CHANGES IN VOLTAGE ACTIVATION, CS+ SENSITIVITY, AND ION PERMEABILITY IN H5 MUTANTS OF THE PLANT K+ CHANNEL KAT1

KAT1 is a voltage-dependent inward rectifying K+ channel cloned from t he higher plant Arabidopsis thaliana [Anderson, J. A., Huprikar, S. S. , Kochian, L. V., Lucas, W. J. & 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 reg ion between Pro-247 and Pro-271 in KAT1 contains 14 additional amino a cids when compared with Shaker [Aldrich, R. W. (1993) Nature (London) 362, 107-108]. We studied various point mutations introduced into H5 t o determine whether voltage-dependent plant and animal K+ channels sha re similar pore structures. Through heterologous expression in Xenopus oocytes and voltage-clamp analysis combined with phenotypic analysis involving a potassium transport-defective Saccharomyces cerevisiae str ain, we investigated the selectivity filter of the mutants and their s usceptibility toward inhibition by cesium and calcium ions. With respe ct to electrophysiological properties, KAT1 mutants segregated into th ree groups: (i) wild-type-like channels, (ii) channels modified in sel ectivity and Cs+ or Ca2+ sensitivity, and (iii) a group that was addit ionally affected in its voltage dependence. Despite the additional 14 amino acids in H5, this motif in K4T1 is also involved in the formatio n of the ion-conducting pore because amino acid substitutions at Leu-2 51, Thr-256, Thr-259, and Thr-260 resulted in functional channels with modified ionic selectivity and inhibition. Creation of Ca2+ sensitivi ty and an increased susceptibility to Cs+ block through mutations with in the narrow pore might indicate that both blockers move deeply into the channel. Furthermore, mutations close to the rim of the pore affec ting the half-activation potential (U-1/2) indicate that amino acids w ithin the pore either interact with the voltage sensor or ion permeati on feeds back on gating.