Caveolin-3 Regulates Protein Kinase A Modulation of the CaV3.2 (α1H) T-type Ca2+ Channels

Yogananda S Markandeya,Jonathan M Fahey,Florentina Pluteanu,Leanne L Cribbs,Ravi C Balijepalli

doi:10.1074/jbc.m110.182550

Yogananda S Markandeya, Jonathan M Fahey + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m110.182550

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Abstract

Voltage-gated T-type Ca(2+) channel Ca(v)3.2 (α(1H)) subunit, responsible for T-type Ca(2+) current, is expressed in different tissues and participates in Ca(2+) entry, hormonal secretion, pacemaker activity, and arrhythmia. The precise subcellular localization and regulation of Ca(v)3.2 channels in native cells is unknown. Caveolae containing scaffolding protein caveolin-3 (Cav-3) localize many ion channels, signaling proteins and provide temporal and spatial regulation of intracellular Ca(2+) in different cells. We examined the localization and regulation of the Ca(v)3.2 channels in cardiomyocytes. Immunogold labeling and electron microscopy analysis demonstrated co-localization of the Ca(v)3.2 channel and Cav-3 relative to caveolae in ventricular myocytes. Co-immunoprecipitation from neonatal ventricular myocytes or transiently transfected HEK293 cells demonstrated that Ca(v)3.1 and Ca(v)3.2 channels co-immunoprecipitate with Cav-3. GST pulldown analysis confirmed that the N terminus region of Cav-3 closely interacts with Ca(v)3.2 channels. Whole cell patch clamp analysis demonstrated that co-expression of Cav-3 significantly decreased the peak Ca(v)3.2 current density in HEK293 cells, whereas co-expression of Cav-3 did not alter peak Ca(v)3.1 current density. In neonatal mouse ventricular myocytes, overexpression of Cav-3 inhibited the peak T-type calcium current (I(Ca,T)) and adenovirus (AdCa(v)3.2)-mediated increase in peak Ca(v)3.2 current, but did not affect the L-type current. The protein kinase A-dependent stimulation of I(Ca,T) by 8-Br-cAMP (membrane permeable cAMP analog) was abolished by siRNA directed against Cav-3. Our findings on functional modulation of the Ca(v)3.2 channels by Cav-3 is important for understanding the compartmentalized regulation of Ca(2+) signaling during normal and pathological processes.

Highlights

T-type Ca2ϩ channels (TTCC)2 are low voltage-activated Ca2ϩ channels, expressed in various tissues including brain and heart and contribute to a variety of physiological functions such as neuronal excitability, hormone secretion, muscle contraction, and pacemaker activity [1,2,3]
T-type Ca2ϩ Channel Isoforms Associate with Cav-3 in Ventricular Myocytes and human embryonic kidney 293 (HEK293) Cells—T-type Ca2ϩ channel isoforms Cav3.1 and Cav3.2 as well as Cav-3 are known to be expressed in neonatal cardiomyocytes
In a converse experiment we used either anti-Cav3.1 or Cav3.2 or a control rabbit IgG antibody for immunoprecipitation from neonatal ventricular myocyte lysates and found that Cav-3 co-immunoprecipitated with either of the TTCC isoform antibodies but not with control IgG. These results suggested that the Cav3.1 and Cav3.2 channels associate with Cav-3 in ventricular myocytes

Summary

Introduction

T-type Ca2ϩ channels (TTCC)2 are low voltage-activated Ca2ϩ channels, expressed in various tissues including brain and heart and contribute to a variety of physiological functions such as neuronal excitability, hormone secretion, muscle contraction, and pacemaker activity [1,2,3]. Co-immunoprecipitation from neonatal ventricular myocytes or transiently transfected HEK293 cells demonstrated that Cav3.1 and Cav3.2 channels co-immunoprecipitate with Cav-3. Non-transfected HEK293 cells did not show protein bands for T-type Ca2ϩ channel isoforms or Cav-3.

Results

Conclusion