title: Modulation of MthK potassium channel activity at the intracellular entrance to the pore.
abstract: We used a bacterial complementation screen with the LB2003 K(+) uptake-deficient strain of Escherichia coli to analyze residues that are critical to Methanobacterium thermoautotrophicum potassium channel (MthK) function. Channel expression and relative structural integrity of mutants were analyzed by SDS-PAGE and Western blot, and mechanisms underlying altered mutant channel function were analyzed using single-channel recording. We observed that wild-type MthK expression complements K(+) uptake deficiency. Although MthK function was previously thought to require Ca(2+) in the millimolar range, we demonstrate that at elevated temperatures the requirement for Ca(2+) becomes much lower. Mutations at the cytoplasmic mouth of the MthK pore can blunt complementation, indicating that those mutant channels cannot support K(+) uptake. In contrast, substitutions at the Ca(2+)-binding site in the MthK RCK domain did not decrease complementation compared with wild-type MthK. We focused on mutations to residues Glu-92 and Glu-96, which may form the narrowest part of the pore in the channel's closed state. Mutations at these residues can yield slight changes in single-channel conductance that do not necessarily correlate with effects on bacterial complementation. However, mutations at Glu-92 could also change channel open probability, and these changes correlated with complementation effects. The most striking of these mutations was E92A, which nearly eliminated bacterial complementation by decreasing the open probability of MthK. Our results suggest that the small, hydrophobic alanine side chain at the K(+) channel bundle crossing may generate an intrinsically stable structure, which in turn shifts the closed-to-open-state equilibrium toward the closed state.
authors.affiliation: Department of Physiology, University of Texas Health Science Center, San Antonio, Texas 78229.
authors.forename: Lyubov V
authors.initials: LV
authors.lastname: Parfenova
authors.affiliation: Department of Physiology, University of Texas Health Science Center, San Antonio, Texas 78229.
authors.forename: Brittany M
authors.initials: BM
authors.lastname: Crane
authors.affiliation: Department of Physiology, University of Texas Health Science Center, San Antonio, Texas 78229. Electronic address: rothberg@uthscsa.edu.
authors.forename: Brad S
authors.initials: BS
authors.lastname: Rothberg
mesh_terms: D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D005816:Genetic Complementation Test; D018698:Glutamic Acid / Q000737:chemistry; D017014:Methanobacterium / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011188:Potassium / Q000737:chemistry; D024681:Potassium Channels, Calcium-Activated / Q000737:chemistry* / Q000378:metabolism; D013696:Temperature
chemical_list: D024681:Potassium Channels, Calcium-Activated; D018698:Glutamic Acid; D000409:Alanine; D011188:Potassium; D002118:Calcium