Spaetgens R L, Zamponi G W
Department of Pharmacology and Therapeutics, Neuroscience Research Group, University of Calgary, Calgary, Alberta T2N 4N1, Canada.
J Biol Chem. 1999 Aug 6;274(32):22428-36. doi: 10.1074/jbc.274.32.22428.
We have investigated the molecular determinants that mediate the differences in voltage-dependent inactivation properties between rapidly inactivating (R-type) alpha(1E) and noninactivating (L-type) alpha(1C) calcium channels. When coexpressed in human embryonic kidney cells with ancillary beta(1b) and alpha(2)-delta subunits, the wild type channels exhibit dramatically different inactivation properties; the half-inactivation potential of alpha(1E) is 45 mV more negative than that observed with alpha(1C), and during a 150-ms test depolarization, alpha(1E) undergoes 65% inactivation compared with only about 15% for alpha(1C). To define the structural determinants that govern these intrinsic differences, we have created a series of chimeric calcium channel alpha(1) subunits that combine the major structural domains of the two wild type channels, and we investigated their voltage-dependent inactivation properties. Each of the four transmembrane domains significantly affected the half-inactivation potential, with domains II and III being most critical. In particular, substitution of alpha(1C) sequence in domains II or III with that of alpha(1E) resulted in 25-mV negative shifts in half-inactivation potential. Similarly, the differences in inactivation rate were predominantly governed by transmembrane domains II and III and to some extent by domain IV. Thus, voltage-dependent inactivation of alpha(1E) channels is a complex process that involves multiple structural domains and possibly a global conformational change in the channel protein.
我们研究了介导快速失活（R型）α(1E)和非失活（L型）α(1C)钙通道电压依赖性失活特性差异的分子决定因素。当与辅助β(1b)和α(2)-δ亚基在人胚肾细胞中共表达时，野生型通道表现出显著不同的失活特性；α(1E)的半失活电位比α(1C)的负45 mV，在150 ms的测试去极化过程中，α(1E)发生65%的失活，而α(1C)仅约15%。为了确定控制这些内在差异的结构决定因素，我们构建了一系列嵌合钙通道α(1)亚基，它们结合了两个野生型通道的主要结构域，并研究了它们的电压依赖性失活特性。四个跨膜结构域中的每一个都显著影响半失活电位，其中结构域II和III最为关键。特别是，将结构域II或III中的α(1C)序列替换为α(1E)的序列会导致半失活电位负移25 mV。同样，失活速率的差异主要由跨膜结构域II和III决定，在一定程度上也由结构域IV决定。因此，α(1E)通道的电压依赖性失活是一个复杂的过程，涉及多个结构域，可能还涉及通道蛋白的整体构象变化。
