title: Structure and dynamics of the pore of inwardly rectifying K(ATP) channels.
abstract: Inwardly rectifying K(+) currents are generated by a complex of four Kir (Kir1-6) subunits. Pore properties are conferred by the second transmembrane domain (M2) of each subunit. Using cadmium ions as a cysteine-interacting probe, we examined the accessibility of substituted cysteines in M2 of the Kir6.2 subunit of inwardly rectifying K(ATP) channels. The ability of Cd(2+) ions to inhibit channels was used as the estimate of accessibility. The distribution of Cd(2+) accessibility is consistent with an alpha-helical structure of M2. The apparent surface of reactivity is broad, and the most reactive residues correspond to the solvent-accessible residues in the bacterial KcsA channel crystal structure. In several mutants, single channel measurements indicated that inhibition occurred by a single transition from the open state to a zero-conductance state. Analysis of currents expressed from mixtures of control and L164C mutant subunits indicated that at least three cysteines are required for coordination of the Cd(2+) ion. Application of phosphatidylinositol 4,5-diphosphate to inside-out membrane patches stabilized the open state of all mutants and also reduced cadmium sensitivity. Moreover, the Cd(2+) sensitivity of several mutants was greatly reduced in the presence of inhibitory ATP concentrations. Taken together, these results are consistent with state-dependent accessibility of single Cd(2+) ions to coordination sites within a relatively narrow inner vestibule.
authors.affiliation: Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
authors.forename: G
authors.initials: G
authors.lastname: Loussouarn
authors.forename: E N
authors.initials: EN
authors.lastname: Makhina
authors.forename: T
authors.initials: T
authors.lastname: Rose
authors.forename: C G
authors.initials: CG
authors.lastname: Nichols
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001426:Bacterial Proteins; D019556:COS Cells; D002104:Cadmium / Q000494:pharmacology; D003545:Cysteine; D007700:Kinetics; D046911:Macromolecular Substances; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D018408:Patch-Clamp Techniques; D026902:Potassium Channel Blockers; D015221:Potassium Channels / Q000737:chemistry* / Q000502:physiology*; D024661:Potassium Channels, Inwardly Rectifying; D011487:Protein Conformation; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
chemical_list: D001426:Bacterial Proteins; D046911:Macromolecular Substances; D026902:Potassium Channel Blockers; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D011994:Recombinant Proteins; C107790:prokaryotic potassium channel; D002104:Cadmium; D000255:Adenosine Triphosphate; D003545:Cysteine