction, and the window current was increased by each of FGF13 isoforms. Most strikingly, FGF13S hastened the rate of NaV1.5 entry into the slow inactivation state and induced a dramatic slowing of recovery from inactivation, which caused a large decrease in current after either low or high frequency stimulation. Overall, these data showed the diversity of the roles of the FGF13 N-termini in NaV1.5 channel modulation and suggested the importance of isoform-specific regulation.

Authors
Yang, J; Wang, Z; Sinden, DS; Wang, X; Shan, B; Yu, X; Zhang, H; Pitt, GS; Wang, C
MLA Citation
Yang, J, Wang, Z, Sinden, DS, Wang, X, Shan, B, Yu, X, Zhang, H, Pitt, GS, and Wang, C. "FGF13 modulates the gating properties of the cardiac sodium channel Nav1.5 in an isoform-specific manner." Channels (Austin, Tex.) 10.5 (September 2016): 410-420.
PMID
27246624
Source
epmc
Published In
Channels (Austin, Tex.)
Volume
10
Issue
5
Publish Date
2016
Start Page
410
End Page
420
DOI
10.1080/19336950.2016.1190055

Targeted Epigenetic Remodeling of Endogenous Loci by CRISPR/Cas9-Based Transcriptional Activators Directly Converts Fibroblasts to Neuronal Cells.

Overexpression of exogenous fate-specifying transcription factors can directly reprogram differentiated somatic cells to target cell types. Here, we show that similar reprogramming can also be achieved through the direct activation of endogenous genes using engineered CRISPR/Cas9-based transcriptional activators. We use this approach to induce activation of the endogenous Brn2, Ascl1, and Myt1l genes (BAM factors) to convert mouse embryonic fibroblasts to induced neuronal cells. This direct activation of endogenous genes rapidly remodeled the epigenetic state of the target loci and induced sustained endogenous gene expression during reprogramming. Thus, transcriptional activation and epigenetic remodeling of endogenous master transcription factors are sufficient for conversion between cell types. The rapid and sustained activation of endogenous genes in their native chromatin context by this approach may facilitate reprogramming with transient methods that avoid genomic integration and provides a new strategy for overcoming epigenetic barriers to cell fate specification.

Authors
Black, JB; Adler, AF; Wang, HG; D'Ippolito, AM; Hutchinson, HA; Reddy, TE; Pitt, GS; Leong, KW; Gersbach, CA
MLA Citation
Black, JB, Adler, AF, Wang, HG, D'Ippolito, AM, Hutchinson, HA, Reddy, TE, Pitt, GS, Leong, KW, and Gersbach, CA. "Targeted Epigenetic Remodeling of Endogenous Loci by CRISPR/Cas9-Based Transcriptional Activators Directly Converts Fibroblasts to Neuronal Cells." Cell stem cell 19.3 (September 2016): 406-414.
PMID
27524438
Source
epmc
Published In
Cell Stem Cell
Volume
19
Issue
3
Publish Date
2016
Start Page
406
End Page
414
DOI
10.1016/j.stem.2016.07.001

Ion Channels in Health and Disease

© 2016 Elsevier Inc. All rights reserved.Ion Channels in Health and Disease provides key insight to allow researchers to generate discoveries across disease states. A single resource that integrates disparate areas of biology and disease ion channel biology, this publication includes cross-referencing for disease, channels, and tissues. Offers a broad view of research of interest to early and experienced researchers across biological and biomedical research. Provides an overview of fundamental concepts in ion channels research to link defects in human disease. Written in an accessible manner, without jargon. Provides a helpful, easy cross-reference for diseases, channels, and tissues.

Authors
Pitt, GS
MLA Citation
Pitt, GS. Ion Channels in Health and Disease. July 29, 2016.
Source
scopus
Publish Date
2016
Start Page
1
End Page
378

Long QT Syndrome and Seizures∗

Authors
Sun, AY; Pitt, GS
MLA Citation
Sun, AY, and Pitt, GS. "Long QT Syndrome and Seizures∗." JACC: Clinical Electrophysiology 2.3 (June 2016): 277-278.
Source
crossref
Published In
JACC. Clinical electrophysiology
Volume
2
Issue
3
Publish Date
2016
Start Page
277
End Page
278
DOI
10.1016/j.jacep.2015.12.015

Genetic variants and disease: correlate or cause?

Authors
Matsui, M; Pitt, GS
MLA Citation
Matsui, M, and Pitt, GS. "Genetic variants and disease: correlate or cause?." European heart journal 37.18 (May 2016): 1476-1478.
PMID
26419624
Source
epmc
Published In
European Heart Journal (Elsevier)
Volume
37
Issue
18
Publish Date
2016
Start Page
1476
End Page
1478
DOI
10.1093/eurheartj/ehv516

Polarized localization of voltage-gated Na+ channels is regulated by concerted FGF13 and FGF14 action.

Clustering of voltage-gated sodium channels (VGSCs) within the neuronal axon initial segment (AIS) is critical for efficient action potential initiation. Although initially inserted into both somatodendritic and axonal membranes, VGSCs are concentrated within the axon through mechanisms that include preferential axonal targeting and selective somatodendritic endocytosis. How the endocytic machinery specifically targets somatic VGSCs is unknown. Here, using knockdown strategies, we show that noncanonical FGF13 binds directly to VGSCs in hippocampal neurons to limit their somatodendritic surface expression, although exerting little effect on VGSCs within the AIS. In contrast, homologous FGF14, which is highly concentrated in the proximal axon, binds directly to VGSCs to promote their axonal localization. Single-point mutations in FGF13 or FGF14 abrogating VGSC interaction in vitro cannot support these specific functions in neurons. Thus, our data show how the concerted actions of FGF13 and FGF14 regulate the polarized localization of VGSCs that supports efficient action potential initiation.

Authors
Pablo, JL; Wang, C; Presby, MM; Pitt, GS
MLA Citation
Pablo, JL, Wang, C, Presby, MM, and Pitt, GS. "Polarized localization of voltage-gated Na+ channels is regulated by concerted FGF13 and FGF14 action." Proceedings of the National Academy of Sciences of the United States of America 113.19 (May 2016): E2665-E2674.
PMID
27044086
Source
epmc
Published In
Proceedings of the National Academy of Sciences of USA
Volume
113
Issue
19
Publish Date
2016
Start Page
E2665
End Page
E2674
DOI
10.1073/pnas.1521194113

A novel NaV1.5 voltage sensor mutation associated with severe atrial and ventricular arrhythmias.

Inherited autosomal dominant mutations in cardiac sodium channels (NaV1.5) cause various arrhythmias, such as long QT syndrome and Brugada syndrome. Although dozens of mutations throughout the protein have been reported, there are few reported mutations within a voltage sensor S4 transmembrane segment and few that are homozygous. Here we report analysis of a novel lidocaine-sensitive recessive mutation, p.R1309H, in the NaV1.5 DIII/S4 voltage sensor in a patient with a complex arrhythmia syndrome.We expressed the wild type or mutant NaV1.5 heterologously for analysis with the patch-clamp and voltage clamp fluorometry (VCF) techniques. p.R1309H depolarized the voltage-dependence of activation, hyperpolarized the voltage-dependence of inactivation, and slowed recovery from inactivation, thereby reducing the channel availability at physiologic membrane potentials. Additionally, p.R1309H increased the "late" Na(+) current. The location of the mutation in DIIIS4 prompted testing for a gating pore current. We observed an inward current at hyperpolarizing voltages that likely exacerbates the loss-of-function defects at resting membrane potentials. Lidocaine reduced the gating pore current.The p.R1309H homozygous NaV1.5 mutation conferred both gain-of-function and loss-of-function effects on NaV1.5 channel activity. Reduction of a mutation-induced gating pore current by lidocaine suggested a therapeutic mechanism.

Authors
Wang, HG; Zhu, W; Kanter, RJ; Silva, JR; Honeywell, C; Gow, RM; Pitt, GS
MLA Citation
Wang, HG, Zhu, W, Kanter, RJ, Silva, JR, Honeywell, C, Gow, RM, and Pitt, GS. "A novel NaV1.5 voltage sensor mutation associated with severe atrial and ventricular arrhythmias." Journal of molecular and cellular cardiology 92 (March 2016): 52-62.
PMID
26801742
Source
epmc
Published In
Journal of Molecular and Cellular Cardiology
Volume
92
Publish Date
2016
Start Page
52
End Page
62
DOI
10.1016/j.yjmcc.2016.01.014

Fibroblast Growth Factor Homologous Factors: New Roles in Neuronal Health and Disease.

Fibroblast growth factor homologous factors (FHFs) are a noncanonical subset of intracellular fibroblast growth factors that have been implicated in a variety of neurobiological processes and in disease. They are most prominently regulators of voltage-gated Na(+) channels (NaVs). In this review, we discuss new insights into how FHFs modulate NaVs. This is followed by a summary of a growing body of evidence that FHFs operate in much broader fashion. Finally, we highlight unknown aspects of FHF function as areas of future interest.

Authors
Pablo, JL; Pitt, GS
MLA Citation
Pablo, JL, and Pitt, GS. "Fibroblast Growth Factor Homologous Factors: New Roles in Neuronal Health and Disease." The Neuroscientist : a review journal bringing neurobiology, neurology and psychiatry 22.1 (February 2016): 19-25. (Review)
PMID
25492945
Source
epmc
Published In
Neuroscientist
Volume
22
Issue
1
Publish Date
2016
Start Page
19
End Page
25
DOI
10.1177/1073858414562217

α1-Syntrophin Variant Identified in Drug-Induced Long QT Syndrome Increases Late Sodium Current.

Drug-induced long-QT syndrome (diLQTS) is often due to drug block of IKr, especially in genetically susceptible patients with subclinical mutations in the IKr-encoding KCHN2. Few variants in the cardiac NaV1.5 Na+ channel complex have been associated with diLQTS. We tested whether a novel SNTA1 (α1-syntrophin) variant (p.E409Q) found in a patient with diLQTS increases late sodium current (INa-L), thereby providing a disease mechanism. Electrophysiological studies were performed in HEK293T cells co-expressing human NaV1.5/nNOS/PMCA4b with either wild type (WT) or SNTA1 variants (A390V-previously reported in congenital LQTS; and E409Q); and in adult rat ventricular cardiomyocytes infected with SNTA1 expressing adenoviruses (WT or one of the two SNTA1 variants). In HEK293T cells and in cardiomyocytes, there was no significant difference in the peak INa densities among the SNTA1 WT and variants. However, both variants increased INa-L (% of peak current) in HEK293T cells (0.58 ± 0.10 in WT vs. 0.90 ± 0.11 in A390V, p = 0.048; vs. 0.88 ± 0.07 in E409Q, p = 0.023). In cardiomyocytes, INa-L was significantly increased by E409Q, but not by A390V compared to WT (0.49 ± 0.14 in WT vs.0.94 ± 0.23 in A390V, p = 0.099; vs. 1.12 ± 0.24 in E409Q, p = 0.019). We demonstrated that a novel SNTA1 variant is likely causative for diLQTS by augmenting INa-L. These data suggest that variants within the NaV1.5-interacting α1-syntrophin are a potential mechanism for diLQTS, thereby expanding the concept that variants within congenital LQTS loci can cause diLQTS.

Authors
Choi, JI; Wang, C; Thomas, MJ; Pitt, GS
MLA Citation
Choi, JI, Wang, C, Thomas, MJ, and Pitt, GS. "α1-Syntrophin Variant Identified in Drug-Induced Long QT Syndrome Increases Late Sodium Current." PloS one 11.3 (January 2016): e0152355-.
PMID
27028743
Source
epmc
Published In
PloS one
Volume
11
Issue
3
Publish Date
2016
Start Page
e0152355
DOI
10.1371/journal.pone.0152355

STIM1-Ca2+ signaling modulates automaticity of the mouse sinoatrial node.

Cardiac pacemaking is governed by specialized cardiomyocytes located in the sinoatrial node (SAN). SAN cells (SANCs) integrate voltage-gated currents from channels on the membrane surface (membrane clock) with rhythmic Ca(2+) release from internal Ca(2+) stores (Ca(2+) clock) to adjust heart rate to meet hemodynamic demand. Here, we report that stromal interaction molecule 1 (STIM1) and Orai1 channels, key components of store-operated Ca(2+) entry, are selectively expressed in SANCs. Cardiac-specific deletion of STIM1 in mice resulted in depletion of sarcoplasmic reticulum (SR) Ca(2+) stores of SANCs and led to SAN dysfunction, as was evident by a reduction in heart rate, sinus arrest, and an exaggerated autonomic response to cholinergic signaling. Moreover, STIM1 influenced SAN function by regulating ionic fluxes in SANCs, including activation of a store-operated Ca(2+) current, a reduction in L-type Ca(2+) current, and enhancing the activities of Na(+)/Ca(2+) exchanger. In conclusion, these studies reveal that STIM1 is a multifunctional regulator of Ca(2+) dynamics in SANCs that links SR Ca(2+) store content with electrical events occurring in the plasma membrane, thereby contributing to automaticity of the SAN.

Authors
Zhang, H; Sun, AY; Kim, JJ; Graham, V; Finch, EA; Nepliouev, I; Zhao, G; Li, T; Lederer, WJ; Stiber, JA; Pitt, GS; Bursac, N; Rosenberg, PB
MLA Citation
Zhang, H, Sun, AY, Kim, JJ, Graham, V, Finch, EA, Nepliouev, I, Zhao, G, Li, T, Lederer, WJ, Stiber, JA, Pitt, GS, Bursac, N, and Rosenberg, PB. "STIM1-Ca2+ signaling modulates automaticity of the mouse sinoatrial node." October 2015.
PMID
26424448
Source
epmc
Published In
Proceedings of the National Academy of Sciences of USA
Volume
112
Issue
41
Publish Date
2015
Start Page
E5618
End Page
E5627
DOI
10.1073/pnas.1503847112

SCN5A variant that blocks fibroblast growth factor homologous factor regulation causes human arrhythmia.

Nav channels are essential for metazoan membrane depolarization, and Nav channel dysfunction is directly linked with epilepsy, ataxia, pain, arrhythmia, myotonia, and irritable bowel syndrome. Human Nav channelopathies are primarily caused by variants that directly affect Nav channel permeability or gating. However, a new class of human Nav channelopathies has emerged based on channel variants that alter regulation by intracellular signaling or cytoskeletal proteins. Fibroblast growth factor homologous factors (FHFs) are a family of intracellular signaling proteins linked with Nav channel regulation in neurons and myocytes. However, to date, there is surprisingly little evidence linking Nav channel gene variants with FHFs and human disease. Here, we provide, to our knowledge, the first evidence that mutations in SCN5A (encodes primary cardiac Nav channel Nav1.5) that alter FHF binding result in human cardiovascular disease. We describe a five*generation kindred with a history of atrial and ventricular arrhythmias, cardiac arrest, and sudden cardiac death. Affected family members harbor a novel SCN5A variant resulting in p.H1849R. p.H1849R is localized in the central binding core on Nav1.5 for FHFs. Consistent with these data, Nav1.5 p.H1849R affected interaction with FHFs. Further, electrophysiological analysis identified Nav1.5 p.H1849R as a gain-of-function for INa by altering steady-state inactivation and slowing the rate of Nav1.5 inactivation. In line with these data and consistent with human cardiac phenotypes, myocytes expressing Nav1.5 p.H1849R displayed prolonged action potential duration and arrhythmogenic afterdepolarizations. Together, these findings identify a previously unexplored mechanism for human Nav channelopathy based on altered Nav1.5 association with FHF proteins.

Authors
Musa, H; Kline, CF; Sturm, AC; Murphy, N; Adelman, S; Wang, C; Yan, H; Johnson, BL; Csepe, TA; Kilic, A; Higgins, RS; Janssen, PM; Fedorov, VV; Weiss, R; Salazar, C; Hund, TJ; Pitt, GS; Mohler, PJ
MLA Citation
Musa, H, Kline, CF, Sturm, AC, Murphy, N, Adelman, S, Wang, C, Yan, H, Johnson, BL, Csepe, TA, Kilic, A, Higgins, RS, Janssen, PM, Fedorov, VV, Weiss, R, Salazar, C, Hund, TJ, Pitt, GS, and Mohler, PJ. "SCN5A variant that blocks fibroblast growth factor homologous factor regulation causes human arrhythmia." Proceedings of the National Academy of Sciences of the United States of America 112.40 (October 2015): 12528-12533.
PMID
26392562
Source
epmc
Published In
Proceedings of the National Academy of Sciences of USA
Volume
112
Issue
40
Publish Date
2015
Start Page
12528
End Page
12533
DOI
10.1073/pnas.1516430112

Calcium Channel Mutations in Cardiac Arrhythmia Syndromes

Authors
Betzenhauser, M; Pitt, G; Antzelevitch, C
MLA Citation
Betzenhauser, M, Pitt, G, and Antzelevitch, C. "Calcium Channel Mutations in Cardiac Arrhythmia Syndromes." Current Molecular Pharmacology 8.2 (September 17, 2015): 133-142.
Source
crossref
Published In
Current Molecular Pharmacology
Volume
8
Issue
2
Publish Date
2015
Start Page
133
End Page
142
DOI
10.2174/1874467208666150518114857

Cardiovascular precision medicine: hope or hype?

Authors
Pitt, GS
MLA Citation
Pitt, GS. "Cardiovascular precision medicine: hope or hype?." European heart journal 36.29 (August 2015): 1842-1843.
PMID
26074465
Source
epmc
Published In
European Heart Journal (Elsevier)
Volume
36
Issue
29
Publish Date
2015
Start Page
1842
End Page
1843
DOI
10.1093/eurheartj/ehv226

Calcium signaling regulates ventricular hypertrophy during development independent of contraction or blood flow.

In utero interventions aimed at restoring left ventricular hemodynamic forces in fetuses with prenatally diagnosed hypoplastic left heart syndrome failed to stimulate ventricular myocardial growth during gestation, suggesting chamber growth during development may not rely upon fluid forces. We therefore hypothesized that ventricular hypertrophy during development may depend upon fundamental Ca(2+)-dependent growth pathways that function independent of hemodynamic forces. To test this hypothesis, zebrafish embryos were treated with inhibitors or activators of Ca(2+) signaling in the presence or absence of contraction during the period of chamber development. Abolishment of contractile function alone in the setting of preserved Ca(2+) signaling did not impair ventricular hypertrophy. In contrast, inhibition of L-type voltage-gated Ca(2+) influx abolished contraction and led to reduced ventricular hypertrophy, whereas increasing L-type voltage-gated Ca(2+) influx led to enhanced ventricular hypertrophy in either the presence or absence of contraction. Similarly, inhibition of the downstream Ca(2+)-sensitive phosphatase calcineurin, a known regulator of adult cardiac hypertrophy, led to reduced ventricular hypertrophy in the presence or absence of contraction, whereas hypertrophy was rescued in the absence of L-type voltage-gated Ca(2+) influx and contraction by expression of a constitutively active calcineurin. These data suggest that ventricular cardiomyocyte hypertrophy during chamber formation is dependent upon Ca(2+) signaling pathways that are unaffected by heart function or hemodynamic forces. Disruption of Ca(2+)-dependent hypertrophy during heart development may therefore represent one mechanism for impaired chamber formation that is not related to impaired blood flow.

Authors
Andersen, ND; Ramachandran, KV; Bao, MM; Kirby, ML; Pitt, GS; Hutson, MR
MLA Citation
Andersen, ND, Ramachandran, KV, Bao, MM, Kirby, ML, Pitt, GS, and Hutson, MR. "Calcium signaling regulates ventricular hypertrophy during development independent of contraction or blood flow." Journal of molecular and cellular cardiology 80 (March 2015): 1-9.
PMID
25536179
Source
epmc
Published In
Journal of Molecular and Cellular Cardiology
Volume
80
Publish Date
2015
Start Page
1
End Page
9
DOI
10.1016/j.yjmcc.2014.12.016

Na+ channel function, regulation, structure, trafficking and sequestration.

This paper is the second of a series of three reviews published in this issue resulting from the University of California Davis Cardiovascular Symposium 2014: Systems approach to understanding cardiac excitation-contraction coupling and arrhythmias: Na(+) channel and Na(+) transport. The goal of the symposium was to bring together experts in the field to discuss points of consensus and controversy on the topic of sodium in the heart. The present review focuses on Na(+) channel function and regulation, Na(+) channel structure and function, and Na(+) channel trafficking, sequestration and complexing.

Authors
Chen-Izu, Y; Shaw, RM; Pitt, GS; Yarov-Yarovoy, V; Sack, JT; Abriel, H; Aldrich, RW; Belardinelli, L; Cannell, MB; Catterall, WA; Chazin, WJ; Chiamvimonvat, N; Deschenes, I; Grandi, E; Hund, TJ; Izu, LT; Maier, LS; Maltsev, VA; Marionneau, C; Mohler, PJ; Rajamani, S; Rasmusson, RL; Sobie, EA; Clancy, CE; Bers, DM
MLA Citation
Chen-Izu, Y, Shaw, RM, Pitt, GS, Yarov-Yarovoy, V, Sack, JT, Abriel, H, Aldrich, RW, Belardinelli, L, Cannell, MB, Catterall, WA, Chazin, WJ, Chiamvimonvat, N, Deschenes, I, Grandi, E, Hund, TJ, Izu, LT, Maier, LS, Maltsev, VA, Marionneau, C, Mohler, PJ, Rajamani, S, Rasmusson, RL, Sobie, EA, Clancy, CE, and Bers, DM. "Na+ channel function, regulation, structure, trafficking and sequestration." The Journal of physiology 593.6 (March 2015): 1347-1360. (Review)
PMID
25772290
Source
epmc
Published In
The Journal of Physiology
Volume
593
Issue
6
Publish Date
2015
Start Page
1347
End Page
1360
DOI
10.1113/jphysiol.2014.281428

The PDZ motif of the α1C subunit is not required for surface trafficking and adrenergic modulation of CaV1.2 channel in the heart.

Voltage-gated Ca(2+) channels play a key role in initiating muscle excitation-contraction coupling, neurotransmitter release, gene expression, and hormone secretion. The association of CaV1.2 with a supramolecular complex impacts trafficking, localization, turnover, and, most importantly, multifaceted regulation of its function in the heart. Several studies hint at an important role for the C terminus of the α1C subunit as a hub for multidimensional regulation of CaV1.2 channel trafficking and function. Recent studies have demonstrated an important role for the four-residue PDZ binding motif at the C terminus of α1C in interacting with scaffold proteins containing PDZ domains, in the subcellular localization of CaV1.2 in neurons, and in the efficient signaling to cAMP-response element-binding protein in neurons. However, the role of the α1C PDZ ligand domain in the heart is not known. To determine whether the α1C PDZ motif is critical for CaV1.2 trafficking and function in cardiomyocytes, we generated transgenic mice with inducible expression of an N-terminal FLAG epitope-tagged dihydropyridine-resistant α1C with the PDZ motif deleted (ΔPDZ). These mice were crossed with α-myosin heavy chain reverse transcriptional transactivator transgenic mice, and the double-transgenic mice were fed doxycycline. The ΔPDZ channels expressed, trafficked to the membrane, and supported robust excitation-contraction coupling in the presence of nisoldipine, a dihydropyridine Ca(2+) channel blocker, providing functional evidence that they appropriately target to dyads. The ΔPDZ Ca(2+) channels were appropriately regulated by isoproterenol and forskolin. These data indicate that the α1C PDZ motif is not required for surface trafficking, localization to the dyad, or adrenergic stimulation of CaV1.2 in adult cardiomyocytes.

Authors
Yang, L; Katchman, A; Weinberg, RL; Abrams, J; Samad, T; Wan, E; Pitt, GS; Marx, SO
MLA Citation
Yang, L, Katchman, A, Weinberg, RL, Abrams, J, Samad, T, Wan, E, Pitt, GS, and Marx, SO. "The PDZ motif of the α1C subunit is not required for surface trafficking and adrenergic modulation of CaV1.2 channel in the heart." The Journal of biological chemistry 290.4 (January 2015): 2166-2174.
PMID
25505241
Source
epmc
Published In
The Journal of biological chemistry
Volume
290
Issue
4
Publish Date
2015
Start Page
2166
End Page
2174
DOI
10.1074/jbc.m114.602508

FGF14 modulates resurgent sodium current in mouse cerebellar Purkinje neurons.

Rapid firing of cerebellar Purkinje neurons is facilitated in part by a voltage-gated Na(+) (NaV) 'resurgent' current, which allows renewed Na(+) influx during membrane repolarization. Resurgent current results from unbinding of a blocking particle that competes with normal channel inactivation. The underlying molecular components contributing to resurgent current have not been fully identified. In this study, we show that the NaV channel auxiliary subunit FGF14 'b' isoform, a locus for inherited spinocerebellar ataxias, controls resurgent current and repetitive firing in Purkinje neurons. FGF14 knockdown biased NaV channels towards the inactivated state by decreasing channel availability, diminishing the 'late' NaV current, and accelerating channel inactivation rate, thereby reducing resurgent current and repetitive spiking. Critical for these effects was both the alternatively spliced FGF14b N-terminus and direct interaction between FGF14b and the NaV C-terminus. Together, these data suggest that the FGF14b N-terminus is a potent regulator of resurgent NaV current in cerebellar Purkinje neurons.

Authors
Yan, H; Pablo, JL; Wang, C; Pitt, GS
MLA Citation
Yan, H, Pablo, JL, Wang, C, and Pitt, GS. "FGF14 modulates resurgent sodium current in mouse cerebellar Purkinje neurons." eLife 3 (September 30, 2014): e04193-.
PMID
25269146
Source
epmc
Published In
eLife
Volume
3
Publish Date
2014
Start Page
e04193
DOI
10.7554/elife.04193

Structural analyses of Ca²⁺/CaM interaction with NaV channel C-termini reveal mechanisms of calcium-dependent regulation.

Ca(2+) regulates voltage-gated Na(+) (NaV) channels, and perturbed Ca(2+) regulation of NaV function is associated with epilepsy syndromes, autism and cardiac arrhythmias. Understanding the disease mechanisms, however, has been hindered by a lack of structural information and competing models for how Ca(2+) affects NaV channel function. Here we report the crystal structures of two ternary complexes of a human NaV cytosolic C-terminal domain (CTD), a fibroblast growth factor homologous factor and Ca(2+)/calmodulin (Ca(2+)/CaM). These structures rule out direct binding of Ca(2+) to the NaV CTD and uncover new contacts between CaM and the NaV CTD. Probing these new contacts with biochemical and functional experiments allows us to propose a mechanism by which Ca(2+) could regulate NaV channels. Further, our model provides hints towards understanding the molecular basis of the neurologic disorders and cardiac arrhythmias caused by NaV channel mutations.

Authors
Wang, C; Chung, BC; Yan, H; Wang, HG; Lee, SY; Pitt, GS
MLA Citation
Wang, C, Chung, BC, Yan, H, Wang, HG, Lee, SY, and Pitt, GS. "Structural analyses of Ca²⁺/CaM interaction with NaV channel C-termini reveal mechanisms of calcium-dependent regulation." Nature communications 5 (September 18, 2014): 4896-.
PMID
25232683
Source
epmc
Published In
Nature Communications
Volume
5
Publish Date
2014
Start Page
4896
DOI
10.1038/ncomms5896

Ion channels under the sun.

Authors
Abbott, GW; Pitt, GS
MLA Citation
Abbott, GW, and Pitt, GS. "Ion channels under the sun." FASEB journal : official publication of the Federation of American Societies for Experimental Biology 28.5 (May 2014): 1957-1962.
PMID
24786286
Source
epmc
Published In
The FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Volume
28
Issue
5
Publish Date
2014
Start Page
1957
End Page
1962
DOI
10.1096/fj.14-0501ufm

A CACNA1C variant associated with reduced voltage-dependent inactivation, increased CaV1.2 channel window current, and arrhythmogenesis.

Mutations in CACNA1C that increase current through the CaV1.2 L-type Ca2+ channel underlie rare forms of long QT syndrome (LQTS), and Timothy syndrome (TS). We identified a variant in CACNA1C in a male child of Filipino descent with arrhythmias and extracardiac features by candidate gene sequencing and performed functional expression studies to electrophysiologically characterize the effects of the variant on CaV1.2 channels. As a baby, the subject developed seizures and displayed developmental delays at 30 months of age. At age 5 years, he displayed a QTc of 520 ms and experienced recurrent VT. Physical exam at 17 years of age was notable for microcephaly, short stature, lower extremity weakness and atrophy with hyperreflexia, spastic diplegia, multiple dental caries and episodes of rhabdomyolysis. Candidate gene sequencing identified a G>C transversion at position 5731 of CACNA1C (rs374528680) predicting a glycine>arginine substitution at residue 1911 (p.G1911R) of CaV1.2. The allele frequency of this variant is 0.01 in Malays, but absent in 984 Caucasian alleles and in the 1000 genomes project. In electrophysiological analyses, the variant decreased voltage-dependent inactivation, thus causing a gain of function of CaV1.2. We also observed a negative shift of V1/2 of activation and positive shift of V1/2 of channel inactivation, resulting in an increase of the window current. Together, these suggest a gain-of-function effect on CaV1.2 and suggest increased susceptibility for arrhythmias in certain clinical settings. The p.G1911R variant was also identified in a case of sudden unexplained infant death (SUID), for which an increasing number of clinical observations have demonstrated can be associated with arrhythmogenic mutations in cardiac ion channels. In summary, the combined effects of the CACNA1C variant to diminish voltage-dependent inactivation of CaV1.2 and increase window current expand our appreciation of mechanisms by which a gain of function of CaV1.2 can contribute to QT prolongation.

Authors
Hennessey, JA; Boczek, NJ; Jiang, YH; Miller, JD; Patrick, W; Pfeiffer, R; Sutphin, BS; Tester, DJ; Barajas-Martinez, H; Ackerman, MJ; Antzelevitch, C; Kanter, R; Pitt, GS
MLA Citation
Hennessey, JA, Boczek, NJ, Jiang, YH, Miller, JD, Patrick, W, Pfeiffer, R, Sutphin, BS, Tester, DJ, Barajas-Martinez, H, Ackerman, MJ, Antzelevitch, C, Kanter, R, and Pitt, GS. "A CACNA1C variant associated with reduced voltage-dependent inactivation, increased CaV1.2 channel window current, and arrhythmogenesis." PloS one 9.9 (January 2014): e106982-.
PMID
25184293
Source
epmc
Published In
PloS one
Volume
9
Issue
9
Publish Date
2014
Start Page
e106982
DOI
10.1371/journal.pone.0106982

FGF12 is a candidate Brugada syndrome locus.

BACKGROUND: Less than 30% of the cases of Brugada syndrome (BrS) have an identified genetic cause. Of the known BrS-susceptibility genes, loss-of-function mutations in SCN5A or CACNA1C and their auxiliary subunits are most common. On the basis of the recent demonstration that fibroblast growth factor (FGF) homologous factors (FHFs; FGF11-FGF14) regulate cardiac Na(+) and Ca(2+) channel currents, we hypothesized that FHFs are candidate BrS loci. OBJECTIVE: The goal of this study was to test whether FGF12 is a candidate BrS locus. METHODS: We used quantitative polymerase chain reaction to identify the major FHF expressed in the human ventricle and then queried a phenotype-positive, genotype-negative BrS biorepository for FHF mutations associated with BrS. We queried the effects of an identified mutant with biochemical analyses combined with electrophysiological assessment. We designed a novel rat ventricular cardiomyocyte system in which we swapped the endogenous FHF with the identified mutant and defined its effects on multiple ionic currents in their native milieu and on the cardiac action potential. RESULTS: We identified FGF12 as the major FHF expressed in the human ventricle. In 102 individuals in the biorepository, we identified a single missense mutation in FGF12-B (Q7R-FGF12). The mutant reduced binding to the NaV1.5 C terminus, but not to junctophilin-2. In adult rat cardiac myocytes, Q7R-FGF12, but not wild-type FGF12, reduced Na(+) channel current density and availability without affecting Ca(2+) channel function. Furthermore, the mutant, but not wild-type FGF12, reduced action potential amplitude, which is consistent with a mutant-induced loss of Na(+) channel function. CONCLUSIONS: These multilevel investigations strongly suggest that Q7R-FGF12 is a disease-associated BrS mutation. Moreover, these data suggest for the first time that FHF effects on Na(+) and Ca(2+) channels are separable. Most significantly, this study establishes a new method to analyze effects of human arrhythmogenic mutations on cardiac ionic currents.

Authors
Hennessey, JA; Marcou, CA; Wang, C; Wei, EQ; Wang, C; Tester, DJ; Torchio, M; Dagradi, F; Crotti, L; Schwartz, PJ; Ackerman, MJ; Pitt, GS
MLA Citation
Hennessey, JA, Marcou, CA, Wang, C, Wei, EQ, Wang, C, Tester, DJ, Torchio, M, Dagradi, F, Crotti, L, Schwartz, PJ, Ackerman, MJ, and Pitt, GS. "FGF12 is a candidate Brugada syndrome locus." Heart Rhythm 10.12 (December 2013): 1886-1894.
PMID
24096171
Source
pubmed
Published In
Heart Rhythm
Volume
10
Issue
12
Publish Date
2013
Start Page
1886
End Page
1894
DOI
10.1016/j.hrthm.2013.09.064

FGF12 is a Novel Brugada Syndrome Locus

Authors
Hennessey, JA; Marcou, CA; Wang, C; Wei, EQ; Wang, C; Crotti, L; Schwartz, P; Ackerman, MJ; Pitt, GS
MLA Citation
Hennessey, JA, Marcou, CA, Wang, C, Wei, EQ, Wang, C, Crotti, L, Schwartz, P, Ackerman, MJ, and Pitt, GS. "FGF12 is a Novel Brugada Syndrome Locus." November 26, 2013.
Source
wos-lite
Published In
Circulation
Volume
128
Issue
22
Publish Date
2013

A CACNA1C Mutation that Causes a Subset of Timothy Syndrome Phenotypes Correlates.

Timothy syndrome (TS) is a rare congenital long QT syndrome (LQTS) associated with extracardiac manifestations including craniofacial dysmorphia and dental abnormalities. The locus for TS is CACNA1C, which encodes the CaV1.2 L-type Ca(2+) channel, for which canonical mutations lead to a decrease in voltage-dependent inactivation (VDI). However, a recent report of a patient with LQTS in isolation and a CACNA1C mutation that did not affect VDI raised the question whether altered VDI is necessary for extracardiac phenotypes. In a patient with a maternally inherited microdeletion with a chromosomal translocation who presented with LQTS and associated ventricular tachyarrhythmias (Figure A), a subset of TS phenotypes, and a skeletal myopathy not readily explained by the translocation, we sought to identify a causative mutation for the TS phenotypes.

Authors
Hennessey, JA; Jiang, Y; Miller, JD; Stadt, HA; Patrick, W; Pfeiffer, R; Antzelevitch, C; Kanter, R; Pitt, GS
MLA Citation
Hennessey, JA, Jiang, Y, Miller, JD, Stadt, HA, Patrick, W, Pfeiffer, R, Antzelevitch, C, Kanter, R, and Pitt, GS. "A CACNA1C Mutation that Causes a Subset of Timothy Syndrome Phenotypes Correlates." Heart Rhythm 10.11 (November 2013): 1745-.
PMID
24210388
Source
pubmed
Published In
Heart Rhythm
Volume
10
Issue
11
Publish Date
2013
Start Page
1745
DOI
10.1016/j.hrthm.2013.09.026

Fibroblast growth factor homologous factors modulate cardiac calcium channels.

RATIONALE: Fibroblast growth factor (FGF) homologous factors (FHFs; FGF11-14) are intracellular modulators of voltage-gated Na+ channels, but their cellular distribution in cardiomyocytes indicated that they performed other functions. OBJECTIVE: We aimed to uncover novel roles for FHFs in cardiomyocytes, starting with a proteomic approach to identify novel interacting proteins. METHODS AND RESULTS: Affinity purification of FGF13 from rodent ventricular lysates followed by mass spectroscopy revealed an interaction with junctophilin-2, a protein that organizes the close apposition of the L-type Ca2+ channel CaV1.2 and the ryanodine receptor 2 in the dyad. Immunocytochemical analysis revealed that overall T-tubule structure and localization of ryanodine receptor 2 were unaffected by FGF13 knockdown in adult ventricular cardiomyocytes but localization of CaV1.2 was affected. FGF13 knockdown decreased CaV1.2 current density and reduced the amount of CaV1.2 at the surface as a result of aberrant localization of the channels. CaV1.2 current density and channel localization were rescued by expression of an shRNA-insensitive FGF13, indicating a specific role for FGF13. Consistent with these newly discovered effects on CaV1.2, we demonstrated that FGF13 also regulated Ca(2+)-induced Ca2+ release, indicated by a smaller Ca2+ transient after FGF13 knockdown. Furthermore, FGF13 knockdown caused a profound decrease in the cardiac action potential half-width. CONCLUSIONS: This study demonstrates that FHFs not only are potent modulators of voltage-gated Na+ channels but also affect Ca2+ channels and their function. We predict that FHF loss-of-function mutations would adversely affect currents through both Na+ and Ca2+ channels, suggesting that FHFs may be arrhythmogenic loci, leading to arrhythmias through a novel, dual-ion channel mechanism.

Authors
Hennessey, JA; Wei, EQ; Pitt, GS
MLA Citation
Hennessey, JA, Wei, EQ, and Pitt, GS. "Fibroblast growth factor homologous factors modulate cardiac calcium channels." Circ Res 113.4 (August 2, 2013): 381-388.
PMID
23804213
Source
pubmed
Published In
Circulation Research
Volume
113
Issue
4
Publish Date
2013
Start Page
381
End Page
388
DOI
10.1161/CIRCRESAHA.113.301215

FGF14 Regulates Presynaptic Ca2+ Channels and Synaptic Transmission

Fibroblast growth factor homologous factors (FHFs) are not growth factors, but instead bind to voltage-gated Na+ channels (NaV) and regulate their function. Mutations in FGF14, an FHF that is the locus for spinocerebellar ataxia 27 (SCA27), are believed to be pathogenic because of a dominant-negative reduction of NaV currents in cerebellar granule cells. Here, we demonstrate that FGF14 also regulates members of the presynaptic CaV2 Ca2+ channel family. Knockdown of FGF14 in granule cells reduced Ca2+ currents and diminished vesicular recycling, a marker for presynaptic Ca2+ influx. As a consequence, excitatory postsynaptic currents (EPSCs) at the granule cell to Purkinje cell synapse were markedly diminished. Expression of the SCA27-causing FGF14 mutant in granule cells exerted adominant-negative reduction in Ca2+ currents, vesicular recycling, and the resultant EPSCs in Purkinje cells. Thus, FHFs are multimodal, regulating several discrete neuronal signaling events. SCA27 most likely results at least in part from dysregulation of Ca2+ channel function. © 2013 The Authors.

Authors
Yan, H; Pablo, JL; Pitt, GS
MLA Citation
Yan, H, Pablo, JL, and Pitt, GS. "FGF14 Regulates Presynaptic Ca2+ Channels and Synaptic Transmission." Cell Reports 4.1 (July 11, 2013): 66-75.
Source
scopus
Published In
Cell Reports
Volume
4
Issue
1
Publish Date
2013
Start Page
66
End Page
75
DOI
10.1016/j.celrep.2013.06.012

FGF14 regulates presynaptic Ca2+ channels and synaptic transmission.

Fibroblast growth factor homologous factors (FHFs) are not growth factors, but instead bind to voltage-gated Na+ channels (NaV) and regulate their function. Mutations in FGF14, an FHF that is the locus for spinocerebellar ataxia 27 (SCA27), are believed to be pathogenic because of a dominant-negative reduction of NaV currents in cerebellar granule cells. Here, we demonstrate that FGF14 also regulates members of the presynaptic CaV2 Ca2+ channel family. Knockdown of FGF14 in granule cells reduced Ca2+ currents and diminished vesicular recycling, a marker for presynaptic Ca2+ influx. As a consequence, excitatory postsynaptic currents (EPSCs) at the granule cell to Purkinje cell synapse were markedly diminished. Expression of the SCA27-causing FGF14 mutant in granule cells exerted a dominant-negative reduction in Ca2+ currents, vesicular recycling, and the resultant EPSCs in Purkinje cells. Thus, FHFs are multimodal, regulating several discrete neuronal signaling events. SCA27 most likely results at least in part from dysregulation of Ca2+ channel function.

Authors
Yan, H; Pablo, JL; Pitt, GS
MLA Citation
Yan, H, Pablo, JL, and Pitt, GS. "FGF14 regulates presynaptic Ca2+ channels and synaptic transmission." Cell Rep 4.1 (July 11, 2013): 66-75.
PMID
23831029
Source
pubmed
Published In
Cell Reports
Volume
4
Issue
1
Publish Date
2013
Start Page
66
End Page
75
DOI
10.1016/j.celrep.2013.06.012

The auxiliary subunit KChIP2 is an essential regulator of homeostatic excitability.

BACKGROUND: The necessity for, or redundancy of, distinctive KChIP proteins is not known. RESULTS: Deletion of KChIP2 leads to increased susceptibility to epilepsy and to a reduction in IA and increased excitability in pyramidal hippocampal neurons. CONCLUSION: KChIP2 is essential for homeostasis in hippocampal neurons. SIGNIFICANCE: Mutations in K(A) channel auxiliary subunits may be loci for epilepsy. The somatodendritic IA (A-type) K(+) current underlies neuronal excitability, and loss of IA has been associated with the development of epilepsy. Whether any one of the four auxiliary potassium channel interacting proteins (KChIPs), KChIP1-KChIP4, in specific neuronal populations is critical for IA is not known. Here we show that KChIP2, which is abundantly expressed in hippocampal pyramidal cells, is essential for IA regulation in hippocampal neurons and that deletion of Kchip2 affects susceptibility to limbic seizures. The specific effects of Kchip2 deletion on IA recorded from isolated hippocampal pyramidal neurons were a reduction in amplitude and shift in the V½ for steady-state inactivation to hyperpolarized potentials when compared with WT neurons. Consistent with the relative loss of IA, hippocampal neurons from Kchip2(-/-) mice showed increased excitability. WT cultured neurons fired only occasional single action potentials, but the average spontaneous firing rate (spikes/s) was almost 10-fold greater in Kchip2(-/-) neurons. In slice preparations, spontaneous firing was detected in CA1 pyramidal neurons from Kchip2(-/-) mice but not from WT. Additionally, when seizures were induced by kindling, the number of stimulations required to evoke an initial class 4 or 5 seizure was decreased, and the average duration of electrographic seizures was longer in Kchip2(-/-) mice compared with WT controls. Together, these data demonstrate that the KChIP2 is essential for physiologic IA modulation and homeostatic stability and that there is a lack of functional redundancy among the different KChIPs in hippocampal neurons.

Authors
Wang, HG; He, XP; Li, Q; Madison, RD; Moore, SD; McNamara, JO; Pitt, GS
MLA Citation
Wang, HG, He, XP, Li, Q, Madison, RD, Moore, SD, McNamara, JO, and Pitt, GS. "The auxiliary subunit KChIP2 is an essential regulator of homeostatic excitability." J Biol Chem 288.19 (May 10, 2013): 13258-13268.
PMID
23536187
Source
pubmed
Published In
The Journal of biological chemistry
Volume
288
Issue
19
Publish Date
2013
Start Page
13258
End Page
13268
DOI
10.1074/jbc.M112.434548

Calcium influx through L-type Ca(V)1.2 Ca2+ channels regulates mandibular development

Authors
Ramachandran, KV; Hennessey, JA; Barnett, AS; Yin, X; Stadt, HA; Foster, E; Shah, RA; Yazawa, M; Dolmetsch, RE; Kirby, ML; Pitt, GS
MLA Citation
Ramachandran, KV, Hennessey, JA, Barnett, AS, Yin, X, Stadt, HA, Foster, E, Shah, RA, Yazawa, M, Dolmetsch, RE, Kirby, ML, and Pitt, GS. "Calcium influx through L-type Ca(V)1.2 Ca2+ channels regulates mandibular development." JOURNAL OF CLINICAL INVESTIGATION 123.4 (April 2013): 1638-1646.
PMID
23549079
Source
wos-lite
Published In
Journal of Clinical Investigation
Volume
123
Issue
4
Publish Date
2013
Start Page
1638
End Page
1646
DOI
10.1172/JCI66903

Calmodulin and CaMKII as Ca<sup>2+</sup> Switches for Cardiac Ion Channels

Changes in intracellular Ca2+ are among the most diverse and important means of cell signaling. In the heart, signaling pathways from excitation-contraction coupling to humoral activation of hypertrophic responses all rely on changes in the concentration of intracellular Ca2+. Ion channels belong to a class of signaling proteins that is particularly sensitive to a change in intracellular Ca2+, which is the final signal of all coordinated ion channel activity. Thus, regulation of ion channel function by Ca2+ provides an essential feedback mechanism for cellular electrical activity. Two intracellular proteins activated by Ca2+, the ubiquitous Ca2+ binding protein calmodulin (CaM), and the Ca2+/CaM-dependent protein kinase CaMKII, dictate most of the actions of intracellular Ca2+ on cardiac ion channels. In this chapter, we highlight key roles for CaM and CaMKII in cardiac myocytes as downstream integrators of Ca2+ signals in the regulation of cardiac ion channels and consequent actions on myocyte excitability. © 2014 Elsevier Inc. All rights reserved.

Authors
Pitt, GS; Marx, SO
MLA Citation
Pitt, GS, and Marx, SO. "Calmodulin and CaMKII as Ca<sup>2+</sup> Switches for Cardiac Ion Channels." Cardiac Electrophysiology: From Cell to Bedside: Sixth Edition. January 1, 2013. 189-195.
Source
scopus
Publish Date
2013
Start Page
189
End Page
195
DOI
10.1016/B978-1-4557-2856-5.00019-4

Long-term in vivo imaging of multiple organs at the single cell level.

Two-photon microscopy has enabled the study of individual cell behavior in live animals. Many organs and tissues cannot be studied, especially longitudinally, because they are located too deep, behind bony structures or too close to the lung and heart. Here we report a novel mouse model that allows long-term single cell imaging of many organs. A wide variety of live tissues were successfully engrafted in the pinna of the mouse ear. Many of these engrafted tissues maintained the normal tissue histology. Using the heart and thymus as models, we further demonstrated that the engrafted tissues functioned as would be expected. Combining two-photon microscopy with fluorescent tracers, we successfully visualized the engrafted tissues at the single cell level in live mice over several months. Four dimensional (three-dimensional (3D) plus time) information of individual cells was obtained from this imaging. This model makes long-term high resolution 4D imaging of multiple organs possible.

Authors
Chen, BJ; Jiao, Y; Zhang, P; Sun, AY; Pitt, GS; Deoliveira, D; Drago, N; Ye, T; Liu, C; Chao, NJ
MLA Citation
Chen, BJ, Jiao, Y, Zhang, P, Sun, AY, Pitt, GS, Deoliveira, D, Drago, N, Ye, T, Liu, C, and Chao, NJ. "Long-term in vivo imaging of multiple organs at the single cell level." PLoS One 8.1 (2013): e52087-.
PMID
23300962
Source
pubmed
Published In
PloS one
Volume
8
Issue
1
Publish Date
2013
Start Page
e52087
DOI
10.1371/journal.pone.0052087

Can polymorphisms predict response to antiarrhythmic drugs in atrial fibrillation?

Authors
Daubert, JP; Pitt, GS
MLA Citation
Daubert, JP, and Pitt, GS. "Can polymorphisms predict response to antiarrhythmic drugs in atrial fibrillation?." J Am Coll Cardiol 60.6 (August 7, 2012): 546-547.
PMID
22726628
Source
pubmed
Published In
Journal of the American College of Cardiology
Volume
60
Issue
6
Publish Date
2012
Start Page
546
End Page
547
DOI
10.1016/j.jacc.2012.02.060

A VGF-derived peptide attenuates development of type 2 diabetes via enhancement of islet β-cell survival and function.

Deterioration of functional islet β-cell mass is the final step in progression to Type 2 diabetes. We previously reported that overexpression of Nkx6.1 in rat islets has the dual effects of enhancing glucose-stimulated insulin secretion (GSIS) and increasing β-cell replication. Here we show that Nkx6.1 strongly upregulates the prohormone VGF in rat islets and that VGF is both necessary and sufficient for Nkx6.1-mediated enhancement of GSIS. Moreover, the VGF-derived peptide TLQP-21 potentiates GSIS in rat and human islets and improves glucose tolerance in vivo. Chronic injection of TLQP-21 in prediabetic ZDF rats preserves islet mass and slows diabetes onset. TLQP-21 prevents islet cell apoptosis by a pathway similar to that used by GLP-1, but independent of the GLP-1, GIP, or VIP receptors. Unlike GLP-1, TLQP-21 does not inhibit gastric emptying or increase heart rate. We conclude that TLQP-21 is a targeted agent for enhancing islet β-cell survival and function.

Authors
Stephens, SB; Schisler, JC; Hohmeier, HE; An, J; Sun, AY; Pitt, GS; Newgard, CB
MLA Citation
Stephens, SB, Schisler, JC, Hohmeier, HE, An, J, Sun, AY, Pitt, GS, and Newgard, CB. "A VGF-derived peptide attenuates development of type 2 diabetes via enhancement of islet β-cell survival and function." Cell Metab 16.1 (July 3, 2012): 33-43.
PMID
22768837
Source
pubmed
Published In
Cell Metabolism
Volume
16
Issue
1
Publish Date
2012
Start Page
33
End Page
43
DOI
10.1016/j.cmet.2012.05.011

Crystal structure of the ternary complex of a NaV C-terminal domain, a fibroblast growth factor homologous factor, and calmodulin.

Voltage-gated Na⁺ (Na(V)) channels initiate neuronal action potentials. Na(V) channels are composed of a transmembrane domain responsible for voltage-dependent Na⁺ conduction and a cytosolic C-terminal domain (CTD) that regulates channel function through interactions with many auxiliary proteins, including fibroblast growth factor homologous factors (FHFs) and calmodulin (CaM). Most ion channel structural studies have focused on mechanisms of permeation and voltage-dependent gating but less is known about how intracellular domains modulate channel function. Here we report the crystal structure of the ternary complex of a human Na(V) CTD, an FHF, and Ca²⁺-free CaM at 2.2 Å. Combined with functional experiments based on structural insights, we present a platform for understanding the roles of these auxiliary proteins in Na(V) channel regulation and the molecular basis of mutations that lead to neuronal and cardiac diseases. Furthermore, we identify a critical interaction that contributes to the specificity of individual Na(V) CTD isoforms for distinctive FHFs.

Authors
Wang, C; Chung, BC; Yan, H; Lee, SY; Pitt, GS
MLA Citation
Wang, C, Chung, BC, Yan, H, Lee, SY, and Pitt, GS. "Crystal structure of the ternary complex of a NaV C-terminal domain, a fibroblast growth factor homologous factor, and calmodulin." Structure 20.7 (July 3, 2012): 1167-1176.
PMID
22705208
Source
pubmed
Published In
Structure
Volume
20
Issue
7
Publish Date
2012
Start Page
1167
End Page
1176
DOI
10.1016/j.str.2012.05.001

Novel Roles of Fibroblast Growth Factor Homologous Factors in Heart

Authors
Amenta, J; Pitt, GS
MLA Citation
Amenta, J, and Pitt, GS. "Novel Roles of Fibroblast Growth Factor Homologous Factors in Heart." BIOPHYSICAL JOURNAL 102.3 (January 31, 2012): 34A-34A.
Source
wos-lite
Published In
Biophysical Journal
Volume
102
Issue
3
Publish Date
2012
Start Page
34A
End Page
34A

Thermosensory and nonthermosensory isoforms of Drosophila melanogaster TRPA1 reveal heat-sensor domains of a thermoTRP Channel.

Specialized somatosensory neurons detect temperatures ranging from pleasantly cool or warm to burning hot and painful (nociceptive). The precise temperature ranges sensed by thermally sensitive neurons is determined by tissue-specific expression of ion channels of the transient receptor potential(TRP) family.We show here that in Drosophila, TRPA1 is required for the sensing of nociceptive heat. We identify two previously unidentified protein isoforms of dTRPA1, named dTRPA1-C and dTRPA1-D, that explain this requirement. A dTRPA1-C/D reporter was exclusively expressed in nociceptors, and dTRPA1-C rescued thermal nociception phenotypes when restored to mutant nociceptors. However,surprisingly, we find that dTRPA1-C is not a direct heat sensor. Alternative splicing generates at least four isoforms of dTRPA1. Our analysis of these isoforms reveals a 37-amino-acid-long intracellular region (encoded by a single exon) that is critical for dTRPA1 temperature responses. The identification of these amino acids opens the door to a biophysical understanding of a molecular thermosensor.

Authors
Zhong, L; Bellemer, A; Yan, H; Ken, H; Jessica, R; Hwang, RY; Pitt, GS; Tracey, WD
MLA Citation
Zhong, L, Bellemer, A, Yan, H, Ken, H, Jessica, R, Hwang, RY, Pitt, GS, and Tracey, WD. "Thermosensory and nonthermosensory isoforms of Drosophila melanogaster TRPA1 reveal heat-sensor domains of a thermoTRP Channel." Cell Rep 1.1 (January 26, 2012): 43-55.
PMID
22347718
Source
pubmed
Published In
Cell Reports
Volume
1
Issue
1
Publish Date
2012
Start Page
43
End Page
55
DOI
10.1016/j.celrep.2011.11.002

Dissection of a quantitative trait locus for PR interval duration identifies Tnni3k as a novel modulator of cardiac conduction.

Atrio-ventricular conduction disease is a common feature in Mendelian rhythm disorders associated with sudden cardiac death and is characterized by prolongation of the PR interval on the surface electrocardiogram (ECG). Prolongation of the PR interval is also a strong predictor of atrial fibrillation, the most prevalent sustained cardiac arrhythmia. Despite the significant genetic component in PR duration variability, the genes regulating PR interval duration remain largely elusive. We here aimed to dissect the quantitative trait locus (QTL) for PR interval duration that we previously mapped in murine F2 progeny of a sensitized 129P2 and FVBN/J cross. To determine the underlying gene responsible for this QTL, genome-wide transcriptional profiling was carried out on myocardial tissue from 109 F2 mice. Expression QTLs (eQTLs) were mapped and the PR interval QTL was inspected for the co-incidence of eQTLs. We further determined the correlation of each of these transcripts to the PR interval. Tnni3k was the only eQTL, mapping to the PR-QTL, with an established abundant cardiac-specific expression pattern and a significant correlation to PR interval duration. Genotype inspection in various inbred mouse strains revealed the presence of at least three independent haplotypes at the Tnni3k locus. Measurement of PR interval duration and Tnni3k mRNA expression levels in six inbred lines identified a positive correlation between the level of Tnni3k mRNA and PR interval duration. Furthermore, in DBA/2J mice overexpressing hTNNI3K, and in DBA.AKR.hrtfm2 congenic mice, which harbor the AKR/J "high-Tnni3k expression" haplotype in the DBA/2J genetic background, PR interval duration was prolonged as compared to DBA/2J wild-type mice ("low-Tnni3k expression" haplotype). Our data provide the first evidence for a role of Tnni3k in controlling the electrocardiographic PR interval indicating a function of Tnni3k in atrio-ventricular conduction.

Authors
Lodder, EM; Scicluna, BP; Milano, A; Sun, AY; Tang, H; Remme, CA; Moerland, PD; Tanck, MW; Pitt, GS; Marchuk, DA; Bezzina, CR
MLA Citation
Lodder, EM, Scicluna, BP, Milano, A, Sun, AY, Tang, H, Remme, CA, Moerland, PD, Tanck, MW, Pitt, GS, Marchuk, DA, and Bezzina, CR. "Dissection of a quantitative trait locus for PR interval duration identifies Tnni3k as a novel modulator of cardiac conduction." PLoS Genet 8.12 (2012): e1003113-.
PMID
23236294
Source
pubmed
Published In
PLoS genetics
Volume
8
Issue
12
Publish Date
2012
Start Page
e1003113
DOI
10.1371/journal.pgen.1003113

Fibroblast growth factor homologous factors in the heart: a potential locus for cardiac arrhythmias.

The four fibroblast growth factor homologous factors (FHFs; FGF11-FGF14) are intracellular proteins that bind and modulate voltage-gated sodium channels (VGSCs). Although FHFs have been well studied in neurons and implicated in neurologic disease, their role in cardiomyocytes was unclear until recently. This review discusses the expression profile and function of FHFs in mouse and rat ventricular cardiomyocytes. Recent data show that FGF13 is the predominant FHF in the murine heart, directly binds the cardiac VGSC α subunit, and is essential for normal cardiac conduction. FHF loss-of-function mutations may be unrecognized causes of cardiac arrhythmias, such as long QT and Brugada syndromes.

Authors
Wei, EQ; Barnett, AS; Pitt, GS; Hennessey, JA
MLA Citation
Wei, EQ, Barnett, AS, Pitt, GS, and Hennessey, JA. "Fibroblast growth factor homologous factors in the heart: a potential locus for cardiac arrhythmias." Trends Cardiovasc Med 21.7 (October 2011): 199-203. (Review)
PMID
22867699
Source
pubmed
Published In
Trends in Cardiovascular Medicine
Volume
21
Issue
7
Publish Date
2011
Start Page
199
End Page
203
DOI
10.1016/j.tcm.2012.05.010

Fibroblast growth factor homologous factor 13 regulates Na+ channels and conduction velocity in murine hearts.

RATIONALE: Fibroblast growth factor homologous factors (FHFs), a subfamily of fibroblast growth factors (FGFs) that are incapable of functioning as growth factors, are intracellular modulators of Na(+) channels and have been linked to neurodegenerative diseases. Although certain FHFs have been found in embryonic heart, they have not been reported in adult heart, and they have not been shown to regulate endogenous cardiac Na(+) channels or to participate in cardiac pathophysiology. OBJECTIVE: We tested whether FHFs regulate Na(+) channels in murine heart. METHODS AND RESULTS: We demonstrated that isoforms of FGF13 are the predominant FHFs in adult mouse ventricular myocytes. FGF13 binds directly to, and colocalizes with, the Na(V)1.5 Na(+) channel in the sarcolemma of adult mouse ventricular myocytes. Knockdown of FGF13 in adult mouse ventricular myocytes revealed a loss of function of Na(V)1.5-reduced Na(+) current density, decreased Na(+) channel availability, and slowed Na(V)1.5-reduced Na(+) current recovery from inactivation. Cell surface biotinylation experiments showed ≈45% reduction in Na(V)1.5 protein at the sarcolemma after FGF13 knockdown, whereas no changes in whole-cell Na(V)1.5 protein or in mRNA level were observed. Optical imaging in neonatal rat ventricular myocyte monolayers demonstrated slowed conduction velocity and a reduced maximum capture rate after FGF13 knockdown. CONCLUSION: These findings show that FHFs are potent regulators of Na(+) channels in adult ventricular myocytes and suggest that loss-of-function mutations in FHFs may underlie a similar set of cardiac arrhythmias and cardiomyopathies that result from Na(V)1.5 loss-of-function mutations.

Authors
Wang, C; Hennessey, JA; Kirkton, RD; Wang, C; Graham, V; Puranam, RS; Rosenberg, PB; Bursac, N; Pitt, GS
MLA Citation
Wang, C, Hennessey, JA, Kirkton, RD, Wang, C, Graham, V, Puranam, RS, Rosenberg, PB, Bursac, N, and Pitt, GS. "Fibroblast growth factor homologous factor 13 regulates Na+ channels and conduction velocity in murine hearts." Circ Res 109.7 (September 16, 2011): 775-782.
PMID
21817159
Source
pubmed
Published In
Circulation Research
Volume
109
Issue
7
Publish Date
2011
Start Page
775
End Page
782
DOI
10.1161/CIRCRESAHA.111.247957

Identification of novel interaction sites that determine specificity between fibroblast growth factor homologous factors and voltage-gated sodium channels.

Fibroblast growth factor homologous factors (FHFs, FGF11-14) bind to the C termini (CTs) of specific voltage-gated sodium channels (VGSC) and thereby regulate their function. The effect of an individual FHF on a specific VGSC varies greatly depending upon the individual FHF isoform. How individual FHFs impart distinctive effects on specific VGSCs is not known and the specificity of these pairwise interactions is not understood. Using several biochemical approaches combined with functional analysis, we mapped the interaction site for FGF12B on the Na(V)1.5 C terminus and discovered previously unknown determinants necessary for FGF12 interaction. Also, we demonstrated that FGF12B binds to some, but not all Na(V)1 CTs, suggesting specificity of interaction. Exploiting a human single nucleotide polymorphism in the core domain of FGF12 (P149Q), we identified a surface proline that contributes a part of this pairwise specificity. This proline is conserved among all FHFs, and mutation of the homologous residue in FGF13 also leads to loss of interaction with a specific VGSC CT (Na(V)1.1) and loss of modulation of the resultant Na(+) channel function. We hypothesized that some of the specificity mediated by this proline may result from differences in the affinity of the binding partners. Consistent with this hypothesis, surface plasmon resonance data showed that the P149Q mutation decreased the binding affinity between FHFs and VGSC CTs. Moreover, immunocytochemistry revealed that the mutation prevented proper subcellular targeting of FGF12 to the axon initial segment in neurons. Together, these results give new insights into details of the interactions between FHFs and Na(V)1.x CTs, and the consequent regulation of Na(+) channels.

Authors
Wang, C; Wang, C; Hoch, EG; Pitt, GS
MLA Citation
Wang, C, Wang, C, Hoch, EG, and Pitt, GS. "Identification of novel interaction sites that determine specificity between fibroblast growth factor homologous factors and voltage-gated sodium channels." J Biol Chem 286.27 (July 8, 2011): 24253-24263.
PMID
21566136
Source
pubmed
Published In
The Journal of biological chemistry
Volume
286
Issue
27
Publish Date
2011
Start Page
24253
End Page
24263
DOI
10.1074/jbc.M111.245803

The S1103Y cardiac sodium channel variant is associated with implantable cardioverter-defibrillator events in blacks with heart failure and reduced ejection fraction.

BACKGROUND: Risk-stratifying heart failure patients for primary prevention implantable cardioverter-defibrillators (ICDs) remains a challenge, especially for blacks, who have an increased incidence of sudden cardiac death but have been underrepresented in clinical trials. We hypothesized that the S1103Y cardiac sodium channel SCN5A variant influences the propensity for ventricular arrhythmias in black patients with heart failure and reduced ejection fraction. METHODS AND RESULTS: Blacks (n=112) with ejection fractions <35% receiving primary prevention ICDs were identified from the Duke Electrophysiology Genetic and Genomic Studies (EPGEN) biorepository and followed for appropriate ICD therapy (either anti tachycardia pacing or shock) for documented sustained ventricular tachycardia or fibrillation. The S1103Y variant was overrepresented in patients receiving appropriate ICD therapy compared with subjects who did not (35% versus 13%, P=0.03). Controlling for baseline characteristics, the adjusted hazard ratio using a Cox proportional hazard model for ICD therapy in Y1103 allele carriers was 4.33 (95% confidence interval, 1.60 to 11.73, P=<0.01). There was no difference in mortality between carriers and noncarriers. CONCLUSIONS: This is the first report that the S1103Y variant is associated with a higher incidence of ventricular arrhythmias in blacks with heart failure and reduced ejection fraction.

Authors
Sun, AY; Koontz, JI; Shah, SH; Piccini, JP; Nilsson, KR; Craig, D; Haynes, C; Gregory, SG; Hranitzky, PM; Pitt, GS
MLA Citation
Sun, AY, Koontz, JI, Shah, SH, Piccini, JP, Nilsson, KR, Craig, D, Haynes, C, Gregory, SG, Hranitzky, PM, and Pitt, GS. "The S1103Y cardiac sodium channel variant is associated with implantable cardioverter-defibrillator events in blacks with heart failure and reduced ejection fraction." Circ Cardiovasc Genet 4.2 (April 2011): 163-168.
PMID
21498565
Source
pubmed
Published In
Circulation: Cardiovascular Genetics
Volume
4
Issue
2
Publish Date
2011
Start Page
163
End Page
168
DOI
10.1161/CIRCGENETICS.110.958652

Pinning down the CaMKII targets in the L-type Ca(2+) channel: an essential step in defining CaMKII regulation.

Authors
Sun, AY; Pitt, GS
MLA Citation
Sun, AY, and Pitt, GS. "Pinning down the CaMKII targets in the L-type Ca(2+) channel: an essential step in defining CaMKII regulation." Heart Rhythm 8.4 (April 2011): 631-633.
PMID
20933100
Source
pubmed
Published In
Heart Rhythm
Volume
8
Issue
4
Publish Date
2011
Start Page
631
End Page
633
DOI
10.1016/j.hrthm.2010.10.001

Rem2-targeted shRNAs reduce frequency of miniature excitatory postsynaptic currents without altering voltage-gated Ca²⁺ currents.

Ca²⁺ influx through voltage-gated Ca²⁺ channels (VGCCs) plays important roles in neuronal cell development and function. Rem2 is a member of the RGK (Rad, Rem, Rem2, Gem/Kir) subfamily of small GTPases that confers potent inhibition upon VGCCs. The physiologic roles of RGK proteins, particularly in the brain, are poorly understood. Rem2 was implicated in synaptogenesis through an RNAi screen and proposed to regulate Ca²⁺ homeostasis in neurons. To test this hypothesis and uncover physiological roles for Rem2 in the brain, we investigated the molecular mechanisms by which Rem2 knockdown affected synaptogenesis and Ca²⁺ homeostasis in cultured rat hippocampal neurons. Expression of a cocktail of shRNAs targeting rat Rem2 (rRem2) reduced the frequency of miniature excitatory postsynaptic currents (mEPSCs) measured 10 d after transfection (14 d in vitro), but did not affect mEPSC amplitude. VGCC current amplitude after rRem2-targeted knockdown was not different from that in control cells, however, at either 4 or 10 d post transfection. Co-expression of a human Rem2 that was insensitive to the shRNAs targeting rRem2 was unable to prevent the reduction in mEPSC frequency after rRem2-targeted knockdown. Over-expression of rRem2 resulted in 50% reduction in VGCC current, but neither the mEPSC frequency nor amplitude was affected. Taken together, the observed effects upon synaptogenesis after shRNA treatment are more likely due to mechanisms other than modulation of VGCCs and Ca²⁺ homeostasis, and may be independent of Rem2. In addition, our results reveal a surprising lack of contribution of VGCCs to synaptogenesis during early development in cultured hippocampal neurons.

Authors
Wang, HG; Wang, C; Pitt, GS
MLA Citation
Wang, HG, Wang, C, and Pitt, GS. "Rem2-targeted shRNAs reduce frequency of miniature excitatory postsynaptic currents without altering voltage-gated Ca²⁺ currents." PLoS One 6.9 (2011): e25741-.
PMID
21980534
Source
pubmed
Published In
PloS one
Volume
6
Issue
9
Publish Date
2011
Start Page
e25741
DOI
10.1371/journal.pone.0025741

The glitter of gold: Biolistic transfection of fresh adult cardiac myocytes. Focus on "normal targeting of a tagged Kv1.5 channel acutely transfected into fresh adult cardiac myocytes by a biolistic method"

Authors
Amenta, JJ; Pitt, GS
MLA Citation
Amenta, JJ, and Pitt, GS. "The glitter of gold: Biolistic transfection of fresh adult cardiac myocytes. Focus on "normal targeting of a tagged Kv1.5 channel acutely transfected into fresh adult cardiac myocytes by a biolistic method"." American Journal of Physiology - Cell Physiology 298.6 (2010): C1305-C1307.
PMID
20357185
Source
scival
Published In
American journal of physiology. Cell physiology
Volume
298
Issue
6
Publish Date
2010
Start Page
C1305
End Page
C1307
DOI
10.1152/ajpcell.00110.2010

Rationale and design of the Duke Electrophysiology Genetic and Genomic Studies (EPGEN) biorepository.

BACKGROUND: Disturbances in cardiac rhythm can lead to significant morbidity and mortality. Many arrhythmias are known to have a heritable component, but the degree to which genetic variation contributes to disease risk and morbidity is poorly understood. METHODS AND RESULTS: The EPGEN is a prospective single-center repository that archives DNA, RNA, and protein samples obtained at the time of an electrophysiologic evaluation or intervention. To identify genes and molecular variants that are associated with risk for arrhythmic phenotypes, EPGEN uses unbiased genomic screening; candidate gene analysis; and both unbiased and targeted transcript, protein, and metabolite profiling. To date, EPGEN has successfully enrolled >1,500 subjects. The median age of the study population is 62.9 years; 35% of the subjects are female and 21% are black. To this point, the study population has been composed of patients who had undergone defibrillator (implantable cardioverter-defibrillator or cardiac resynchronization therapy defibrillator) implantation (45%), electrophysiology studies or ablation procedures (35%), and pacemaker implantation or other procedures (20%). The cohort has a high prevalence of comorbidities, including diabetes (33%), hypertension (73%), chronic kidney disease (26%), and peripheral vascular disease (13%). CONCLUSIONS: We have established a biorepository and clinical database composed of patients with electrophysiologic diseases. EPGEN will seek to (1) improve risk stratification, (2) elucidate mechanisms of arrhythmogenesis, and (3) identify novel pharmacologic targets for the treatment of heart rhythm disorders.

Authors
Koontz, JI; Haithcock, D; Cumbea, V; Waldron, A; Stricker, K; Hughes, A; Nilsson, K; Sun, A; Piccini, JP; Kraus, WE; Pitt, GS; Shah, SH; Hranitzky, P
MLA Citation
Koontz, JI, Haithcock, D, Cumbea, V, Waldron, A, Stricker, K, Hughes, A, Nilsson, K, Sun, A, Piccini, JP, Kraus, WE, Pitt, GS, Shah, SH, and Hranitzky, P. "Rationale and design of the Duke Electrophysiology Genetic and Genomic Studies (EPGEN) biorepository." Am Heart J 158.5 (November 2009): 719-725.
PMID
19853688
Source
pubmed
Published In
American Heart Journal
Volume
158
Issue
5
Publish Date
2009
Start Page
719
End Page
725
DOI
10.1016/j.ahj.2009.08.011

Channeling a New Focus for Heart Failure: Insights Into Ion Channels

Authors
Pitt, GS
MLA Citation
Pitt, GS. "Channeling a New Focus for Heart Failure: Insights Into Ion Channels." JOURNAL OF CARDIOVASCULAR PHARMACOLOGY 54.2 (August 2009): 95-97.
PMID
19597367
Source
wos-lite
Published In
Journal of Cardiovascular Pharmacology
Volume
54
Issue
2
Publish Date
2009
Start Page
95
End Page
97

Accessory subunit KChIP2 modulates the cardiac L-type calcium current.

Complex modulation of voltage-gated Ca2+ currents through the interplay among Ca2+ channels and various Ca(2+)-binding proteins is increasingly being recognized. The K+ channel interacting protein 2 (KChIP2), originally identified as an auxiliary subunit for K(V)4.2 and a component of the transient outward K+ channel (I(to)), is a Ca(2+)-binding protein whose regulatory functions do not appear restricted to K(V)4.2. Consequently, we hypothesized that KChIP2 is a direct regulator of the cardiac L-type Ca2+ current (I(Ca,L)). We found that I(Ca,L) density from KChIP2(-/-) myocytes is reduced by 28% compared to I(Ca,L) recorded from wild-type myocytes (P<0.05). This reduction in current density results from loss of a direct effect on the Ca2+ channel current, as shown in a transfected cell line devoid of confounding cardiac ion currents. I(Ca,L) regulation by KChIP2 was independent of Ca2+ binding to KChIP2. Biochemical analysis suggested a direct interaction between KChIP2 and the Ca(V)1.2 alpha(1C) subunit N terminus. We found that KChIP2 binds to the N-terminal inhibitory module of alpha(1C) and augments I(Ca,L) current density without increasing Ca(V)1.2 protein expression or trafficking to the plasma membrane. We propose a model in which KChIP2 impedes the N-terminal inhibitory module of Ca(V)1.2, resulting in increased I(Ca,L). In the context of recent reports that KChIP2 modulates multiple K(V) and Na(V) currents, these results suggest that KChIP2 is a multimodal regulator of cardiac ionic currents.

Authors
Thomsen, MB; Wang, C; Ozgen, N; Wang, HG; Rosen, MR; Pitt, GS
MLA Citation
Thomsen, MB, Wang, C, Ozgen, N, Wang, HG, Rosen, MR, and Pitt, GS. "Accessory subunit KChIP2 modulates the cardiac L-type calcium current." Circ Res 104.12 (June 19, 2009): 1382-1389.
PMID
19461043
Source
pubmed
Published In
Circulation Research
Volume
104
Issue
12
Publish Date
2009
Start Page
1382
End Page
1389
DOI
10.1161/CIRCRESAHA.109.196972

Genetics of cardiac repolarization.

Prolongation of the electrocardiographic QT interval, a measure of cardiac repolarization, is associated with arrhythmogenic disorders and is a risk factor for sudden cardiac death. Two genome-wide association studies (GWAS) of variation in the QT interval in population-based cohorts now report association with variants in a subset of ion channel genes and other new associations.

Authors
Shah, SH; Pitt, GS
MLA Citation
Shah, SH, and Pitt, GS. "Genetics of cardiac repolarization." Nat Genet 41.4 (April 2009): 388-389.
PMID
19338079
Source
pubmed
Published In
Nature Genetics
Volume
41
Issue
4
Publish Date
2009
Start Page
388
End Page
389

Solution structure of the NaV1.2 C-terminal EF-hand domain.

Voltage-gated sodium channels initiate the rapid upstroke of action potentials in many excitable tissues. Mutations within intracellular C-terminal sequences of specific channels underlie a diverse set of channelopathies, including cardiac arrhythmias and epilepsy syndromes. The three-dimensional structure of the C-terminal residues 1777-1882 of the human NaV1.2 voltage-gated sodium channel has been determined in solution by NMR spectroscopy at pH 7.4 and 290.5 K. The ordered structure extends from residues Leu-1790 to Glu-1868 and is composed of four alpha-helices separated by two short anti-parallel beta-strands; a less well defined helical region extends from residue Ser-1869 to Arg-1882, and a disordered N-terminal region encompasses residues 1777-1789. Although the structure has the overall architecture of a paired EF-hand domain, the NaV1.2 C-terminal domain does not bind Ca2+ through the canonical EF-hand loops, as evidenced by monitoring 1H,15N chemical shifts during aCa2+ titration. Backbone chemical shift resonance assignments and Ca2+ titration also were performed for the NaV1.5 (1773-1878) isoform, demonstrating similar secondary structure architecture and the absence of Ca2+ binding by the EF-hand loops. Clinically significant mutations identified in the C-terminal region of NaV1 sodium channels cluster in the helix I-IV interface and the helix II-III interhelical segment or in helices III and IV of the NaV1.2 (1777-1882) structure.

Authors
Miloushev, VZ; Levine, JA; Arbing, MA; Hunt, JF; Pitt, GS; Palmer, AG
MLA Citation
Miloushev, VZ, Levine, JA, Arbing, MA, Hunt, JF, Pitt, GS, and Palmer, AG. "Solution structure of the NaV1.2 C-terminal EF-hand domain." J Biol Chem 284.10 (March 6, 2009): 6446-6454.
PMID
19129176
Source
pubmed
Published In
The Journal of biological chemistry
Volume
284
Issue
10
Publish Date
2009
Start Page
6446
End Page
6454
DOI
10.1074/jbc.M807401200

Editorial: Channeling a new focus for heart failure: Insights into ion channels

Authors
Pitt, GS
MLA Citation
Pitt, GS. "Editorial: Channeling a new focus for heart failure: Insights into ion channels." Journal of Cardiovascular Pharmacology 54.2 (2009): 95-97.
Source
scival
Published In
Journal of Cardiovascular Pharmacology
Volume
54
Issue
2
Publish Date
2009
Start Page
95
End Page
97
DOI
10.1097/FJC.0b013e3181b2b6bd

Calcineurin finds a new partner in the L-type Ca2+ channel

Authors
Pitt, GS
MLA Citation
Pitt, GS. "Calcineurin finds a new partner in the L-type Ca2+ channel." Circulation Research 105.1 (2009): 7-8.
PMID
19571266
Source
scival
Published In
Circulation Research
Volume
105
Issue
1
Publish Date
2009
Start Page
7
End Page
8
DOI
10.1161/CIRCRESAHA.109.201236

Ca2+/CaM controls Ca2+-dependent inactivation of NMDA receptors by dimerizing the NR1 C termini.

Ca2+ influx through NMDA receptors (NMDARs) leads to channel inactivation, which limits Ca2+ entry and protects against excitotoxicity. Extensive functional data suggests that this Ca2+-dependent inactivation (CDI) requires both calmodulin (CaM) binding to the C0 cassette of the NR1 subunit's C terminus (CT) and regulation by alpha-actinin-2, but a molecular understanding of CDI has been elusive. Here we used a number of methods to analyze the molecular nature of the interaction among CaM, alpha-actinin-2, and the NR1 CT. We found that a single CaM binds to two NR1 CTs in a Ca2+-dependent manner and promotes their reversible "dimerization." Expressed NMDARs containing NR1 concatamers in which the NR1 C termini are "uncoupled" display markedly reduced CDI. In contrast to current models, alpha-actinin-2 does not bind to the NR1 CT. We propose a new model for CDI in which the noncanonical Ca2+/CaM-dependent dimerization of the two NR1 subunits inactivates the channel by propagating a conformational change from the short NR1 CT to the nearby channel pore.

Authors
Wang, C; Wang, HG; Xie, H; Pitt, GS
MLA Citation
Wang, C, Wang, HG, Xie, H, and Pitt, GS. "Ca2+/CaM controls Ca2+-dependent inactivation of NMDA receptors by dimerizing the NR1 C termini." J Neurosci 28.8 (February 20, 2008): 1865-1870.
PMID
18287503
Source
pubmed
Published In
The Journal of neuroscience : the official journal of the Society for Neuroscience
Volume
28
Issue
8
Publish Date
2008
Start Page
1865
End Page
1870
DOI
10.1523/JNEUROSCI.5417-07.2008

Ca2+/calmodulin regulates trafficking of Ca(V)1.2 Ca2+ channels in cultured hippocampal neurons.

As the Ca2+-sensor for Ca2+-dependent inactivation, calmodulin (CaM) has been proposed, but never definitively demonstrated, to be a constitutive Ca(V)1.2 Ca2+ channel subunit. Here we show that CaM is associated with the Ca(V)1.2 pore-forming alpha1C subunit in brain in a Ca2+-independent manner. Within its CaM binding pocket, alpha1C has been proposed to contain a membrane targeting domain. Because ion channel subunits assemble early during channel biosynthesis, we postulated that this association with CaM could afford the opportunity for Ca2+-dependent regulation of membrane targeting. We showed that the isolated domain functioned as a Ca2+/CaM regulated trafficking determinant for CD8 (a model transmembrane protein) using fluorescent-activated cell sorting analysis and, using green fluorescent protein-tagged alpha1C subunits expressed in cultured hippocampal neurons, that Ca2+/CaM interaction with this domain accelerated trafficking of Ca(V)1.2 channels to distal regions of the dendritic arbor. Furthermore, this Ca2+/CaM-accelerated trafficking was activity dependent. Thus, CaM imparts Ca2+-dependent regulation not only to mature Ca(V)1.2 channels at the cell surface but also to steps during channel biosynthesis.

Authors
Wang, HG; George, MS; Kim, J; Wang, C; Pitt, GS
MLA Citation
Wang, HG, George, MS, Kim, J, Wang, C, and Pitt, GS. "Ca2+/calmodulin regulates trafficking of Ca(V)1.2 Ca2+ channels in cultured hippocampal neurons." J Neurosci 27.34 (August 22, 2007): 9086-9093.
PMID
17715345
Source
pubmed
Published In
The Journal of neuroscience : the official journal of the Society for Neuroscience
Volume
27
Issue
34
Publish Date
2007
Start Page
9086
End Page
9093
DOI
10.1523/JNEUROSCI.1720-07.2007

Added benefit of mineralocorticoid receptor blockade in the primary prevention of sudden cardiac death.

Authors
Pitt, B; Pitt, GS
MLA Citation
Pitt, B, and Pitt, GS. "Added benefit of mineralocorticoid receptor blockade in the primary prevention of sudden cardiac death." Circulation 115.23 (June 12, 2007): 2976-2982. (Review)
PMID
17562968
Source
pubmed
Published In
Circulation
Volume
115
Issue
23
Publish Date
2007
Start Page
2976
End Page
2982
DOI
10.1161/CIRCULATIONAHA.106.670109

Calmodulin and CaMKII as molecular switches for cardiac ion channels.

Because changes in intracellular Ca(2+) concentration are the final signals of electrical activity in excitable cells, many mechanisms have evolved to regulate Ca(2+) influx. Among the most important are those pathways that directly regulate the ion channels responsible for regulating and generating the Ca(2+) influx signal. Recent work has demonstrated that the Ca(2+) binding protein calmodulin (CaM) and the Ca(2+)/CaM-sensitive kinase CaMKII are important modulators of cardiac ion channels. Thus, Ca(2+) participates in feedback modulation to control electrical activity. This review highlights various mechanisms by which CaM and CaMKII regulate cardiovascular ion channel activity and presents a novel model for CaMKII regulation of Ca(V)1.2 Ca(2+) channel function.

Authors
Pitt, GS
MLA Citation
Pitt, GS. "Calmodulin and CaMKII as molecular switches for cardiac ion channels." Cardiovasc Res 73.4 (March 1, 2007): 641-647. (Review)
PMID
17137569
Source
pubmed
Published In
Cardiovascular Research
Volume
73
Issue
4
Publish Date
2007
Start Page
641
End Page
647
DOI
10.1016/j.cardiores.2006.10.019

Response to Kloner and Cannom

Authors
Pitt, B; Pitt, GS
MLA Citation
Pitt, B, and Pitt, GS. "Response to Kloner and Cannom." Circulation 115.23 (2007): 2989--.
Source
scival
Published In
Circulation
Volume
115
Issue
23
Publish Date
2007
Start Page
2989-
DOI
10.1161/CIRCULATIONAHA.106.684522

Dose-dependent and isoform-specific modulation of Ca2+ channels by RGK GTPases.

Although inhibition of voltage-gated calcium channels by RGK GTPases (RGKs) represents an important mode of regulation to control Ca(2+) influx in excitable cells, their exact mechanism of inhibition remains controversial. This has prevented an understanding of how RGK regulation can be significant in a physiological context. Here we show that RGKs-Gem, Rem, and Rem2-decreased Ca(V)1.2 Ca(2+) current amplitude in a dose-dependent manner. Moreover, Rem2, but not Rem or Gem, produced dose-dependent alterations on gating kinetics, uncovering a new mode by which certain RGKs can precisely modulate Ca(2+) currents and affect Ca(2+) influx during action potentials. To explore how RGKs influence gating kinetics, we separated the roles mediated by the Ca(2+) channel accessory beta subunit's interaction with its high affinity binding site in the pore-forming alpha(1C) subunit (AID) from its other putative contact sites by utilizing an alpha(1C)*beta3 concatemer in which the AID was mutated to prevent beta subunit interaction. This mutant concatemer generated currents with all the hallmarks of beta subunit modulation, demonstrating that AID-beta-independent interactions are sufficient for beta subunit modulation. Using this construct we found that although inhibition of current amplitude was still partially sensitive to RGKs, Rem2 no longer altered gating kinetics, implicating different determinants for this specific mode of Rem2-mediated regulation. Together, these results offer new insights into the molecular mechanism of RGK-mediated Ca(2+) channel current modulation.

Authors
Seu, L; Pitt, GS
MLA Citation
Seu, L, and Pitt, GS. "Dose-dependent and isoform-specific modulation of Ca2+ channels by RGK GTPases." J Gen Physiol 128.5 (November 2006): 605-613.
PMID
17074979
Source
pubmed
Published In
The Journal of General Physiology
Volume
128
Issue
5
Publish Date
2006
Start Page
605
End Page
613
DOI
10.1085/jgp.200609631

Remodeled cardiac calcium channels.

Cardiac calcium channels play a pivotal role in the proper functioning of cardiac cells. In response to various pathologic stimuli, they become remodeled, changing how they function, as they adapt to their new environment. Specific features of remodeled channels depend upon the particular disease state. This review will summarize what is known about remodeled cardiac calcium channels in three disease states: hypertrophy, heart failure and atrial fibrillation. In addition, it will review the recent advances made in our understanding of the function of the various molecular building blocks that contribute to the proper functioning of the cardiac calcium channel.

Authors
Pitt, GS; Dun, W; Boyden, PA
MLA Citation
Pitt, GS, Dun, W, and Boyden, PA. "Remodeled cardiac calcium channels." J Mol Cell Cardiol 41.3 (September 2006): 373-388. (Review)
PMID
16901502
Source
pubmed
Published In
Journal of Molecular and Cellular Cardiology
Volume
41
Issue
3
Publish Date
2006
Start Page
373
End Page
388
DOI
10.1016/j.yjmcc.2006.06.071

Aldosterone, ion channels, and sudden death: another piece of the circle?

Authors
Pitt, GS; Pitt, B
MLA Citation
Pitt, GS, and Pitt, B. "Aldosterone, ion channels, and sudden death: another piece of the circle?." Am J Physiol Heart Circ Physiol 290.6 (June 2006): H2176-H2177.
PMID
16517947
Source
pubmed
Published In
American journal of physiology. Heart and circulatory physiology
Volume
290
Issue
6
Publish Date
2006
Start Page
H2176
End Page
H2177
DOI
10.1152/ajpheart.00186.2006

The real estate of cardiac signaling: location, location, location.

Authors
George, MS; Pitt, GS
MLA Citation
George, MS, and Pitt, GS. "The real estate of cardiac signaling: location, location, location." Proc Natl Acad Sci U S A 103.20 (May 16, 2006): 7535-7536.
PMID
16682624
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
103
Issue
20
Publish Date
2006
Start Page
7535
End Page
7536
DOI
10.1073/pnas.0602389103

KCNQ1 assembly and function is blocked by long-QT syndrome mutations that disrupt interaction with calmodulin.

Calmodulin (CaM) has been recognized as an obligate subunit for many ion channels in which its function has not been clearly established. Because channel subunits associate early during channel biosynthesis, CaM may provide a mechanism for Ca(2+)-dependent regulation of channel formation. Here we show that CaM is a constitutive component of KCNQ1 K+ channels, the most commonly mutated long-QT syndrome (LQTS) locus. CaM not only acts as a regulator of channel gating, relieving inactivation in a Ca(2+)-dependent manner, but it also contributes to control of channel assembly. Formation of functional tetramers requires CaM interaction with the KCNQ1 C-terminus. This CaM-regulated process is essential: LQTS mutants that disrupt CaM interaction prevent functional assembly of channels in a dominant-negative manner. These findings offer a new mechanism for LQTS defects and provide a basis for understanding novel ways that intracellular Ca2+ and CaM regulate ion channels.

Authors
Ghosh, S; Nunziato, DA; Pitt, GS
MLA Citation
Ghosh, S, Nunziato, DA, and Pitt, GS. "KCNQ1 assembly and function is blocked by long-QT syndrome mutations that disrupt interaction with calmodulin." Circ Res 98.8 (April 28, 2006): 1048-1054.
PMID
16556866
Source
pubmed
Published In
Circulation Research
Volume
98
Issue
8
Publish Date
2006
Start Page
1048
End Page
1054
DOI
10.1161/01.RES.0000218863.44140.f2

CaMKII tethers to L-type Ca2+ channels, establishing a local and dedicated integrator of Ca2+ signals for facilitation.

Ca2+-dependent facilitation (CDF) of voltage-gated calcium current is a powerful mechanism for up-regulation of Ca2+ influx during repeated membrane depolarization. CDF of L-type Ca2+ channels (Ca(v)1.2) contributes to the positive force-frequency effect in the heart and is believed to involve the activation of Ca2+/calmodulin-dependent kinase II (CaMKII). How CaMKII is activated and what its substrates are have not yet been determined. We show that the pore-forming subunit alpha(1C) (Ca(v)alpha1.2) is a CaMKII substrate and that CaMKII interaction with the COOH terminus of alpha1C is essential for CDF of L-type channels. Ca2+ influx triggers distinct features of CaMKII targeting and activity. After Ca2+-induced targeting to alpha1C, CaMKII becomes tightly tethered to the channel, even after calcium returns to normal levels. In contrast, activity of the tethered CaMKII remains fully Ca2+/CaM dependent, explaining its ability to operate as a calcium spike frequency detector. These findings clarify the molecular basis of CDF and demonstrate a novel enzymatic mechanism by which ion channel gating can be modulated by activity.

Authors
Hudmon, A; Schulman, H; Kim, J; Maltez, JM; Tsien, RW; Pitt, GS
MLA Citation
Hudmon, A, Schulman, H, Kim, J, Maltez, JM, Tsien, RW, and Pitt, GS. "CaMKII tethers to L-type Ca2+ channels, establishing a local and dedicated integrator of Ca2+ signals for facilitation." J Cell Biol 171.3 (November 7, 2005): 537-547.
PMID
16275756
Source
pubmed
Published In
The Journal of Cell Biology
Volume
171
Issue
3
Publish Date
2005
Start Page
537
End Page
547
DOI
10.1083/jcb.200505155

Essential Ca(V)beta modulatory properties are AID-independent.

Voltage-gated Ca(2+) channel beta (Ca(v)beta) subunits have a highly conserved core consisting of interacting Src homology 3 and guanylate kinase domains, and are postulated to exert their effects through AID, the major interaction site in the pore-forming alpha(1) subunit. This stereotypical interaction does not explain how individual Ca(v)beta subunits modulate alpha(1) subunits differentially. Here we show that AID is neither necessary nor sufficient for critical Ca(v)beta regulatory properties. Complete modulation depends on additional contacts that are exclusive of AID and not revealed in recent crystal structures. These data offer a new context for understanding Ca(v)beta modulation, suggesting that the AID interaction orients the Ca(v)beta core so as to permit additional isoform-specific Ca(v)alpha(1)-Ca(v)beta interactions that underlie the particular regulation seen with each Ca(v)alpha(1)-Ca(v)beta pair, rather than as the main site of regulation.

Authors
Maltez, JM; Nunziato, DA; Kim, J; Pitt, GS
MLA Citation
Maltez, JM, Nunziato, DA, Kim, J, and Pitt, GS. "Essential Ca(V)beta modulatory properties are AID-independent." Nat Struct Mol Biol 12.4 (April 2005): 372-377.
PMID
15750602
Source
pubmed
Published In
Nature Structural & Molecular Biology
Volume
12
Issue
4
Publish Date
2005
Start Page
372
End Page
377
DOI
10.1038/nsmb909

Calmodulin mediates Ca2+ sensitivity of sodium channels.

Ca2+ has been proposed to regulate Na+ channels through the action of calmodulin (CaM) bound to an IQ motif or through direct binding to a paired EF hand motif in the Nav1 C terminus. Mutations within these sites cause cardiac arrhythmias or autism, but details about how Ca2+ confers sensitivity are poorly understood. Studies on the homologous Cav1.2 channel revealed non-canonical CaM interactions, providing a framework for exploring Na+ channels. In contrast to previous reports, we found that Ca2+ does not bind directly to Na+ channel C termini. Rather, Ca2+ sensitivity appears to be mediated by CaM bound to the C termini in a manner that differs significantly from CaM regulation of Cav1.2. In Nav1.2 or Nav1.5, CaM bound to a localized region containing the IQ motif and did not support the large Ca(2+)-dependent conformational change seen in the Cav1.2.CaM complex. Furthermore, CaM binding to Nav1 C termini lowered Ca2+ binding affinity and cooperativity among the CaM-binding sites compared with CaM alone. Nonetheless, we found suggestive evidence for Ca2+/CaM-dependent effects upon Nav1 channels. The R1902C autism mutation conferred a Ca(2+)-dependent conformational change in Nav1.2 C terminus.CaM complex that was absent in the wild-type complex. In Nav1.5, CaM modulates the Cterminal interaction with the III-IV linker, which has been suggested as necessary to stabilize the inactivation gate, to minimize sustained channel activity during depolarization, and to prevent cardiac arrhythmias that lead to sudden death. Together, these data offer new biochemical evidence for Ca2+/CaM modulation of Na+ channel function.

Authors
Kim, J; Ghosh, S; Liu, H; Tateyama, M; Kass, RS; Pitt, GS
MLA Citation
Kim, J, Ghosh, S, Liu, H, Tateyama, M, Kass, RS, and Pitt, GS. "Calmodulin mediates Ca2+ sensitivity of sodium channels." J Biol Chem 279.43 (October 22, 2004): 45004-45012.
PMID
15316014
Source
pubmed
Published In
The Journal of biological chemistry
Volume
279
Issue
43
Publish Date
2004
Start Page
45004
End Page
45012
DOI
10.1074/jbc.M407286200

Calcium channel function regulated by the SH3-GK module in beta subunits.

beta subunits of voltage-gated calcium channels (VGCCs) regulate channel trafficking and function, thereby shaping the intensity and duration of intracellular changes in calcium. beta subunits share limited sequence homology with the Src homology 3-guanylate kinase (SH3-GK) module of membrane-associated guanylate kinases (MAGUKs). Here, we show biochemical similarities between beta subunits and MAGUKs, revealing important aspects of beta subunit structure and function. Similar to MAGUKs, an SH3-GK interaction within beta subunits can occur both intramolecularly and intermolecularly. Mutations that disrupt the SH3-GK interaction in beta subunits alter channel inactivation and can inhibit binding between the alpha(1) and beta subunits. Coexpression of beta subunits with complementary mutations in their SH3 and GK domains rescues these deficits through intermolecular beta subunit assembly. In MAGUKs, the SH3-GK module controls protein scaffolding. In beta subunits, this module regulates the inactivation of VGCCs and provides an additional mechanism for tuning calcium responsiveness.

Authors
McGee, AW; Nunziato, DA; Maltez, JM; Prehoda, KE; Pitt, GS; Bredt, DS
MLA Citation
McGee, AW, Nunziato, DA, Maltez, JM, Prehoda, KE, Pitt, GS, and Bredt, DS. "Calcium channel function regulated by the SH3-GK module in beta subunits." Neuron 42.1 (April 8, 2004): 89-99.
PMID
15066267
Source
pubmed
Published In
Neuron
Volume
42
Issue
1
Publish Date
2004
Start Page
89
End Page
99

Identification of the components controlling inactivation of voltage-gated Ca2+ channels.

Ca(2+)-dependent inactivation (CDI) of L-type voltage-gated Ca(2+) channels limits Ca(2+) entry into neurons, thereby regulating numerous cellular events. Here we present the isolation and purification of the Ca(2+)-sensor complex, consisting of calmodulin (CaM) and part of the channel's pore-forming alpha(1C) subunit, and demonstrate the Ca(2+)-dependent conformational shift that underlies inactivation. Dominant-negative CaM mutants that prevent CDI block the sensor's Ca(2+)-dependent conformational change. We show how Ile1654 in the CaM binding IQ motif of alpha(1C) forms the link between the Ca(2+) sensor and the downstream inactivation machinery, using the alpha(1C) EF hand motif as a signal transducer to activate the putative pore-occluder, the alpha(1C) I-II intracellular linker.

Authors
Kim, J; Ghosh, S; Nunziato, DA; Pitt, GS
MLA Citation
Kim, J, Ghosh, S, Nunziato, DA, and Pitt, GS. "Identification of the components controlling inactivation of voltage-gated Ca2+ channels." Neuron 41.5 (March 4, 2004): 745-754.
PMID
15003174
Source
pubmed
Published In
Neuron
Volume
41
Issue
5
Publish Date
2004
Start Page
745
End Page
754

Molecular basis of calmodulin tethering and Ca2+-dependent inactivation of L-type Ca2+ channels.

Ca(2+)-dependent inactivation (CDI) of L-type Ca(2+) channels plays a critical role in controlling Ca(2+) entry and downstream signal transduction in excitable cells. Ca(2+)-insensitive forms of calmodulin (CaM) act as dominant negatives to prevent CDI, suggesting that CaM acts as a resident Ca(2+) sensor. However, it is not known how the Ca(2+) sensor is constitutively tethered. We have found that the tethering of Ca(2+)-insensitive CaM was localized to the C-terminal tail of alpha(1C), close to the CDI effector motif, and that it depended on nanomolar Ca(2+) concentrations, likely attained in quiescent cells. Two stretches of amino acids were found to support the tethering and to contain putative CaM-binding sequences close to or overlapping residues previously shown to affect CDI and Ca(2+)-independent inactivation. Synthetic peptides containing these sequences displayed differences in CaM-binding properties, both in affinity and Ca(2+) dependence, leading us to propose a novel mechanism for CDI. In contrast to a traditional disinhibitory scenario, we suggest that apoCaM is tethered at two sites and signals actively to slow inactivation. When the C-terminal lobe of CaM binds to the nearby CaM effector sequence (IQ motif), the braking effect is relieved, and CDI is accelerated.

Authors
Pitt, GS; Zühlke, RD; Hudmon, A; Schulman, H; Reuter, H; Tsien, RW
MLA Citation
Pitt, GS, Zühlke, RD, Hudmon, A, Schulman, H, Reuter, H, and Tsien, RW. "Molecular basis of calmodulin tethering and Ca2+-dependent inactivation of L-type Ca2+ channels." J Biol Chem 276.33 (August 17, 2001): 30794-30802.
PMID
11408490
Source
pubmed
Published In
The Journal of biological chemistry
Volume
276
Issue
33
Publish Date
2001
Start Page
30794
End Page
30802
DOI
10.1074/jbc.M104959200

Erratum: Molecular basis of calmodulin tethering and Ca 2+-dependent inactivation of L-type Ca2+ channels (Journal of Biological Chemistry (2001) 276 (30794-30802))

Authors
Pitt, GS; Zühlke, RD; Hudmon, A; Schulman, H; Reuter, H; Tsien, RW
MLA Citation
Pitt, GS, Zühlke, RD, Hudmon, A, Schulman, H, Reuter, H, and Tsien, RW. "Erratum: Molecular basis of calmodulin tethering and Ca 2+-dependent inactivation of L-type Ca2+ channels (Journal of Biological Chemistry (2001) 276 (30794-30802))." Journal of Biological Chemistry 276.39 (2001): 36862--.
Source
scival
Published In
Journal of Biological Chemistry
Volume
276
Issue
39
Publish Date
2001
Start Page
36862-

Ca2+-sensitive inactivation and facilitation of L-type Ca2+ channels both depend on specific amino acid residues in a consensus calmodulin-binding motif in the(alpha)1C subunit.

L-type Ca(2+) channels are unusual in displaying two opposing forms of autoregulatory feedback, Ca(2+)-dependent inactivation and facilitation. Previous studies suggest that both involve direct interactions between calmodulin (CaM) and a consensus CaM-binding sequence (IQ motif) in the C terminus of the channel's alpha(1C) subunit. Here we report the functional effects of an extensive series of modifications of the IQ motif aimed at dissecting the structural determinants of the different forms of modulation. Although the combined substitution by alanine at five key positions (Ile(1624), Gln(1625), Phe(1628), Arg(1629), and Lys(1630)) abolished all Ca(2+) dependence, corresponding single alanine replacements behaved similarly to the wild-type channel (77wt) in four of five cases. The mutant I1624A stood out in displaying little or no Ca(2+)-dependent inactivation, but clear Ca(2+)- and frequency-dependent facilitation. An even more pronounced tilt in favor of facilitation was seen with the double mutant I1624A/Q1625A: overt facilitation was observed even during a single depolarizing pulse, as confirmed by two-pulse experiments. Replacement of Ile(1624) by 13 other amino acids produced graded and distinct patterns of change in the two forms of modulation. The extent of Ca(2+)-dependent facilitation was monotonically correlated with the affinity of CaM for the mutant IQ motif, determined in peptide binding experiments in vitro. Ca(2+)-dependent inactivation also depended on strong CaM binding to the IQ motif, but showed an additional requirement for a bulky, hydrophobic side chain at position 1624. Abolition of Ca(2+)-dependent modulation by IQ motif modifications mimicked and occluded the effects of overexpressing a dominant-negative CaM mutant.

Authors
Zühlke, RD; Pitt, GS; Tsien, RW; Reuter, H
MLA Citation
Zühlke, RD, Pitt, GS, Tsien, RW, and Reuter, H. "Ca2+-sensitive inactivation and facilitation of L-type Ca2+ channels both depend on specific amino acid residues in a consensus calmodulin-binding motif in the(alpha)1C subunit." J Biol Chem 275.28 (July 14, 2000): 21121-21129.
PMID
10779517
Source
pubmed
Published In
The Journal of biological chemistry
Volume
275
Issue
28
Publish Date
2000
Start Page
21121
End Page
21129
DOI
10.1074/jbc.M002986200

Calmodulin supports both inactivation and facilitation of L-type calcium channels.

L-type Ca2+ channels support Ca2+ entry into cells, which triggers cardiac contraction, controls hormone secretion from endocrine cells and initiates transcriptional events that support learning and memory. These channels are examples of molecular signal-transduction units that regulate themselves through their own activity. Among the many types of voltage-gated Ca2+ channel, L-type Ca2+ channels particularly display inactivation and facilitation, both of which are closely linked to the earlier entry of Ca2+ ions. Both forms of autoregulation have a significant impact on the amount of Ca2+ that enters the cell during repetitive activity, with major consequences downstream. Despite extensive biophysical analysis, the molecular basis of autoregulation remains unclear, although a putative Ca2+-binding EF-hand motif and a nearby consensus calmodulin-binding isoleucine-glutamine ('IQ') motif in the carboxy terminus of the alpha1C channel subunit have been implicated. Here we show that calmodulin is a critical Ca2+ sensor for both inactivation and facilitation, and that the nature of the modulatory effect depends on residues within the IQ motif important for calmodulin binding. Replacement of the native isoleucine by alanine removed Ca2+-dependent inactivation and unmasked a strong facilitation; conversion of the same residue to glutamate eliminated both forms of autoregulation. These results indicate that the same calmodulin molecule may act as a Ca2+ sensor for both positive and negative modulation.

Authors
Zühlke, RD; Pitt, GS; Deisseroth, K; Tsien, RW; Reuter, H
MLA Citation
Zühlke, RD, Pitt, GS, Deisseroth, K, Tsien, RW, and Reuter, H. "Calmodulin supports both inactivation and facilitation of L-type calcium channels." Nature 399.6732 (May 13, 1999): 159-162.
PMID
10335846
Source
pubmed
Published In
Nature
Volume
399
Issue
6732
Publish Date
1999
Start Page
159
End Page
162
DOI
10.1038/20200

Role of cAMP-dependent protein kinase in controlling aggregation and postaggregative development in dictyostelium

We have examined the role of cAMP-dependent protein kinase (PKA) in controlling aggregation and postaggregative development in Dictyostelium. We previously showed that cells in which the gene encoding the PKA catalytic subunit has been disrupted (pkacat- cells) are unable to aggregate [S. K. O. Mann and R. A. Firtel (1991). A developmentally regulated, putative serine/threonine protein kinase is essential for development in Dictyostelium. Mech. Dev. 35, 89-102]. We show that pkacat- cells are unable to activate adenylyl cyclase in response to cAMP stimulation due to the inability to express the aggregation-stage, G-protein-stimulated adenylyl cyclase (ACA). Constitutive expression of ACA from an actin promoter results in a high level of Mn2+-stimulated adenylyl cyclase activity and restores chemoattractant- and GTPγS,stimulated adenylyl cyclase activity but not the ability to aggregate. Similarly, expression of the constitutively active, non-G protein-coupled adenylyl cyclase ACG in pkacat- cells also does not restore the ability to aggregate, although ACG can complement cells in which the ACA gene has been disrupted. These results indicate that pkacat- cells lack multiple, essential aggregation-stage functions. As the mound forms, high, continuous levels of extracellular cAMP functioning through the cAMP serpentine receptors activate a transcriptional cascade that leads to cell-type differentiation and morphogenesis. The first step is the induction and activation of the transcription factor GBF and downstream postaggregative genes, followed by the induction of prestalk- and prespore-specific genes. We show that pkacat- cells induce postaggregative gene expression in response to exogenous cAMP, but the level of induction of some of these genes, including GBF is reduced. SP60 (a prespore-specific gene) is not induced and ecmA (a prestalk-specific gene) is induced to very low levels. Expressing GBF constitutively in pkacat- cells restores ecmA expression to a moderate level, but SP60 is not detectably induced. Overexpression of PKAcat from the Actin 15 (Act15) ecmA prestalk, and the PKAcat promoters in pkacat- cells results in significant aberrant spatial patterning of prestalk and prespore cells, as determined by lacZ reporter studies. Our studies identify new, essential regulatory roles for PKA in mediating multicellular development.

Authors
Mann, SKO; Brown, JM; Briscoe, C; Parent, C; Pitt, G; Devreotes, PN; Firtel, RA
MLA Citation
Mann, SKO, Brown, JM, Briscoe, C, Parent, C, Pitt, G, Devreotes, PN, and Firtel, RA. "Role of cAMP-dependent protein kinase in controlling aggregation and postaggregative development in dictyostelium." Developmental Biology 183.2 (1997): 208-221.
PMID
9126295
Source
scival
Published In
Developmental Biology
Volume
183
Issue
2
Publish Date
1997
Start Page
208
End Page
221
DOI
10.1006/dbio.1996.8499

Adenylyl cyclase G, an osmosensor controlling germination of Dictyostelium spores.

Dictyostelium cells express a G-protein-coupled adenylyl cyclase, ACA, during aggregation and an atypical adenylyl cyclase, ACG, in mature spores. The ACG gene was disrupted by homologous recombination. acg- cells developed into normal fruiting bodies with viable spores, but spore germination was no longer inhibited by high osmolarity, a fairly universal constraint for spore and seed germination. ACG activity, measured in aca-/ACG cells, was strongly stimulated by high osmolarity with optimal stimulation occurring at 200 milliosmolar. RdeC mutants, which display unrestrained protein kinase A (PKA) activity and a cell line, which overexpresses PKA under a prespore specific promoter, germinate very poorly, both at high and low osmolarity. These data indicate that ACG is an osmosensor controlling spore germination through activation of protein kinase A.

Authors
van Es, S; Virdy, KJ; Pitt, GS; Meima, M; Sands, TW; Devreotes, PN; Cotter, DA; Schaap, P
MLA Citation
van Es, S, Virdy, KJ, Pitt, GS, Meima, M, Sands, TW, Devreotes, PN, Cotter, DA, and Schaap, P. "Adenylyl cyclase G, an osmosensor controlling germination of Dictyostelium spores." J Biol Chem 271.39 (September 27, 1996): 23623-23625.
PMID
8798577
Source
pubmed
Published In
The Journal of biological chemistry
Volume
271
Issue
39
Publish Date
1996
Start Page
23623
End Page
23625

Extracellular cAMP is sufficient to restore developmental gene expression and morphogenesis in Dictyostelium cells lacking the aggregation adenylyl cyclase (ACA).

Cell movement and cell-type-specific gene expression during Dictyostelium development are regulated by cAMP, which functions both as an extracellular hormone-like signal and an intracellular second messenger. Previous data indicated that aca- mutants, which lack adenylyl cyclase activity, fail to aggregate and do not express cell-type-specific genes. We show here that overexpression of ACG, a constitutively active adenylyl cyclase, which in wild-type cells is only expressed during spore germination, partially restores the coordination of cell movement and completely restores developmental gene expression. The aca- cells can also be induced to develop into viable spores by synergy with wild-type cells and, furthermore, form small but normal fruiting bodies, after a developmentally relevant regimen of stimulation with nanomolar cAMP pulses followed by micromolar cAMP concentrations. 2'-Deoxy cAMP, a cAMP analog that activates the cell-surface cAMP receptors but not cAMP-dependent protein kinase (PKA), also induces fruiting body formation as well as expression of prespore-specific and prestalk-enriched genes in aca- cells. Intracellular cAMP levels were not altered in aca- cells after stimulation with 2'-deoxy cAMP. Our data indicate that ACA is not required to provide intracellular cAMP for PKA activation but is essential to produce extracellular cAMP for coordination of cell movement during all stages of development and for induction of developmental gene expression.

Authors
Pitt, GS; Brandt, R; Lin, KC; Devreotes, PN; Schaap, P
MLA Citation
Pitt, GS, Brandt, R, Lin, KC, Devreotes, PN, and Schaap, P. "Extracellular cAMP is sufficient to restore developmental gene expression and morphogenesis in Dictyostelium cells lacking the aggregation adenylyl cyclase (ACA)." Genes Dev 7.11 (November 1993): 2172-2180.
PMID
8224844
Source
pubmed
Published In
Genes & development
Volume
7
Issue
11
Publish Date
1993
Start Page
2172
End Page
2180

Multiple cyclic AMP receptors are linked to adenylyl cyclase in Dictyostelium.

cAMP receptor 1 and G-protein alpha-subunit 2 null cell lines (car1- and g alpha 2-) were examined to assess the roles that these two proteins play in cAMP stimulated adenylyl cyclase activation in Dictyostelium. In intact wild-type cells, cAMP stimulation elicited a rapid activation of adenylyl cyclase that peaked in 1-2 min and subsided within 5 min; in g alpha 2- cells, this activation did not occur; in car1- cells an activation occurred but it rose and subsided more slowly. cAMP also induced a persistent activation of adenylyl cyclase in growth stage cells that contain only low levels of cAMP receptor 1 (cAR1). In lysates of untreated wild-type, car1-, or g alpha 2- cells, guanosine 5'-O-'(3-thiotriphosphate) (GTP gamma S) produced a similar 20-fold increase in adenylyl cyclase activity. Brief treatment of intact cells with cAMP reduced this activity by 75% in control and g alpha 2- cells but by only 8% in the car1- cells. These observations suggest several conclusions regarding the cAMP signal transduction system. 1) cAR1 and another cAMP receptor are linked to activation of adenylyl cyclase in intact cells. Both excitation signals require G alpha 2. 2) cAR1 is required for normal adaptation of adenylyl cyclase. The adaptation reaction caused by cAR1 is not mediated via G alpha 2. 3) Neither cAR1 nor G alpha 2 is required for GTP gamma S-stimulation of adenylyl cyclase in cell lysates. The adenylyl cyclase is directly coupled to an as yet unidentified G-protein.

Authors
Pupillo, M; Insall, R; Pitt, GS; Devreotes, PN
MLA Citation
Pupillo, M, Insall, R, Pitt, GS, and Devreotes, PN. "Multiple cyclic AMP receptors are linked to adenylyl cyclase in Dictyostelium." Mol Biol Cell 3.11 (November 1992): 1229-1234.
PMID
1333842
Source
pubmed
Published In
Molecular Biology of the Cell
Volume
3
Issue
11
Publish Date
1992
Start Page
1229
End Page
1234

Amino acid substitutions in the Dictyostelium G alpha subunit G alpha 2 produce dominant negative phenotypes and inhibit the activation of adenylyl cyclase, guanylyl cyclase, and phospholipase C.

Previous studies have demonstrated that the Dictyostelium G alpha subunit G alpha 2 is essential for the cAMP-activation of adenylyl cyclase and guanylyl cyclase and that g alpha 2 null mutants do not aggregate. In this manuscript, we extend the analysis of the function of G alpha 2 in regulating downstream effectors by examining the in vivo developmental and physiological phenotypes of both wild-type and g alpha 2 null cells carrying a series of mutant G alpha 2 subunits expressed from the cloned G alpha 2 promoter. Our results show that wild-type cells expressing G alpha 2 subunits carrying mutations G40V and Q208L in the highly conserved GAGESG (residues 38-43) and GGQRS (residues 206-210) domains, which are expected to reduce the intrinsic GTPase activity, are blocked in multicellular development. Analysis of down-stream effector pathways essential for mediating aggregation indicates that cAMP-mediated activation of guanylyl cyclase and phosphatidylinositol-phospholipase C (PI-PLC) is almost completely inhibited and that there is a substantial reduction of cAMP-mediated activation of adenylyl cyclase. Moreover, neither mutant G alpha 2 subunit can complement g alpha 2 null mutants. Expression of G alpha 2(G43V) and G alpha 2(G207V) have little or no effect on the effector pathways and can partially complement g alpha 2 null cells. Our results suggest a model in which the dominant negative phenotypes resulting from the expression of G alpha 2(G40V) and G alpha 2(Q208L) are due to a constitutive adaptation of the effectors through a G alpha 2-mediated pathway. Analysis of PI-PLC in g alpha 2 null mutants and in cell lines expressing mutant G alpha 2 proteins also strongly suggests that G alpha 2 is the G alpha subunit that directly activates PI-PLC during aggregation. Moreover, overexpression of wild-type G alpha 2 results in the ability to precociously activate guanylyl cyclase by cAMP in vegetative cells, suggesting that G alpha 2 may be rate limiting in the developmental regulation of guanylyl cyclase activation. In agreement with previous results, the activation of adenylyl cyclase, while requiring G alpha 2 function in vivo, does not appear to be directly carried out by the G alpha 2 subunit. Our data are consistent with adenylyl cyclase being directly activated by either another G alpha subunit or by beta gamma subunits released on activation of the G protein containing G alpha 2.

Authors
Okaichi, K; Cubitt, AB; Pitt, GS; Firtel, RA
MLA Citation
Okaichi, K, Cubitt, AB, Pitt, GS, and Firtel, RA. "Amino acid substitutions in the Dictyostelium G alpha subunit G alpha 2 produce dominant negative phenotypes and inhibit the activation of adenylyl cyclase, guanylyl cyclase, and phospholipase C." Mol Biol Cell 3.7 (July 1992): 735-747.
PMID
1355376
Source
pubmed
Published In
Molecular Biology of the Cell
Volume
3
Issue
7
Publish Date
1992
Start Page
735
End Page
747

Structurally distinct and stage-specific adenylyl cyclase genes play different roles in Dictyostelium development.

We have isolated two adenylyl cyclase genes, designated ACA and ACG, from Dictyostelium. The proposed structure for ACA resembles that proposed for mammalian adenylyl cyclases: two large hydrophilic domains and two sets of six transmembrane spans. ACG has a novel structure, reminiscent of the membrane-bound guanylyl cyclases. An aca- mutant, created by gene disruption, has little detectable adenylyl cyclase activity and fails to aggregate, demonstrating that cAMP is required for cell-cell communication. cAMP is not required for motility, chemotaxis, growth, and cell division, which are unaffected. Constitutive expression in aca- cells of either ACA or ACG, which is normally expressed only during germination, restores aggregation and the ability to complete the developmental program. ACA expression restores receptor and guanine nucleotide-regulated adenylyl cyclase activity, while activity in cells expressing ACG is insensitive to these regulators. Although they lack ACA, which has a transporter-like structure, the cells expressing ACG secrete cAMP constitutively.

Authors
Pitt, GS; Milona, N; Borleis, J; Lin, KC; Reed, RR; Devreotes, PN
MLA Citation
Pitt, GS, Milona, N, Borleis, J, Lin, KC, Reed, RR, and Devreotes, PN. "Structurally distinct and stage-specific adenylyl cyclase genes play different roles in Dictyostelium development." Cell 69.2 (April 17, 1992): 305-315.
PMID
1348970
Source
pubmed
Published In
Cell
Volume
69
Issue
2
Publish Date
1992
Start Page
305
End Page
315

G-protein-linked signaling pathways mediate development in Dictyostelium.

Authors
Johnson, RL; Gundersen, R; Hereld, D; Pitt, GS; Tugendreich, S; Saxe, CL; Kimmel, AR; Devreotes, PN
MLA Citation
Johnson, RL, Gundersen, R, Hereld, D, Pitt, GS, Tugendreich, S, Saxe, CL, Kimmel, AR, and Devreotes, PN. "G-protein-linked signaling pathways mediate development in Dictyostelium." Cold Spring Harb Symp Quant Biol 57 (1992): 169-176. (Review)
PMID
1339656
Source
pubmed
Published In
Cold Spring Harbor Laboratory: Symposia on Quantitative Biology
Volume
57
Publish Date
1992
Start Page
169
End Page
176

Mechanisms of excitation and adaptation in Dictyostelium.

A G-protein linked signal transduction mechanism controls chemotaxis in eukaryotes. During development the social amoeba Dictyostelium directs chemotaxis towards external cAMP with its G-protein linked cAMP receptor. Interactions of the receptor and G-proteins transduce the chemotactic signal to the interior of the cell and eventually to the motor apparatus. Phosphorylation of the cAMP receptor has been correlated with the cell's ability to adapt to the external cAMP signal. This signal transduction pathway may help to explain the ability of eukaryotic cells to orient within a chemical gradient by the use of spatial cues.

Authors
Pitt, GS; Gundersen, RE; Devreotes, PN
MLA Citation
Pitt, GS, Gundersen, RE, and Devreotes, PN. "Mechanisms of excitation and adaptation in Dictyostelium." Semin Cell Biol 1.2 (April 1990): 99-104. (Review)
PMID
2129339
Source
pubmed
Published In
Seminars in cell biology
Volume
1
Issue
2
Publish Date
1990
Start Page
99
End Page
104

G protein-linked signal transduction in aggregating Dictyostelium.

Authors
Pitt, GS; Gundersen, RE; Lilly, PJ; Pupillo, MB; Vaughan, RA; Devreotes, PN
MLA Citation
Pitt, GS, Gundersen, RE, Lilly, PJ, Pupillo, MB, Vaughan, RA, and Devreotes, PN. "G protein-linked signal transduction in aggregating Dictyostelium." Soc Gen Physiol Ser 45 (1990): 125-131. (Review)
PMID
2116034
Source
pubmed
Published In
Society of General Physiologists Series
Volume
45
Publish Date
1990
Start Page
125
End Page
131

G protein-linked signal transduction in aggregating Dictyostelium.

Authors
Pitt, GS; Gundersen, RE; Lilly, PJ; Pupillo, MB; Vaughan, RA; Devreotes, PN
MLA Citation
Pitt, GS, Gundersen, RE, Lilly, PJ, Pupillo, MB, Vaughan, RA, and Devreotes, PN. "G protein-linked signal transduction in aggregating Dictyostelium." Society of General Physiologists series 45 (1990): 125-131.
Source
scival
Published In
Society of General Physiologists series
Volume
45
Publish Date
1990
Start Page
125
End Page
131

Multiple alpha subunits of guanine nucleotide-binding proteins in Dictyostelium.

Previous results have shown that chemotaxis and the expression of several classes of genes in Dictyostelium discoideum are regulated through a cell surface cAMP receptor interacting with guanine nucleotide-binding proteins (G proteins). We now describe cloning and sequencing of cDNAs encoding two G alpha protein subunits from Dictyostelium. The derived amino acid sequences show that they are 45% identical to each other and to G alpha protein subunits from mammals and yeast. Both cDNAs are complementary to multiple mRNAs that are differentially expressed during development. This evidence and analysis of mutants presented elsewhere suggest that they have distinct physiological functions.

Authors
Pupillo, M; Kumagai, A; Pitt, GS; Firtel, RA; Devreotes, PN
MLA Citation
Pupillo, M, Kumagai, A, Pitt, GS, Firtel, RA, and Devreotes, PN. "Multiple alpha subunits of guanine nucleotide-binding proteins in Dictyostelium." Proc Natl Acad Sci U S A 86.13 (July 1989): 4892-4896.
PMID
2500658
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
86
Issue
13
Publish Date
1989
Start Page
4892
End Page
4896

G-protein-linked signal transduction systems control development in Dictyostelium.

G-protein-linked cAMP receptors play an essential role in Dictyostelium development. The cAMP receptors are proposed to have seven transmembrane domains and a cytoplasmic C-terminal region. Overexpression of the receptor in cells, when the endogenous receptor is not present, results in a 10- to 50-fold increase in cAMP-binding sites. Antisense cell lines, which lack cAMP receptors, do not enter the developmental program. Ligand-induced phosphorylation is proposed to occur on serine and threonine residues in the receptor C-terminus. The kinetics of receptor phosphorylation and dephosphorylation correlate closely with the shift of receptor mobility and the adaptation of several cAMP-induced responses. Two alpha-subunits, G-alpha-1 and G-alpha-2, have been cloned and specific antisera developed against each. Both subunits are expressed as multiple RNAs with different developmental time courses. The mutant Frigid A has a functional defect in G-alpha-2 which prevents it from entering development. We propose that G-protein-linked receptor systems will be a major component in the development of many organisms.

Authors
Johnson, RL; Gundersen, R; Lilly, P; Pitt, GS; Pupillo, M; Sun, TJ; Vaughan, RA; Devreotes, PN
MLA Citation
Johnson, RL, Gundersen, R, Lilly, P, Pitt, GS, Pupillo, M, Sun, TJ, Vaughan, RA, and Devreotes, PN. "G-protein-linked signal transduction systems control development in Dictyostelium." Development 107 Suppl (1989): 75-80.
PMID
2517628
Source
pubmed
Published In
Development (Cambridge)
Volume
107 Suppl
Publish Date
1989
Start Page
75
End Page
80

A simple BASIC program allows the rapid entry of DNA nucleotide sequences into personal computers.

Authors
Pitt, GS; Devreotes, PN
MLA Citation
Pitt, GS, and Devreotes, PN. "A simple BASIC program allows the rapid entry of DNA nucleotide sequences into personal computers." Biotechniques 6.2 (February 1988): 122-123.
PMID
3273178
Source
pubmed
Published In
BioTechniques
Volume
6
Issue
2
Publish Date
1988
Start Page
122
End Page
123

cAMP receptor and G-protein interactions control development in Dictyostelium.

Authors
Pupillo, M; Klein, P; Vaughan, R; Pitt, G; Lilly, P; Sun, T; Devreotes, P; Kumagai, A; Firtel, R
MLA Citation
Pupillo, M, Klein, P, Vaughan, R, Pitt, G, Lilly, P, Sun, T, Devreotes, P, Kumagai, A, and Firtel, R. "cAMP receptor and G-protein interactions control development in Dictyostelium." January 1988.
PMID
3151181
Source
epmc
Published In
Cold Spring Harbor Laboratory: Symposia on Quantitative Biology
Volume
53 Pt 2
Publish Date
1988
Start Page
657
End Page
665

A molecular analysis of G proteins and control of early gene expression by the cell-surface cAMP receptor in Dictyostelium.

Authors
Kumagai, A; Mann, SK; Pupillo, M; Pitt, G; Devreotes, PN; Firtel, RA
MLA Citation
Kumagai, A, Mann, SK, Pupillo, M, Pitt, G, Devreotes, PN, and Firtel, RA. "A molecular analysis of G proteins and control of early gene expression by the cell-surface cAMP receptor in Dictyostelium." January 1988.
PMID
3151182
Source
epmc
Published In
Cold Spring Harbor Laboratory: Symposia on Quantitative Biology
Volume
53 Pt 2
Publish Date
1988
Start Page
675
End Page
685

A molecular analysis of G proteins and control of early gene expression by the cell-surface cAMP receptor in Dictyostelium

Authors
Kumagai, A; Mann, SKO; Pupillo, M; Pitt, G; Devreotes, PN; Firtel, RA
MLA Citation
Kumagai, A, Mann, SKO, Pupillo, M, Pitt, G, Devreotes, PN, and Firtel, RA. "A molecular analysis of G proteins and control of early gene expression by the cell-surface cAMP receptor in Dictyostelium." Cold Spring Harbor Symposia on Quantitative Biology 53.2 (1988): 675-685.
Source
scival
Published In
Cold Spring Harbor Laboratory: Symposia on Quantitative Biology
Volume
53
Issue
2
Publish Date
1988
Start Page
675
End Page
685

cAMP receptor and G-protein interactions control development in Dictyostelium

Authors
Pupillo, M; Klein, P; Vaughan, R; Pitt, G; Lilly, P; Sun, T; Devreotes, P; Kumagai, A; Firtel, R
MLA Citation
Pupillo, M, Klein, P, Vaughan, R, Pitt, G, Lilly, P, Sun, T, Devreotes, P, Kumagai, A, and Firtel, R. "cAMP receptor and G-protein interactions control development in Dictyostelium." Cold Spring Harbor Symposia on Quantitative Biology 53.2 (1988): 657-665.
Source
scival
Published In
Cold Spring Harbor Laboratory: Symposia on Quantitative Biology
Volume
53
Issue
2
Publish Date
1988
Start Page
657
End Page
665

Development and characterization of a monoclonal antibody to human embryonal carcinoma.

A monoclonal anti-testicular carcinoma antibody was obtained via the somatic cell fusion technique by immunization of BALB/c mice with freshly prepared single cell suspension from a patient with testicular embryonal carcinoma with choriocarcinoma components. The hybridoma supernates were screened against the testicular carcinoma cells used in the immunization as well as normal mononuclear white blood cells isolated from the same patient. An antibody (5F9) was selected which bound to fresh tumor cells from two patients with embryonal testicular carcinoma and failed to bind to fresh tumor cells from 24 patients (2 seminoma, 2 melanoma, 3 neck, 2 esophageal, 1 ovarian, 3 colon, 1 prostate, 2 breast, 1 liposarcoma, 3 endometrial, 1 kidney, 1 adrenal, 1 larynx and 1 bladder tumors) or cell suspensions prepared from normal liver, lung, spleen, ovary, testes, kidney, red blood cells or white blood cells. The antibody was tested for its binding to several well established cancer cell lines, and was found to bind to the BeWo human choriocarcinoma and two human embryonal carcinoma cell lines. The antibody did not react with 22 other cell lines or with hCG. The antibody was labeled with 131I and injected into nude mice bearing BeWo tumors and evaluated for tumor localization by performing whole body scans with a gamma camera 5 days later. Six mice injected with the antibody showed positive tumor localization without the need for background subtraction while six mice injected with MOPC-21, a murine myeloma immunoglobulin, demonstrated much less tumor localization. Tissue distribution studies performed after scanning showed specific tumor localization (8:1 tumor: muscle) for the monoclonal antibody and no specific localization for MOPC-21. This antibody thus has selective reactivity with the surface of tumor cells from embryonal carcinoma (testicle) and choriocarcinoma both in vitro and in vivo.

Authors
Khazaeli, MB; Beierwaltes, WH; Pitt, GS; Kabza, GA; Rogers, KJ; LoBuglio, AF
MLA Citation
Khazaeli, MB, Beierwaltes, WH, Pitt, GS, Kabza, GA, Rogers, KJ, and LoBuglio, AF. "Development and characterization of a monoclonal antibody to human embryonal carcinoma." J Urol 137.6 (June 1987): 1295-1299.
PMID
3035238
Source
pubmed
Published In
The Journal of Urology
Volume
137
Issue
6
Publish Date
1987
Start Page
1295
End Page
1299
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