Abbott G W, Mercer E A, Miller R T, Ramesh B, Srai S K
Department of Biochemistry and Molecular Biology, Royal Free Hospital School of Medicine, London, U.K.
Biochemistry. 1998 Feb 10;37(6):1640-5. doi: 10.1021/bi972350c.
Fast inactivation is restored in inactivation deletion mutant voltage-gated potassium (Kv) channels by application of synthetic inactivation 'ball' peptide. Using Fourier transform infrared and circular dichroism spectroscopy, we have investigated the structure of synthetic Kv3.4 channel ball peptide, in a range of environments relevant to the function of the ball domain. The ball peptide contains no alpha-helix or beta-sheet in reducing conditions in aqueous solution, but when cosolubilized with anionic lipid or detergent in order to mimic the environment which the ball domain encounters during channel inactivation, the ball peptide adopts a partial beta-sheet structure. Oxidation of the Kv3.4 ball peptide facilitates formation of a disulfide bond between Cys6 and Cys24 and adoption of a partial beta-sheet structure in aqueous solution; the tendency of the oxidized ball peptide to adopt beta-sheet is generally greater than that of the reduced ball peptide in a given environment. THREADER modeling of the Kv3.4 ball peptide structure predicts a beta-hairpin-like conformation which corresponds well to the structure suggested by spectroscopic analysis of the ball peptide in its cyclic arrangement. A V7E mutant Kv3.4 ball peptide analogue of the noninactivating Shaker B L7E mutant ball peptide cannot adopt beta-structure whatever the environment, and regardless of oxidation state. The results suggest that the Kv3.4 ball domain undergoes a conformational change during channel inactivation and may implicate a novel regulatory role for intramolecular disulfide bond formation in the Kv3.4 ball domain in vivo.
通过应用合成的失活“球”肽，失活缺失突变体电压门控钾（Kv）通道中的快速失活得以恢复。我们使用傅里叶变换红外光谱和圆二色光谱，在一系列与球结构域功能相关的环境中研究了合成的Kv3.4通道球肽的结构。在水溶液的还原条件下，球肽不包含α-螺旋或β-折叠，但当与阴离子脂质或去污剂共溶解以模拟球结构域在通道失活过程中遇到的环境时，球肽会形成部分β-折叠结构。Kv3.4球肽的氧化促进了半胱氨酸6和半胱氨酸24之间二硫键的形成，并在水溶液中形成部分β-折叠结构；在给定环境中，氧化球肽形成β-折叠的趋势通常大于还原球肽。Kv3.4球肽结构的THREADER建模预测了一种β-发夹样构象，这与球肽在其环状排列中的光谱分析所表明的结构非常吻合。非失活的Shaker B L7E突变体球肽的V7E突变体Kv3.4球肽类似物无论在何种环境下，也无论氧化状态如何，都不能形成β-结构。结果表明，Kv3.4球结构域在通道失活过程中发生了构象变化，这可能暗示了体内Kv3.4球结构域中分子内二硫键形成的一种新的调节作用。
