Dirksen R T, Nakai J, Gonzalez A, Imoto K, Beam K G
Department of Anatomy and Neurobiology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins 80523, USA.
Biophys J. 1997 Sep;73(3):1402-9. doi: 10.1016/S0006-3495(97)78172-4.
The alpha1-subunits of the skeletal and cardiac L-type calcium channels (L-channels) contain nearly identical pore regions (P-regions) in each of the four internal homology repeats. In spite of this high conservation of the P-regions, native skeletal L-channels exhibit a unitary conductance that is only about half that of native cardiac L-channels. To identify structural determinants of this difference in L-channel conductance, we have characterized unitary activity in cell-attached patches of dysgenic myotubes expressing skeletal, cardiac, and chimeric L-channel alpha1-subunits. Our results demonstrate that the S5-S6 linker of repeat I (IS5-IS6 linker) is a critical determinant of the difference in skeletal and cardiac unitary conductance. The unitary conductances attributable to the wild-type skeletal (CAC6; approximately 14 pS) and cardiac (CARD1; approximately 25 pS) alpha1-subunits expressed in dysgenic myotubes are identical to those observed in native tissues. Chimeric alpha1-subunits containing skeletal sequence for the first internal repeat and all of the putative intracellular loops (SkC15), the IS5-IS6 linker and the intracellular loops (SkC51), or only the IS5-IS6 linker (SkC49) each exhibit a low, skeletal-like unitary conductance (< or = 17 pS). Constructs in which the IS5-IS6 linker is of cardiac origin (CARD1 and CSk9) display cardiac-like conductance (approximately 25 pS). Unitary conductance and the rate of channel activation are apparently independent processes, since both SkC51 and SkC49 exhibit low, skeletal-like conductance and rapid, cardiac-like rates of ensemble activation. These results demonstrate that the IS5-IS6 linker strongly influences the single channel conductance of L-channels in a manner that is independent from the rate of channel activation.
骨骼肌和心肌L型钙通道（L通道）的α1亚基在四个内部同源重复序列中的每一个中都包含几乎相同的孔区域（P区域）。尽管P区域具有高度保守性，但天然骨骼肌L通道的单位电导仅约为天然心肌L通道的一半。为了确定L通道电导差异的结构决定因素，我们对表达骨骼肌、心肌和嵌合L通道α1亚基的发育不良肌管的细胞贴附膜片上的单位活性进行了表征。我们的结果表明，重复序列I的S5 - S6连接子（IS5 - IS6连接子）是骨骼肌和心肌单位电导差异的关键决定因素。在发育不良肌管中表达的野生型骨骼肌（CAC6；约14 pS）和心肌（CARD1；约25 pS）α1亚基的单位电导与在天然组织中观察到的相同。包含第一个内部重复序列的骨骼肌序列和所有假定的细胞内环（SkC15）、IS5 - IS6连接子和细胞内环（SkC51）或仅IS5 - IS6连接子（SkC49）的嵌合α1亚基均表现出低的、类似骨骼肌的单位电导（≤17 pS）。IS5 - IS6连接子源自心肌的构建体（CARD1和CSk9）表现出类似心肌的电导（约25 pS）。单位电导和通道激活速率显然是独立的过程，因为SkC51和SkC49均表现出低的、类似骨骼肌的电导以及快速的、类似心肌的整体激活速率。这些结果表明，IS5 - IS6连接子以与通道激活速率无关的方式强烈影响L通道的单通道电导。
