title: Functional consequences of leucine and tyrosine mutations in the dual pore motifs of the yeast K(+) channel, Tok1p.
abstract: 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 MP(1)M-MP(2)M 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 P(1) and Y424 in P(2)) 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 P(1) mutation (only) accelerates channel opening about threefold in response to depolarizing voltage shifts. The more pronounced effect at P(1) than at P(2) appears paradoxical in relation to evolution, because a comparison of fungal Tok1p sequences (from 28 ascomycetes) shows the filter sequence of P(2) (overwhelmingly TIGYGD) to be much stabler than that of P(1) (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).
authors.affiliation: Botanisches Institut I, Universität Karlsruhe (TH), Kaiserstrasse 12, 76128, Karlsruhe, Germany.
authors.forename: Anja
authors.initials: A
authors.lastname: Roller
authors.forename: Gabriel
authors.initials: G
authors.lastname: Natura
authors.forename: Hermann
authors.initials: H
authors.lastname: Bihler
authors.forename: Clifford L
authors.initials: CL
authors.lastname: Slayman
authors.forename: Adam
authors.initials: A
authors.lastname: Bertl
mesh_terms: D000595:Amino Acid Sequence; D005816:Genetic Complementation Test; D007930:Leucine / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D018408:Patch-Clamp Techniques; D010802:Phylogeny; D015221:Potassium Channels / Q000145:classification / Q000235:genetics* / Q000378:metabolism; D017433:Protein Structure, Secondary; D029701:Saccharomyces cerevisiae Proteins / Q000145:classification / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment; D014443:Tyrosine / Q000235:genetics*
chemical_list: D015221:Potassium Channels; D029701:Saccharomyces cerevisiae Proteins; C095220:TOK1 protein, S cerevisiae; D014443:Tyrosine; D007930:Leucine