Caveolin-3 regulates protein kinase A modulation of the Ca(V)3.2 (alpha1H) T-type Ca2+ channels

J Biol Chem. 2011 Jan 28;286(4):2433-44. doi: 10.1074/jbc.M110.182550. Epub 2010 Nov 17.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenoviridae
  • Animals
  • Calcium / metabolism
  • Calcium Channels, T-Type / genetics
  • Calcium Channels, T-Type / metabolism*
  • Caveolin 3 / genetics
  • Caveolin 3 / metabolism*
  • Cyclic AMP-Dependent Protein Kinases / genetics
  • Cyclic AMP-Dependent Protein Kinases / metabolism*
  • HEK293 Cells
  • Heart Ventricles / cytology
  • Heart Ventricles / metabolism*
  • Humans
  • Mice
  • Myocytes, Cardiac / cytology
  • Myocytes, Cardiac / metabolism*
  • Transduction, Genetic

Substances

  • CACNA1H protein, human
  • CAV3 protein, human
  • Cacna1h protein, mouse
  • Calcium Channels, T-Type
  • Cav3 protein, mouse
  • Caveolin 3
  • Cyclic AMP-Dependent Protein Kinases
  • Calcium