Kavanaugh M P, Varnum M D, Osborne P B, Christie M J, Busch A E, Adelman J P, North R A
Vollum Institute, Oregon Health Sciences University, Portland 97201.
J Biol Chem. 1991 Apr 25;266(12):7583-7.
Extracellular tetraethylammonium (TEA) inhibits currents in Xenopus oocytes that have been injected with mRNAs encoding voltage-dependent potassium channels. Concentration-response curves were used to measure the affinity of TEA; this differed up to 700-fold among channels RBK1 (KD 0.3 mM), RGK5 (KD 11 mM), and RBK2 (KD greater than 200 mM). Studies in which chimeric channels were expressed localized TEA binding to the putative extracellular loop between trans-membrane domains S5 and S6. Site-directed mutagenesis of residues in this region identified the residue Tyr379 of RBK1 as a crucial determinant of TEA sensitivity; substitution of Tyr in the equivalent positions of RBK2 (Val381) and RGK5 (His401) made these channels as sensitive to TEA as RBK1. Nonionic forces are involved in TEA binding because (i) substitution of the Phe for Tyr379 in RBK1 increased its affinity, (ii) protonation of His401 in RGK5 selectively reduced its affinity, and (iii) the affinity of TEA was unaffected by changes in ionic strength. The results suggest an explanation for the marked differences in TEA sensitivity that have been observed among naturally occurring and cloned potassium channels and indicate that the amino acid corresponding to residue 379 in RBK1 lies within the external mouth of the ion channel.
细胞外四乙铵（TEA）可抑制已注射编码电压依赖性钾通道mRNA的非洲爪蟾卵母细胞中的电流。利用浓度-反应曲线来测量TEA的亲和力；在RBK1（解离常数KD为0.3 mM）、RGK5（KD为11 mM）和RBK2（KD大于200 mM）这几种通道之间，其亲和力差异高达700倍。对嵌合通道进行表达的研究将TEA结合定位到跨膜结构域S5和S6之间假定的细胞外环上。对该区域残基进行定点诱变确定了RBK1的Tyr379残基是TEA敏感性的关键决定因素；将RBK2（Val381）和RGK5（His401）等效位置的Tyr替换后，使这些通道对TEA的敏感性与RBK1相同。TEA结合涉及非离子力，原因如下：（i）将RBK1中的Tyr379替换为Phe会增加其亲和力；（ii）RGK5中His401的质子化选择性降低了其亲和力；（iii）TEA的亲和力不受离子强度变化的影响。这些结果为在天然存在的和克隆的钾通道之间观察到的TEA敏感性的显著差异提供了解释，并表明与RBK1中379位残基相对应的氨基酸位于离子通道的外口处。
