Monteil A, Chemin J, Bourinet E, Mennessier G, Lory P, Nargeot J
IGH-CNRS UPR 1142, 141 rue de la Cardonille, F-34396 Montpellier cedex 05, France.
J Biol Chem. 2000 Mar 3;275(9):6090-100. doi: 10.1074/jbc.275.9.6090.
We describe here several novel properties of the human alpha(1G) subunit that forms T-type calcium channels. The partial intron/exon structure of the corresponding gene CACNA1G was defined and several alpha(1G) isoforms were identified, especially two isoforms that exhibit a distinct III-IV loop: alpha(1G-a) and alpha(1G-b). Northern blot and dot blot analyses indicated that alpha(1G) mRNA is predominantly expressed in the brain, especially in thalamus, cerebellum, and substantia nigra. Additional experiments have also provided evidence that alpha(1G) mRNA is expressed at a higher level during fetal life in nonneuronal tissues (i.e. kidney, heart, and lung). Functional expression in HEK 293 cells of a full-length cDNA encoding the shortest alpha(1G) isoform identified to date, alpha(1G-b), resulted in transient, low threshold activated Ca(2+) currents with the expected permeability ratio (I(Sr) > I(Ca) >/= I(Ba)) and channel conductance ( approximately 7 pS). These properties, together with slowly deactivating tail currents, are typical of those of native T-type Ca(2+) channels. This alpha(1G)-related current was inhibited by mibefradil (IC(50) = 2 microM) and weakly blocked by Ni(2+) ions (IC(50) = 148 microM) and amiloride (IC(50) > 1 mM). We showed that steady state activation and inactivation properties of this current can generate a "window current" in the range of -65 to -55 mV. Using neuronal action potential waveforms, we show that alpha(1G) channels produce a massive and sustained Ca(2+) influx due to their slow deactivation properties. These latter properties would account for the specificity of Ca(2+) influx via T-type channels that occurs in the range of physiological resting membrane potentials, differing considerably from the behavior of other Ca(2+) channels.
我们在此描述了构成T型钙通道的人α(1G)亚基的几个新特性。确定了相应基因CACNA1G的部分内含子/外显子结构，并鉴定了几种α(1G)亚型，特别是两种具有独特III-IV环的亚型：α(1G-a)和α(1G-b)。Northern印迹和斑点印迹分析表明，α(1G) mRNA主要在脑中表达，尤其是在丘脑、小脑和黑质中。额外的实验还提供了证据，表明α(1G) mRNA在胎儿期的非神经组织（即肾脏、心脏和肺）中表达水平更高。在HEK 293细胞中对编码迄今为止鉴定出的最短α(1G)亚型α(1G-b)的全长cDNA进行功能表达，产生了瞬时、低阈值激活的Ca(2+)电流，具有预期的通透率（I(Sr) > I(Ca) ≥ I(Ba)）和通道电导（约7 pS）。这些特性，连同缓慢失活的尾电流，是天然T型Ca(2+)通道的典型特性。这种与α(1G)相关的电流被米贝地尔抑制（IC(50) = 2 μM），被Ni(2+)离子（IC(50) = 148 μM）和阿米洛利（IC(50) > 1 mM）微弱阻断。我们表明，该电流的稳态激活和失活特性可在-65至-55 mV范围内产生“窗口电流”。使用神经元动作电位波形，我们表明α(1G)通道由于其缓慢失活特性而产生大量且持续的Ca(2+)内流。后一种特性将解释通过T型通道在生理静息膜电位范围内发生的Ca(2+)内流特异性，这与其他Ca(2+)通道的行为有很大不同。
