Subbiah Rajesh N, Kondo Mari, Campbell Terence J, Vandenberg Jamie I
Victor Chang Cardiac Research Institute, 384 Victoria Street, Darlinghurst, NSW 2010, Australia.
J Physiol. 2005 Dec 1;569(Pt 2):367-79. doi: 10.1113/jphysiol.2005.097386. Epub 2005 Sep 15.
Inherited mutations or drug-induced block of voltage-gated ion channels, including the human ether-à-go-go-related gene (HERG) K+ channel, are significant causes of malignant arrhythmias and sudden death. The fourth transmembrane domain (S4) of these channels contains multiple positive charges that move across the membrane electric field in response to changes in transmembrane voltage. In HERG K+ channels, the movement of the S4 domain across the transmembrane electric field is particularly slow. To examine the basis of the slow movement of the HERG S4 domain and specifically to probe the relationship between the S4 domain with the lipid bilayer and rest of the channel protein, we individually mutated each of the S4 amino acids in HERG (L524-L539) to tryptophan, and characterized the activation and deactivation properties of the mutant channels in Xenopus oocytes, using two-electrode voltage-clamp methods. Tryptophan has a large bulky hydrophobic sidechain and so should be tolerated at positions that interact with lipid, but not at positions involved in close protein-protein interactions. Significantly, we found that all S4 tryptophan mutants were functional. These data indicate that the S4 domain is loosely packed within the rest of the voltage sensor domain and is likely to be lipid exposed. Further, we identified residues K525, R528 and K538 as being the most important for slow activation of the channels.
遗传性突变或药物诱导的电压门控离子通道阻滞，包括人类醚 - 去极化相关基因（HERG）钾通道，是恶性心律失常和猝死的重要原因。这些通道的第四个跨膜结构域（S4）包含多个正电荷，它们会随着跨膜电压的变化在膜电场中移动。在HERG钾通道中，S4结构域在跨膜电场中的移动特别缓慢。为了研究HERG S4结构域缓慢移动的基础，并特别探究S4结构域与脂质双层以及通道蛋白其余部分之间的关系，我们将HERG中S4的每个氨基酸（L524 - L539）分别突变为色氨酸，并使用双电极电压钳方法在非洲爪蟾卵母细胞中表征突变通道的激活和失活特性。色氨酸具有大的疏水性侧链，因此在与脂质相互作用的位置应该是可以耐受的，但在涉及紧密蛋白质 - 蛋白质相互作用的位置则不然。值得注意的是，我们发现所有S4色氨酸突变体都具有功能。这些数据表明，S4结构域在电压传感器结构域的其余部分内堆积松散，并且可能暴露于脂质中。此外，我们确定残基K525、R528和K538是通道缓慢激活最重要的残基。
