Functional consequences of leucine and tyrosine mutations in the dual pore motifs of the yeast K+ channel, Tok1p
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Tandem pore-loop potassium channels differ from the majority of K+ channels in that a single polypeptide chain carries two K+-specific segments (P) each sandwiched between two transmembrane helices (M) to form an MP1M–MP2M series. Two of these peptide molecules assemble to form one functional potassium channel, which is expected to have biaxial symmetry (commonly described as asymmetric) due to independent mutation in the two MPM units. The resulting intrinsic asymmetry is exaggerated in fungal 2P channels, especially in Tok1p of Saccharomyces, by the N-terminal presence of four more transmembrane helices. Functional implications of such structural asymmetry have been investigated via mutagenesis of residues (L290 in P1 and Y424 in P2) that are believed to provide the outermost ring of carbonyl oxygen atoms for coordination with potassium ions. Both complementary mutations (L290Y and Y424L) yield functional potassium channels having quasi-normal conductance when expressed in Saccharomyces itself, but the P1 mutation (only) accelerates channel opening about threefold in response to depolarizing voltage shifts. The more pronounced effect at P1 than at P2 appears paradoxical in relation to evolution, because a comparison of fungal Tok1p sequences (from 28 ascomycetes) shows the filter sequence of P2 (overwhelmingly TIGYGD) to be much stabler than that of P1 (mostly TIGLGD). Profound functional asymmetry is revealed by the fact that combining mutations (L290Y + Y424L)—which inverts the order of residues from the wild-type channel—reduces the expressed channel conductance by a large factor (20-fold, cf. <twofold for the single mutants).
KeywordsVoltage dependence Conductance Potassium channel Whole-cell recording Activation kinetics
The authors are indebted to Drs. T. Miosga and P. Ljungdahl for yeast strains. The work was supported by Grants from the Deutsche Forschungsgemeinschaft (to A.B.) and from the US National Institutes of Health (GM-60696, to C.L.S.).
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