Xie Cheng, Zhen Xiao-Guang, Yang Jian
Department of Biological Sciences, Columbia University, New York, NY 10027, USA.
J Gen Physiol. 2005 Sep;126(3):205-12. doi: 10.1085/jgp.200509293.
Ion channels open and close in response to changes in transmembrane voltage or ligand concentration. Recent studies show that K+ channels possess two gates, one at the intracellular end of the pore and the other at the selectivity filter. In this study we determined the location of the activation gate in a voltage-gated Ca2+ channel (VGCC) by examining the open/closed state dependence of the rate of modification by intracellular methanethiosulfonate ethyltrimethylammonium (MTSET) of pore-lining cysteines engineered in the S6 segments of the alpha1 subunit of P/Q type Ca2+ channels. We found that positions above the putative membrane/cytoplasm interface, including two positions below the corresponding S6 bundle crossing in K+ channels, showed pronounced state-dependent accessibility to internal MTSET, reacting approximately 1,000-fold faster with MTSET in the open state than in the closed state. In contrast, a position at or below the putative membrane/cytoplasm interface was modified equally rapidly in both the open and closed states. Our results suggest that the S6 helices of the alpha1 subunit of VGCCs undergo conformation changes during gating and the activation gate is located at the intracellular end of the pore.
离子通道会根据跨膜电压或配体浓度的变化而打开和关闭。最近的研究表明，钾离子通道有两个门控，一个在孔道的细胞内末端，另一个在选择性过滤器处。在本研究中，我们通过检测细胞内甲硫基磺酸乙酯三甲基铵（MTSET）对P/Q型钙离子通道α1亚基S6段中工程改造的孔道内衬半胱氨酸的修饰速率的开放/关闭状态依赖性，来确定电压门控钙离子通道（VGCC）中激活门的位置。我们发现，在假定的膜/细胞质界面上方的位置，包括钾离子通道中相应S6束交叉下方的两个位置，对内部MTSET表现出明显的状态依赖性可及性，在开放状态下与MTSET的反应速度比关闭状态下快约1000倍。相比之下，在假定的膜/细胞质界面或其下方的一个位置在开放和关闭状态下被修饰的速度相同。我们的结果表明，VGCCs的α1亚基的S6螺旋在门控过程中发生构象变化，激活门位于孔道的细胞内末端。
