title: Residues beyond the selectivity filter of the K+ channel kir2.1 regulate permeation and block by external Rb+ and Cs+.
abstract: 1. Kir2.1 channels are blocked by Rb+ and Cs+ in a voltage-dependent manner, characteristic of many inward rectifier K+ channels. Mutation of Ser165 in the transmembrane domain M2 to Leu (S165L) abolished Rb+ blockage and lowered Cs+ blocking affinity. At negative voltages Rb+ carried large inward currents. 2. A model of the Kir2.1 channel, built by homology with the structure of the Streptomyces lividans K+ channel KcsA, suggested the existence of an intersubunit hydrogen bond between Ser165 and Thr141 in the channel pore-forming P-region that helps stabilise the structure of this region. However, mutations of Thr141 and Ser165 did not produce effects consistent with a hydrogen bond between these residues being essential for blockage. 3. An alternative alignment between the M2 regions of Kir2.1 and KcsA suggested that Ser165 is itself a pore-lining residue, more directly affecting blockage. We were able to replace Ser165 with a variety of polar and non-polar residues, consistent with this residue being pore lining. Some of these changes affected channel blockage. 4. We tested the hypothesis that Asp172 - a residue implicated in channel gating by polyamines - formed an additional selectivity filter by using the triple mutant T141A/S165L/D172N. Large Rb+ and Cs+ currents were measured in this mutant. 5. We propose that both Thr141 and Ser165 are likely to provide binding sites for monovalent blocking cations in wild-type channels. These residues lie beyond the carbonyl oxygen tunnel thought to form the channel selectivity filter, which the blocking cations must therefore traverse.
authors.affiliation: Ion Channel Group, Department of Cell Physiology and Pharmacology and Department of Chemistry, University of Leicester, PO Box 138, Leicester LE1 9HN, UK.
authors.forename: G A
authors.initials: GA
authors.lastname: Thompson
authors.forename: M L
authors.initials: ML
authors.lastname: Leyland
authors.forename: I
authors.initials: I
authors.lastname: Ashmole
authors.forename: M J
authors.initials: MJ
authors.lastname: Sutcliffe
authors.forename: P R
authors.initials: PR
authors.lastname: Stanfield
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry; D016466:CHO Cells; D002463:Cell Membrane Permeability / Q000187:drug effects / Q000502:physiology*; D002586:Cesium / Q000494:pharmacology*; D006224:Cricetinae; D007930:Leucine; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015221:Potassium Channels / Q000737:chemistry / Q000187:drug effects / Q000502:physiology*; D024661:Potassium Channels, Inwardly Rectifying; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012413:Rubidium / Q000494:pharmacology*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012694:Serine; D014162:Transfection
chemical_list: D001426:Bacterial Proteins; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D011994:Recombinant Proteins; C107790:prokaryotic potassium channel; D002586:Cesium; D012694:Serine; D007930:Leucine; D012413:Rubidium