期刊
BIOCHEMICAL JOURNAL
卷 393, 期 -, 页码 645-655出版社
PORTLAND PRESS LTD
DOI: 10.1042/BJ20051380
关键词
cation selectivity gating; outward-rectifying potassium channel; protein domain interaction; Saccharomyces cerevisiae; voltage- and potassium-dependent channel gating
资金
- Biotechnology and Biological Sciences Research Council [BB/D001528/1, P12750, BB/C500595/1] Funding Source: researchfish
- BBSRC [BB/D001528/1] Funding Source: UKRI
- Biotechnology and Biological Sciences Research Council [BB/C500595/1, BB/D001528/1, P12750] Funding Source: Medline
Gating of the outward-rectifying K+ channel TOKI of Saccharomyces cerevisiae is controlled by membrane voltage and extracellular K+ concentration. Previous studies identified two kinetically distinct effects of K+, and site-mutagenic analysis associated these K+-dependencies with domains of the extracellular turrets of the channel protein. We have mapped the TOK1 pore domains to extant K+ channel crystal structures to target additional residues contributing to TOK1 gating. Leu(270), located in the first pore domain of TOK1, was found to be critical for gating and its K+ sensitivity. Analysis of amino acid substitutions indicated that spatial position of the polypeptide backbone is a primary factor determining gating sensitivity to K+. The strongest effects, with L270Y, L270F and L270W, led to more than a 30-fold decrease in apparent K+ affinity and an inversion in the apparent K+-dependence of voltage-dependent gating compared with the wild-type current. A partial rescue of wild-type gating was obtained on substitution in the second pore domain with the double mutant L270D/A428Y. These, and additional results, demarcate extracellular domains that are associated with the K+-sensitivity of TOK1 and they offer primary evidence for a synergy in gating between the two pore domains of TOK1, demonstrating an unexpected degree of long-distance interaction across the mouth of the K+ channel.
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