VOLTAGE INDUCED S4 CONFORMATION CHANGES BY NMR

泰国项目的长期目标是调查结构和 离子通道过程中S4电压传感器的函数关系 通过核磁共振（NMR）进行活化和失活。 电压门控Na+、K+和Ca 2+通道包含高度保守的 带正电荷的54段，以及其在电压期间的运动 门控被认为控制离子通道的激活。两 可能的模型--“滑动螺旋”（或螺旋螺旋）和“传播 提出了用“螺旋”来描述S4的平移运动， 其构象在膜去极化过程中发生变化。然而，在这方面， 没有结构证据支持这些模型。 我们 建议表征构象变化和电荷 Shaker钾s4片段（及其突变体）的运动 通道中存在和不存在电场的多- 三维NMR方法，如COSY，NOSEY，和 电泳核磁共振将实现三个具体目标： (l)为了了解S4在细胞内的构象变化， 去极化，S4肽的三维结构， 将在存在下测定Shaker钾通道蛋白 （2）54个样品的电泳迁移率 肽（和突变体）的振动钾通道将是 与膜片钳中测量的门控电流相关 实验;扩散系数的54（和突变体）测量， 电场的存在和不存在将与 肽段的构象变化;（3）54个肽段的增殖 54-55接头的构象变化将通过以下方法监测： 确定含有54和54-55接头的肽的结构， 电场的存在和不存在; 将检查该区域在介导信号中的作用 在振荡器钾通道中从54转导至54-55接头。 离子通道的分子机制 信号转导可用于控制与离子相关疾病 心脏兴奋-收缩神经通道异常 传导，肌肉收缩

The long term goal of thais project is to investigate the structure and functional relationship of S4 voltage sensor during ion channel activation and inactivation by nuclear magnetic resonance (NMR). Voltage-gated Na+, K+, and Ca2+ channels contain highly conserved and positively charged 54 segments, and its movement during voltage gating is believed to control the activation of ion channels. Two possible models --"sliding helix" (or helix screwii) and "propagating helix" are proposed to describe the translational movement of S4 and its conformation changes during membrane depolarization. However, no structural evidence is available to support those models. We propose to characterize the conformation changes and charge movement of s4 segment (and its mutants) of the Shaker potassium channel in the presence and absence of electric field by the multi- dimensional NMR methods such as COSY, NOSEY, and electrophoretic NMR. Three specific aims will be accomplished: (l) To understand S4 conformational changes during cell depolarization, three-dimensional structures of S4 peptides of the Shaker potassium channel protein will be determined in the presence and absence of electric field; (2) The electrophoretic mobilities of 54 peptide (and mutants) of the shaker potassium channel will be correlated with the gating currents measured in the patch clamp experiments; the diffusion coefficients of 54 (and mutants) measured in the presence and absence of electric field will be correlated with the conformation changes of the peptides; (3)The propagation of the 54 conformation changes to the 54-55 linker will be monitored by determining structures of peptide that contains 54 and 54-55 linker in the presence and absence of electric field; the leucine-heptad-repeat in this region will be examined for its role in mediating the signal transduction from 54 to the 54-55 linker in shaker potassium channel. The knowledge gained on the molecular mechanism of ion channel signal transduction may be useful to control diseases related to ion channel abnormalities in cardiac excitation-contraction, nerve conduction, muscle contraction.