Sodium blocking induced by a point mutation at the C-terminal end of the pore helix of the KAT1 channel

A plant hyperpolarization-activating KC channel, KAT1, is highly selective for K+ over Na+ and is little affected by external Na+, which is crucial to take up K+ effectively in a Naf-containing environment. It has been shown that a mutation at the location (Thr256) preceding the selectivity signatur e sequence dramatically enhanced the sensitivity of the KAT1 channel to ext ernal Na+. We report here electrophysiological experiments for the mechanis m of action of external Na+ on KAT1 channels. The Thr256 residue was substi tuted with either glutamine (Q) or glutamate (E). The wild-type channel was insensitive to external Naf. However, the activity of both mutant channels was significantly depressed by Na+ with apparent dissociation constants of 6.7 mM and 11.3 mM for T256Q and T256E, respectively. The instantaneous cu rrent-voltage relationships revealed distinct blocking mechanisms for these mutants. For T256Q a typical voltage-dependent fast blocking was shown. On the other hand, the blocking for the T256E mutant was voltage-independent at low Na+ concentrations and became voltage-dependent at higher concentrat ions. At extreme hyperpolarization the blocking was relieved significantly. These data strongly suggest that the mutation at the end of the pore helix rearranged the selectivity filter and allows Na+ to penetrate into the por e.