Institute of Pharmacy and Center for Molecular Biosciences, University of Innsbruck, Austria.
Channels (Austin). 2012 May-Jun;6(3):197-205. doi: 10.4161/chan.21002. Epub 2012 May 1.
A C-terminal modulatory domain (CTM) tightly regulates the biophysical properties of Ca(v)1.3 L-type Ca(2+) channels, in particular the voltage dependence of activation (V(0.5)) and Ca(2+) dependent inactivation (CDI). A functional CTM is present in the long C-terminus of human and mouse Ca(v)1.3 (Ca(v)1.3(L)), but not in a rat long cDNA clone isolated from superior cervical ganglia neurons (rCa(v)1.3(scg)). We therefore addressed the question if this represents a species-difference and compared the biophysical properties of rCa(v)1.3(scg) with a rat cDNA isolated from rat pancreas (rCa(v)1.3(L)). When expressed in tsA-201 cells under identical experimental conditions rCa(v)1.3(L) exhibited Ca(2+) current properties indistinguishable from human and mouse Ca(v)1.3(L), compatible with the presence of a functional CTM. In contrast, rCa(v)1.3(scg) showed gating properties similar to human short splice variants lacking a CTM. rCa(v)1.3(scg) differs from rCa(v)1.3(L) at three single amino acid (aa) positions, one alternative spliced exon (exon31), and a N-terminal polymethionine stretch with two additional lysines. Two aa (S244, A2075) in rCa(v)1.3(scg) explained most of the functional differences to rCa(v)1.3(L). Their mutation to the corresponding residues in rCa(v)1.3(L) (G244, V2075) revealed that both contributed to the more negative V 0.5, but caused opposite effects on CDI. A2075 (located within a region forming the CTM) additionally permitted higher channel open probability. The cooperative action in the double-mutant restored gating properties similar to rCa(v)1.3(L). We found no evidence for transcripts containing one of the single rCa(v)1.3(scg) mutations in rat superior cervical ganglion preparations. However, the rCa(v)1.3(scg) variant provided interesting insight into the structural machinery involved in Ca(v)1.3 gating.
C 端调节域（CTM）可紧密调节 Ca(v)1.3 L 型钙通道的生物物理特性，特别是激活的电压依赖性（V(0.5)）和钙依赖性失活（CDI）。人类和小鼠 Ca(v)1.3（Ca(v)1.3(L)）的长 C 端具有功能性 CTM，但分离自大鼠颈上神经节神经元的大鼠长 cDNA 克隆（rCa(v)1.3(scg)）中却没有。因此，我们提出了这是否代表种属差异的问题，并比较了 rCa(v)1.3(scg)与从大鼠胰腺分离的大鼠 cDNA（rCa(v)1.3(L)）的生物物理特性。当在相同的实验条件下在 tsA-201 细胞中表达时，rCa(v)1.3(L)表现出与人类和小鼠 Ca(v)1.3(L) 相同的钙电流特性，这与存在功能性 CTM 相符。相比之下，rCa(v)1.3(scg)的门控特性与缺乏 CTM 的人类短剪接变体相似。rCa(v)1.3(scg)在三个单一氨基酸（aa）位置、一个替代剪接外显子（exon31）和一个 N 端多甲硫氨酸区与两个额外的赖氨酸存在差异，与 rCa(v)1.3(L) 不同。rCa(v)1.3(scg)中的两个 aa（S244，A2075）解释了与 rCa(v)1.3(L) 相比大部分功能差异的原因。将其突变为 rCa(v)1.3(L)中的相应残基（G244，V2075）表明这两个残基都有助于 V 0.5 更负，但对 CDI 产生相反的影响。A2075（位于形成 CTM 的区域内）还允许更高的通道开放概率。双突变体中的协同作用恢复了与 rCa(v)1.3(L) 相似的门控特性。我们在大鼠颈上神经节制剂中没有发现包含大鼠 Ca(v)1.3(scg) 单个突变之一的转录本的证据。然而，rCa(v)1.3(scg)变体为 Ca(v)1.3 门控涉及的结构机制提供了有趣的见解。
