Department of Medicine, Cellular and Molecular Arrhythmia Research Program, University of Wisconsin, Madison, Wisconsin 53706, USA.
J Biol Chem. 2011 Jan 28;286(4):2433-44. doi: 10.1074/jbc.M110.182550. Epub 2010 Nov 17.
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.
电压门控 T 型钙 (Ca(2+)) 通道 Ca(v)3.2（α(1H)）亚基，负责 T 型 Ca(2+)电流，在不同组织中表达，并参与 Ca(2+)内流、激素分泌、起搏活动和心律失常。在天然细胞中，Ca(v)3.2 通道的精确亚细胞定位和调节尚不清楚。含有支架蛋白 caveolin-3（Cav-3）的 caveolae 定位许多离子通道、信号蛋白，并为不同细胞内 Ca(2+)的时空调节提供作用。我们研究了心肌细胞中 Ca(v)3.2 通道的定位和调节。免疫金标记和电子显微镜分析表明，在心室肌细胞中，Ca(v)3.2 通道与 Cav-3 相对于 caveolae 共定位。从新生心室肌细胞或瞬时转染的 HEK293 细胞中进行的共免疫沉淀表明，Ca(v)3.1 和 Ca(v)3.2 通道与 Cav-3 共免疫沉淀。GST 下拉分析证实，Cav-3 的 N 端区域与 Ca(v)3.2 通道紧密相互作用。全细胞 patch clamp 分析表明，Cav-3 的共表达显著降低了 HEK293 细胞中 Ca(v)3.2 电流密度的峰值，而 Cav-3 的共表达并不改变 Ca(v)3.1 电流密度的峰值。在新生小鼠心室肌细胞中，Cav-3 的过表达抑制了峰值 T 型钙电流（I(Ca,T)）和腺病毒（AdCa(v)3.2）介导的 Ca(v)3.2 电流峰值的增加，但不影响 L 型电流。PKA 依赖性刺激 I(Ca,T)的 8-Br-cAMP（膜可渗透 cAMP 类似物）被针对 Cav-3 的 siRNA 完全阻断。我们关于 Cav-3 对 Ca(v)3.2 通道的功能调节的发现对于理解正常和病理过程中 Ca(2+)信号的分隔调节很重要。
