Cherbavaz D B
Graduate Program in Biophysics, Brandeis University, Waltham, Massachusetts 02254, USA.
Biophys J. 1995 Apr;68(4):1337-46. doi: 10.1016/S0006-3495(95)80306-1.
The extracellular side of single batrachotoxin-activated voltage-dependent Na channels isolated from rat skeletal muscle membranes incorporated into neutral planar lipid bilayers were treated in situ with the carboxyl methylating reagent, trimethyloxonium (TMO). These experiments were designed to determine whether TMO alters Na channel function by a general through-space electrostatic mechanism or by methylating specific carboxyl groups essential to channel function. TMO modification reduced single-channel conductance by decreasing the maximal turnover rate. Modification increased channel selectivity for sodium ions relative to potassium ions as measured under biionic conditions. TMO modification increased the mu-conotoxin (muCTX) off-rate by three orders of magnitude. Modification did not alter the muCTX on-rate at low ionic strength or Na channel voltage-dependent gating characteristics. These data demonstrate that TMO does not act via a general electrostatic mechanism. Instead, TMO targets protein residues specifically involved in ion conduction, ion selectivity, and muCTX binding. These data support the hypothesis that muCTX blocks open-channel current by physically obstructing the ion channel pore.
从大鼠骨骼肌膜分离出来并整合到中性平面脂质双分子层中的单个蛙皮毒素激活的电压依赖性钠通道的细胞外侧，原位用羧甲基化试剂三甲基氧鎓（TMO）处理。这些实验旨在确定TMO是通过一般的空间静电机制还是通过甲基化对通道功能至关重要的特定羧基来改变钠通道功能。TMO修饰通过降低最大周转率降低了单通道电导。在双离子条件下测量，修饰增加了通道对钠离子相对于钾离子的选择性。TMO修饰使μ-芋螺毒素（μCTX）解离速率增加了三个数量级。修饰在低离子强度下不改变μCTX结合速率或钠通道电压依赖性门控特性。这些数据表明TMO不是通过一般的静电机制起作用。相反，TMO靶向特异性参与离子传导、离子选择性和μCTX结合的蛋白质残基。这些数据支持μCTX通过物理阻塞离子通道孔来阻断开放通道电流的假说。
