Caprini M, Ferroni S, Planells-Cases R, Rueda J, Rapisarda C, Ferrer-Montiel A, Montal M
Department of Human and General Physiology, University of Bologna, Via San Donato 19/2, 40127 Bologna, Italy.
J Biol Chem. 2001 Jun 15;276(24):21070-6. doi: 10.1074/jbc.M100487200. Epub 2001 Mar 26.
Sequence similarity among and electrophysiological studies of known potassium channels, along with the three-dimensional structure of the Streptomyces lividans K(+) channel (KcsA), support the tenet that voltage-gated K(+) channels (Kv channels) consist of two distinct modules: the "voltage sensor" module comprising the N-terminal portion of the channel up to and including the S4 transmembrane segment and the "pore" module encompassing the C-terminal portion from the S5 transmembrane segment onward. To substantiate this modular design, we investigated whether the pore module of Kv channels may be replaced with the pore module of the prokaryotic KcsA channel. Biochemical and immunocytochemical studies showed that chimeric channels were expressed on the cell surface of Xenopus oocytes, demonstrating that they were properly synthesized, glycosylated, folded, assembled, and delivered to the plasma membrane. Unexpectedly, surface-expressed homomeric chimeras did not exhibit detectable voltage-dependent channel activity upon both hyperpolarization and depolarization regardless of the expression system used. Chimeras were, however, strongly dominant-negative when coexpressed with wild-type Kv channels, as evidenced by the complete suppression of wild-type channel activity. Notably, the dominant-negative phenotype correlated well with the formation of stable, glycosylated, nonfunctional, heteromeric channels. Collectively, these findings imply a structural compatibility between the prokaryotic pore module and the eukaryotic voltage sensor domain that leads to the biogenesis of non-responsive channels. Our results lend support to the notion that voltage-dependent channel gating depends on the precise coupling between both protein domains, probably through a localized interaction surface.
已知钾通道之间的序列相似性及电生理研究，以及变铅青链霉菌钾通道（KcsA）的三维结构，均支持这样一个原则：电压门控钾通道（Kv通道）由两个不同的模块组成：“电压传感器”模块，包括通道的N端部分直至并包括S4跨膜段；“孔道”模块，涵盖从S5跨膜段开始的C端部分。为了证实这种模块化设计，我们研究了Kv通道的孔道模块是否可以被原核KcsA通道的孔道模块所取代。生化和免疫细胞化学研究表明，嵌合通道在非洲爪蟾卵母细胞的细胞表面表达，这表明它们能够正确合成、糖基化、折叠、组装并转运到质膜。出乎意料的是，无论使用何种表达系统，表面表达的同聚体嵌合体在超极化和去极化时均未表现出可检测到的电压依赖性通道活性。然而，当与野生型Kv通道共表达时，嵌合体具有强烈的显性负性作用，野生型通道活性完全被抑制就证明了这一点。值得注意的是，显性负性表型与稳定的、糖基化的、无功能的异聚体通道的形成密切相关。总体而言，这些发现意味着原核孔道模块与真核电压传感器结构域之间存在结构兼容性，导致了无反应性通道的生物发生。我们的结果支持了这样一种观点，即电压依赖性通道门控可能依赖于两个蛋白质结构域之间的精确偶联，可能是通过一个局部相互作用表面。
