Random mutagenesis reveals a region important for gating of the yeast K+ channel Ykc1

Abstract

YKC1 (TOK1, DUK1, YORK) encodes the outwardly rectifying K+ channel of the yeast plasma membrane. Non‐targeted mutations of YKC1 were isolated by their ability to completely block proliferation when expressed in yeast. All such mutations examined occurred near the cytoplasmic ends of the transmembrane segments following either of the duplicated P loops, which we termed the ‘post‐P loop’ (PP) regions. These PP mutations specifically caused marked defects in the ‘C1’ states, a set of interrelated closed states that Ykc1 enters and exits at rates of tens to hundreds of milliseconds. These results indicate that the Ykc1 PP region plays a role in determining closed state conformations and that non‐targeted mutagenesis and microbial selection can be a valuable tool for probing structure–function relationships of ion channels.

The carboxyl tail forms a discrete functional domain that blocks closure of the yeast K + channel

“…Genetic analysis, coupled with biophysical analysis, of TOK1 has assisted in the identification of what we call the PP region, the cytoplasmic end of the P-region-following membrane domain, which has been found to be intimately involved with gating of cation channels (1,2,3). Our analyses have also pointed to the existence of functional domains that, on first glance, appeared unique to TOK1, including a filter-specific gating ( Fig.…”

“…All RNA injected into oocytes was produced in vitro from plasmid templates as described previously (1). Shortened TOK1 templates, both carboxyl and͞or amino deletions, were produced by using standard PCR techniques to generate ORFs that were inserted between the 5Ј and 3Ј untranslated sequences of the Xenopus ␤-globulin gene in the oocyte expression vector, pGH19 (9).…”

“…Although commonly referred to as ''voltage-regulated'' (21), TOK1's gating is regulated by the K ϩ electrochemical potential (⌬ K ϩ ) rather than merely voltage alone (19,20). The front line of this ⌬ K ϩ -dependent regulation is a nearinstantaneous gating process that prevents inward current flow, the ''R'' state (1). Because it is the only place where the transmembrane ⌬ K ϩ can be efficiently monitored, the R state is most readily accounted for as an intrinsic gating property of the filter region (22).…”

“…Mutations exclusively in the ''PP'' region (the end of M6 and M8 in the case of TOK1) specifically disrupt the C states (1) suggesting that they result from a constriction of the inner mouth of the pore, akin to deactivation-type gating in other cation channels (2,3,23,24). We have proposed that the K ext ϩ ͞Vm dependence of C could simply result from the open noncollapsed filter preventing inner pore gating transitions (4).…”

“…§1734 solely to indicate this fact. Our model was partly deduced from the behavior of tailless mutant channels (1). A deletion can be effective by removing only one of several necessary parts.…”