SINGLE MUTATIONS STRONGLY ALTER THE K-SELECTIVE PORE OF THE K-IN CHANNEL KAT1()

Voltage-dependent potassium uptake channels represent the major pathwa y for K+ accumulation underlying guard cell swelling and stomatal open ing, The core structure of these Shaker-like channels is represented b y six transmembrane domains and an amphiphilic pore-forming region bet ween the fifth and sixth domain. To explore the effect of point mutati ons within the stretch of amino acids lining the K+ conducting pore of KAT1, an Arabidopsis thaliana guard cell K-in channel, we selected re sidues deep inside and in the periphery of the pore. The mutations on positions 256 and 267 strongly altered the interaction of the permeati on pathway with external Ca2+ ions. Point mutations on position 256 in KAT1 affected the affinity towards Ca2+, the voltage dependence as we ll as kinetics of the Ca2+ blocking reaction. Among these T256S showed a Ca2+ phenotype reminiscent of an inactivation-like process, a pheno menon unknown for K-in channels so far. Mutating histidine 267 to alan ine, a substitution strongly affecting C-type inactivation in Shaker, this apparent inactivation could be linked to a very slow calcium bloc k. The mutation H267A did not affect gating but hastened the Ca2+ bloc k/unblock kinetics and increased the Ca2+ affinity of KAT1, From the a nalysis of the presented data we conclude that even moderate point mut ations in the pore of KAT1 seem to affect the pore geometry rather tha n channel gating. (C) 1998 Federation of European Biochemical Societie s.