title: Differential roles of blocking ions in KirBac1.1 tetramer stability.
abstract: Potassium channels are tetrameric proteins that mediate K(+)-selective transmembrane diffusion. For KcsA, tetramer stability depends on interactions between permeant ions and the channel pore. We have examined the role of pore blockers on the tetramer stability of KirBac1.1. In 150 mm KCl, purified KirBac1.1 protein migrates as a monomer (approximately 40 kDa) on SDS-PAGE. Addition of Ba(2+) (K(1/2) approximately 50 microm) prior to loading results in an additional tetramer band (approximately 160 kDa). Mutation A109C, at a residue located near the expected Ba(2+)-binding site, decreased tetramer stabilization by Ba(2+) (K(1/2) approximately 300 microm), whereas I131C, located nearby, stabilized tetramers in the absence of Ba(2+). Neither mutation affected Ba(2+) block of channel activity (using (86)Rb(+) flux assay). In contrast to Ba(2+), Mg(2+) had no effect on tetramer stability (even though Mg(2+) was a potent blocker). Many studies have shown Cd(2+) block of K(+) channels as a result of cysteine substitution of cavity-lining M2 (S6) residues, with the implicit interpretation that coordination of a single ion by cysteine side chains along the central axis effectively blocks the pore. We examined blocking and tetramer-stabilizing effects of Cd(2+) on KirBac1.1 with cysteine substitutions in M2. Cd(2+) block potency followed an alpha-helical pattern consistent with the crystal structure. Significantly, Cd(2+) strongly stabilized tetramers of I138C, located in the center of the inner cavity. This stabilization was additive with the effect of Ba(2+), consistent with both ions simultaneously occupying the channel: Ba(2+) at the selectivity filter entrance and Cd(2+) coordinated by I138C side chains in the inner cavity.
authors.affiliation: Department of Pharmacological and Physiological Science, Saint Louis University School of Medicine, St. Louis, Missouri 63104; Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110.
authors.forename: Shizhen
authors.initials: S
authors.lastname: Wang
authors.affiliation: Department of Pharmacological and Physiological Science, Saint Louis University School of Medicine, St. Louis, Missouri 63104.
authors.forename: Yewande
authors.initials: Y
authors.lastname: Alimi
authors.affiliation: Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110.
authors.forename: Ailing
authors.initials: A
authors.lastname: Tong
authors.affiliation: Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110.
authors.forename: Colin G
authors.initials: CG
authors.lastname: Nichols
authors.affiliation: Department of Pharmacological and Physiological Science, Saint Louis University School of Medicine, St. Louis, Missouri 63104. Electronic address: denkvetc@slu.edu.
authors.forename: Decha
authors.initials: D
authors.lastname: Enkvetchakul
mesh_terms: D001704:Biopolymers / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D015221:Potassium Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D001704:Biopolymers; D015221:Potassium Channels