Changes in voltage activation, Cs+ sensitivity, and ion permeability in H5 mutants of the plant K+ channel KAT1

• Published in: Proceedings of the National Academy of Sciences - PNAS 1996-07, Vol.93 (15), p.8123-8128
• Main Authors: Becker, D. (Universitat Hannover, Hannover, Germany.), Dreyer, I, Hoth, S, Reid, J.D, Busch, H, Lehnen, M, Palme, K, Hedrich, R
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Amino acids; Animals; Arabidopsis - genetics; Arabidopsis - physiology; Arabidopsis Proteins; ARABIDOPSIS THALIANA; Base Sequence; CALCIO; CALCIUM; Calcium - pharmacology; CATION; CATIONES; Cell Membrane Permeability; CESIO; CESIUM; Cesium - pharmacology; Cloning, Molecular; Electric current; Electric potential; ESTRUCTURA CELULAR; Female; ION; Ion Channel Gating; IONES; Kinetics; Leucine; Membrane Potentials - drug effects; Molecular Sequence Data; MUTACION INDUCIDA; Mutagenesis, Site-Directed; MUTANT; MUTANTES; MUTATION PROVOQUEE; Oocytes; Oocytes - drug effects; Oocytes - physiology; Patch-Clamp Techniques; PERMEABILIDAD; PERMEABILITE; Plant Proteins - physiology; Polymerase chain reaction; POTASIO; POTASSIUM; Potassium Channels - biosynthesis; Potassium Channels - chemistry; Potassium Channels - physiology; Potassium Channels, Inwardly Rectifying; Protein Structure, Secondary; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Rectifiers; Saccharomyces cerevisiae; Sensors; STRUCTURE CELLULAIRE; Threonine; Xenopus; Yeasts
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.93.15.8123

KAT1 is a voltage-dependent inward rectifying K+ channel cloned from the higher plant Arabidopsis thaliana [Anderson, J. A., Huprikar, S. S., Kochian, L. V., Lucas, W. J. and Gaber, R. F. (1992) Proc. Natl. Acad. Sci. USA 89, 3736-3740]. It is related to the Shaker superfamily of K+ channels characterized by six transmembrane spanning domains (S1-S6) and a putative pore-forming region between S5 and S6 (H5). The H5 region between Pro-247 and Pro-271 in KAT1 contains 14 additional amino acids when compared with Shaker [Aldrich, R. W. (1993) Nature (London) 362, 107-108]. We studied various point mutations introduced into H5 to determine whether voltage-dependent plant and animal K+ channels share similar pore structure. Through heterologous expression in Xenopus oocytes and voltage-clamp analysis combined with phenotypic analysis involving a potassium transport-defective Saccharomyces cerevisiae strain, we investigated the selectivity filter of the mutants and their susceptibility toward inhibition by cesium and calcium ions. With respect to electrophysiological properties, KAT1 mutants segregated into three groups: (i) wild-type-like channels, (ii) channels modified in selectivity and Cs+ or Ca2+ sensitivity, and (iii) a group that was additionally affected in its voltage dependence. Despite the additional 14 amino acids in H5, this motif in KAT1 is also involved in the formation of the ion-conducting pore because amino acid substitutions at Leu-251, Thr-256, Thr-259, and Thr-260 resulted in functional channels with modified ionic selectivity and inhibition. Creation of Ca2+ sensitivity and an increased susceptibility to Cs + block through mutations within the narrow pore might indicate that both blockers move deeply into the channel. Furthermore, mutations close to the rim of the pore affecting the half-activation potential (U1/2) indicate that amino acids within the pore either interact with the voltage sensor or ion permeation feeds back on gating