Abstract:
Shaker potassium channels have been an essential model for studying inactivation of ion channels and shaped our earliest understanding of N-type vs. C-type mechanisms. In early work describing C-type inactivation, López-Barneo and colleagues systematically characterized numerous mutations of Shaker residue T449, demonstrating that this position was a key determinant of C-type inactivation rate. In most of the closely related mammalian Kv1 channels, however, a persistent enigma has been that residue identity at this position has relatively modest effects on the rate of inactivation in response to long depolarizations. In this study, we report alternative ways to measure or elicit conformational changes in the outer pore associated with C-type inactivation. Using a strategically substituted cysteine in the outer pore, we demonstrate that mutation of Kv1.2 V381 (equivalent to Shaker T449) or W366 (Shaker W434) markedly increases susceptibility to modification by extracellularly applied MTSET. Moreover, due to the cooperative nature of C-type inactivation, Kv1.2 assembly in heteromeric channels markedly inhibits MTSET modification of this substituted cysteine in neighboring subunits. The identity of Kv1.2 residue V381 also markedly influences function in conditions that bias channels toward C-type inactivation, namely when Na+ is substituted for K+ as the permeant ion or when channels are blocked by an N-type inactivation particle (such as Kvβ1.2). Overall, our findings illustrate that in mammalian Kv1 channels, the identity of the T449-equivalent residue can strongly influence function in certain experimental conditions, even while having modest effects on apparent inactivation during long depolarizations. These findings contribute to reconciling differences in experimental outcomes in many Kv1 channels vs. Shaker.
摘要:
摇床钾通道是研究离子通道失活的重要模型,并塑造了我们对N型与C型机制。在描述C型失活的早期工作中,López-Barneo及其同事系统地表征了Shaker残基T449的许多突变,表明该位置是C型失活率的关键决定因素。然而,在大多数密切相关的哺乳动物Kv1通道中,一个持续的谜团是,该位置的残基同一性对响应于长时间去极化的失活速率具有相对适度的影响。在这项研究中,我们报告了测量或引起与C型失活相关的外孔构象变化的替代方法。在外孔中使用战略性取代的半胱氨酸,我们证明Kv1.2V381(相当于振动筛T449)或W366(振动筛W434)的突变显著增加了细胞外应用MTSET修饰的易感性.此外,由于C型失活的协同性质,异聚通道中的Kv1.2组装明显抑制了相邻亚基中这种取代的半胱氨酸的MTSET修饰。Kv1.2残基V381的身份也显着影响在偏向C型失活通道的条件下的功能,即当Na代替K作为渗透离子时,或当通道被N型失活颗粒(例如Kvβ1.2)阻断时。总的来说,我们的发现表明,在哺乳动物Kv1通道中,在某些实验条件下,T449当量残基的身份可以强烈影响函数,即使在长时间去极化过程中对明显的失活有适度的影响。这些发现有助于调和许多Kv1通道与Shaker的实验结果差异。