Taglialatela M, Vandongen A M, Drewe J A, Joho R H, Brown A M, Kirsch G E
Department of Molecular Physiology, Baylor College of Medicine, Houston 77030.
Mol Pharmacol. 1991 Aug;40(2):299-307.
Tetraethylammonium (TEA) is a small ion that is thought to block open K+ channels by binding either to an internal or to an external site. For this reason, it has been used to probe the ion conduction pathway or pore of K+ channel mutants and a K+ channel chimera. The results suggested that the region between transmembrane segments 5 and 6 (S5-S6 linker) was involved in the formation of both the internal and the external TEA binding sites and the K+ conduction pathway. Therefore, we compared internal and external TEA block of the currents expressed in Xenopus oocytes injected with RNAs from four related K+ channel clones, DRK1, RCK1, RCK2, and r-NGK2, which have only subtle structural differences in the S5-S6 linker. r-NGK2 was the most sensitive to external TEA and the least sensitive to internal TEA application. For DRK1 the profile was reversed. RCK1 was blocked equally well from either side, whereas RCK2 was more strongly blocked by internal TEA. The internal block was voltage dependent, whereas the external block was virtually voltage independent. As predicted from block of whole-oocyte currents, internal TEA produced a slow block of DRK1 and RCK2 single-channel currents but had almost no effect on r-NGK2 single-channel currents. Tetrapentylammonium produced a stronger block than TEA at the internal site, and the block was relieved by inward K+ currents, therefore suggesting that the internal TEA binding site is located within the K+ conduction pathway. These results, together with the TEA block of single-channel currents, establish what has until now been inferred by extrapolation from other studies, i.e., that TEA is an open-channel blocker in K+ channel clones. DRK1 mutants with extensive amino- and carboxyl-terminal deletions showed the same blocking profile as the parent DRK1. We conclude that TEA blocks these K+ channels at two sites, which define the inner and outer mouths of the channel pores. Comparison of the primary amino acid sequences in the S5-S6 linker suggests which residues may be responsible for the different patterns of TEA block.
四乙铵（TEA）是一种小离子，被认为通过结合内部或外部位点来阻断开放的钾离子通道。因此，它已被用于探究钾离子通道突变体和钾离子通道嵌合体的离子传导途径或孔道。结果表明，跨膜片段5和6之间的区域（S5-S6连接区）参与了内部和外部TEA结合位点以及钾离子传导途径的形成。因此，我们比较了注射了来自四个相关钾离子通道克隆DRK1、RCK1、RCK2和r-NGK2的RNA的非洲爪蟾卵母细胞中所表达电流的内部和外部TEA阻断情况，这四个克隆在S5-S6连接区仅有细微的结构差异。r-NGK2对外部TEA最敏感，对内部施加的TEA最不敏感。对于DRK1，情况则相反。RCK1从两侧被阻断的效果相同，而RCK2被内部TEA阻断的程度更强。内部阻断依赖电压，而外部阻断实际上不依赖电压。正如从全卵母细胞电流阻断所预测的那样，内部TEA对DRK1和RCK2单通道电流产生缓慢阻断，但对r-NGK2单通道电流几乎没有影响。四戊铵在内部位点产生的阻断比TEA更强，并且这种阻断可被内向钾离子电流缓解，因此表明内部TEA结合位点位于钾离子传导途径内。这些结果，连同单通道电流的TEA阻断情况，证实了到目前为止一直通过从其他研究推断得出的结论，即TEA是钾离子通道克隆中的开放通道阻断剂。具有广泛氨基和羧基末端缺失的DRK1突变体显示出与亲本DRK1相同的阻断模式。我们得出结论，TEA在两个位点阻断这些钾离子通道，这两个位点界定了通道孔的内口和外口。S5-S6连接区中主要氨基酸序列的比较表明哪些残基可能导致TEA阻断的不同模式。
