Study of membrane protein structure by NMR spectroscopy: the KcsA channel

KcsA是一种68 kda的同四聚体膜相关钾通道，它选择性地控制钾离子在膜上的通量。已知该通道经历了一个依赖于ph值的开放到封闭的转变。在对完整的四聚体通道进行研究之后，我们对通道的单体亚基（KcsAM）进行了核磁共振研究，溶解在SDS胶束中。化学位移、溶剂交换、主链15N弛豫和残余偶极耦合（RDC）数据表明，TM1螺旋保持完整，但TM2螺旋包含明显的扭结，在微秒时间尺度上受浓度无关但ph依赖的构象交换的影响。以G99为中心的扭结区先前与四聚体KcsA通道的门控有关。在酸性pH下，基于rdc的KcsAM模型将TM1和缠绕的TM2的两个螺旋段定向为一种构型，使人联想到通道的开放构象。因此，状态之间的过渡似乎是单体的固有能力，四聚体组装对过渡施加调节作用，使通道具有生理门控轮廓。

KcsA is a homotetrameric 68-kDa membrane-associated potassium channel which selectively gates the flux of potassium ions across the membrane. The channel is known to undergo a pH-dependent open-to-closed transition. Following a study of the intact tetrameric channel, we have carried out an NMR study of the monomeric subunit of the channel (KcsAM), solubilized in SDS micelles. Chemical shift, solvent exchange, backbone 15N relaxation and residual dipolar coupling (RDC) data show the TM1 helix to remain intact, but the TM2 helix contains a distinct kink, which is subject to concentration-independent but pH-dependent conformational exchange on a microsecond time scale. The kink region, centered at G99, was previously implicated in gating of the tetrameric KcsA channel. An RDC-based model of KcsAM at acidic pH orients TM1 and the two helical segments of the kinked TM2 in a configuration reminiscent of the open conformation of the channel. Thus, the transition between states appears to be an inherent capability of the monomer, with the tetrameric assembly exerting a modulatory effect upon the transition which gives the channel its physiological gating profile.