Song Haitao, Nie Liping, Rodriguez-Contreras Adrian, Sheng Zu-Hang, Yamoah Ebenezer N
Center for Neuroscience, Department of Otolaryngology, University of California, Davis, California 95616, USA.
J Neurophysiol. 2003 Feb;89(2):1143-9. doi: 10.1152/jn.00482.2002.
We assessed the functional determinants of the properties of L-type Ca(2+) currents in hair cells by co-expressing the pore-forming Ca(V)1.3alpha(1) subunit with the auxiliary subunits beta(1A) and/or alpha(2delta). Because Ca(2+) channels in hair cells are poised to interact with synaptic proteins, we also co-expressed the Ca(V)1.3alpha(1) subunit with syntaxin, vesicle-associated membrane protein (VAMP), and synaptosome associated protein of 25 kDa (SNAP25). Expression of the Ca(V)1.3alpha(1) subunit in human embryonic kidney cells (HEK 293) produced a dihydropyridine (DHP)-sensitive Ca(2+) current (peak current density -2.0 +/- 0.2 pA/pF; n = 11). Co-expression with beta(1A) and alpha(2delta) subunits enhanced the magnitude of the current (peak current density: Ca(V)1.3alpha(1) + beta(1A) = -4.3 +/- 0.8 pA/pF, n = 10; Ca(V)1.3alpha(1) + beta(1A) + alpha(2delta) = -4.1 +/- 0.6 pA/pF, n = 9) and produced a leftward shift of approximately 9 mV in the voltage-dependent activation of the currents. Furthermore, co-expression of Ca(V)1.3alpha(1) with syntaxin/VAMP/SNAP resulted in at least a twofold increase in the peak current density (-4.7 +/- 0.2 pA/pF; n = 11) and reduced the extent of inactivation of the Ca(2+) currents. Botulinum toxin, an inhibitor of syntaxin, accelerated the inactivation profile of Ca(2+) currents in hair cells. Immunocytochemical data also indicated that the Ca(2+) channels and syntaxin are co-localized in hair cells, suggesting there is functional interaction of the Ca(V)1.3alpha(1) with auxiliary subunits and synaptic proteins, that may contribute to the distinct properties of the DHP-sensitive channels in hair cells.
我们通过将形成孔道的Ca(V)1.3α(1)亚基与辅助亚基β(1A)和/或α2δ共表达，评估了毛细胞中L型Ca(2+)电流特性的功能决定因素。由于毛细胞中的Ca(2+)通道准备与突触蛋白相互作用，我们还将Ca(V)1.3α(1)亚基与 syntaxin、囊泡相关膜蛋白（VAMP）和25 kDa的突触体相关蛋白（SNAP25）共表达。在人胚肾细胞（HEK 293）中表达Ca(V)1.3α(1)亚基产生了一种对二氢吡啶（DHP）敏感的Ca(2+)电流（峰值电流密度-2.0±0.2 pA/pF；n = 11）。与β(1A)和α2δ亚基共表达增强了电流幅度（峰值电流密度：Ca(V)1.3α(1)+β(1A)=-4.3±0.8 pA/pF，n = 10；Ca(V)1.3α(1)+β(1A)+α2δ=-4.1±0.6 pA/pF，n = 9），并使电流的电压依赖性激活向左移动了约9 mV。此外，Ca(V)1.3α(1)与syntaxin/VAMP/SNAP共表达导致峰值电流密度至少增加两倍（-4.7±0.2 pA/pF；n = 11），并降低了Ca(2+)电流的失活程度。肉毒杆菌毒素是一种syntaxin抑制剂，加速了毛细胞中Ca(2+)电流的失活曲线。免疫细胞化学数据还表明，Ca(2+)通道和syntaxin在毛细胞中共定位，表明Ca(V)1.3α(1)与辅助亚基和突触蛋白之间存在功能相互作用，这可能有助于毛细胞中对DHP敏感通道的独特特性。
