Determination of Key Structural Requirements of a K+ Channel Pore*

Among the highly conserved sites in K+ channel pores, the tyrosine-glycine sequence is believed to play an important role in selectivity. Here we describe a novel approach in which comprehensive mutagenesis of the YG sites of the voltage-gated K+ channel, Kat1, is combined with phenotypic screening in Saccharomyces cerevisiae and electrophysiological analysis in Xenopus oocytes to determine the roles of these sites in K+ selectivity. We show that structural constraints necessitate a tyrosine or phenylalanine at the first position to confer full K+ selectivity. Substitution to arginine creates a channel titratable by external pH, suggesting that the side group at this position may line the channel pore. Permeation is abolished by any increase in bulk at the adjacent glycine position unless accompanied by a compensatory mutation at the tyrosine site. These results suggest a model in which the selectivity filter of the K+ channel requires an aromatic residue paired with glycine within the pore loop in order to maintain maximal K+ selectivity.

[1]  David E. Clapham,et al.  Nonselective and Gβγ-Insensitive weaver K+ Channels , 1996, Science.

[2]  D. Cox,et al.  Functional Effects of the Mouse weaver Mutation on G Protein–Gated Inwardly Rectifying K+ Channels , 1996, Neuron.

[3]  Roderick MacKinnon,et al.  Agitoxin Footprinting the Shaker Potassium Channel Pore , 1996, Neuron.

[4]  Rama Ranganathan,et al.  Spatial Localization of the K+ Channel Selectivity Filter by Mutant Cycle–Based Structure Analysis , 1996, Neuron.

[5]  Christopher Miller,et al.  A Strongly Interacting Pair of Residues on the Contact Surface of Charybdotoxin and a Shaker K+ Channel , 1996, Neuron.

[6]  James E. Hall,et al.  Topology of the pore-region of a K+ channel revealed by the NMR-derived structures of scorpion toxins , 1995, Neuron.

[7]  J. Schroeder,et al.  Identification of Strong Modifications in Cation Selectivity in an Arabidopsis Inward Rectifying Potassium Channel by Mutant Selection in Yeast (*) , 1995, The Journal of Biological Chemistry.

[8]  J. Sedbrook,et al.  Expression of an Arabidopsis Potassium Channel Gene in Guard Cells , 1995, Plant physiology.

[9]  H. Fozzard,et al.  A structural motif for the voltage-gated potassium channel pore. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[10]  N. Crawford,et al.  Amino Terminus and the First Four Membrane-spanning Segments of the Arabidopsis K Channel KAT1 Confer Inward-rectification Property of Plant-Animal Chimeric Channels (*) , 1995, The Journal of Biological Chemistry.

[11]  A. Brown,et al.  K+ pore structure revealed by reporter cysteines at inner and outer surfaces , 1995, Neuron.

[12]  C. Miller,et al.  Silver as a probe of pore-forming residues in a potassium channel. , 1995, Science.

[13]  C. Slayman,et al.  Use of Saccharomyces cerevisiae for patch-clamp analysis of heterologous membrane proteins: characterization of Kat1, an inward-rectifying K+ channel from Arabidopsis thaliana, and comparison with endogeneous yeast channels and carriers. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[14]  T. Hoshi,et al.  Regulation of voltage dependence of the KAT1 channel by intracellular factors , 1995, The Journal of general physiology.

[15]  F. Gaymard,et al.  Expression of a cloned plant K+ channel in Xenopus oocytes: analysis of macroscopic currents. , 1995, The Plant journal : for cell and molecular biology.

[16]  J. Warmke,et al.  A family of potassium channel genes related to eag in Drosophila and mammals. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[17]  R. MacKinnon,et al.  Mutations in the K+ channel signature sequence. , 1994, Biophysical journal.

[18]  F. Rubio,et al.  The protein phosphatase calcineurin is essential for NaCl tolerance of Saccharomyces cerevisiae. , 1994, The Journal of biological chemistry.

[19]  J. Schroeder.  Heterologous Expression and Functional Analysis of Higher Plant Transport Proteins in Xenopus Oocytes , 1994 .

[20]  D A Dougherty,et al.  A mechanism for ion selectivity in potassium channels: computational studies of cation-pi interactions. , 1993, Science.

[21]  W. J. Lucas,et al.  Expression of an inward-rectifying potassium channel by the Arabidopsis KAT1 cDNA. , 1992, Science.

[22]  Y. Jan,et al.  Tracing the roots of ion channels , 1992, Cell.

[23]  W. J. Lucas,et al.  Functional expression of a probable Arabidopsis thaliana potassium channel in Saccharomyces cerevisiae. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[24]  F. Gaymard,et al.  Cloning and expression in yeast of a plant potassium ion transport system. , 1992, Science.

[25]  H. Guy,et al.  Atomic scale structure and functional models of voltage-gated potassium channels. , 1992, Biophysical journal.

[26]  J. Schroeder,et al.  Inward-rectifying K+ channels in guard cells provide a mechanism for low-affinity K+ uptake. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[27]  N. Atkinson,et al.  A component of calcium-activated potassium channels encoded by the Drosophila slo locus. , 1991, Science.

[28]  R. Gaber,et al.  TRK1 and TRK2 encode structurally related K+ transporters in Saccharomyces cerevisiae , 1991, Molecular and cellular biology.

[29]  R. Drysdale,et al.  A distinct potassium channel polypeptide encoded by the Drosophila eag locus , 1991, Science.

[30]  Y. Jan,et al.  Alteration of voltage-dependence of Shaker potassium channel by mutations in the S4 sequence , 1991, Nature.

[31]  A M Buckley,et al.  TRK2 is required for low affinity K+ transport in Saccharomyces cerevisiae. , 1990, Genetics.

[32]  L. Salkoff,et al.  K+ current diversity is produced by an extended gene family conserved in Drosophila and mouse. , 1990, Science.

[33]  W. Bönigk,et al.  Primary structure and functional expression from complementary DNA of the rod photoreceptor cyclic GMP-gated channel , 1989, Nature.

[34]  L. Salkoff,et al.  A family of putative potassium channel genes in Drosophila. , 1989, Science.

[35]  J. Schroeder.  K+ transport properties of K+ channels in the plasma membrane of Vicia faba guard cells , 1988, The Journal of general physiology.

[36]  G. Fink,et al.  TRK1 encodes a plasma membrane protein required for high-affinity potassium transport in Saccharomyces cerevisiae , 1988, Molecular and cellular biology.

[37]  Y. Jan,et al.  Sequence of a probable potassium channel component encoded at Shaker locus of Drosophila. , 1987, Science.

[38]  F. Sanger,et al.  DNA sequencing with chain-terminating inhibitors. , 1977, Proceedings of the National Academy of Sciences of the United States of America.

[39]  F Bezanilla,et al.  Inactivation of the sodium channel. II. Gating current experiments , 1977, The Journal of general physiology.

[40]  L. Guarente,et al.  High-efficiency transformation of yeast by electroporation. , 1991, Methods in enzymology.

[41]  Thomas J. White,et al.  PCR protocols: a guide to methods and applications. , 1990 .

[42]  R. Keynes.  The ionic channels in excitable membranes. , 1975, Ciba Foundation symposium.