A CONSERVED ARGININE RESIDUE IN THE PORE REGION OF AN INWARD RECTIFIER K-CHANNEL (IRK1) AS AN EXTERNAL BARRIER FOR CATIONIC BLOCKERS

The number, sign, and distribution of charged residues in the pore-for ming H5 domain for inward rectifying K channels (IRK1) are different f rom the otherwise homologous H5 domains of other voltage-gated K chann els. We have mutated Arg(148), which is perfectly conserved in all inw ard rectifiers, to His in the H5 of IRK1 (Kir2.1). Channel activity wa s lost by the mutation, but coexpression of the mutant (R148H) along w ith the wild-type (WT) mRNA revealed populations of channels with redu ced single-channel conductances. Long-lasting and flickery sublevels w ere detected exclusively for the coexpressed channels. These findings indicated that the mutant subunit formed hetero-oligomers with the WT subunit. The permeability ratio was altered by the mutation, while the selectivity sequence (K+ > Rb+ > NH4+ >> Na+) was preserved. The coex pression made the IRK1 channel more sensitive to extracellular block b y Mg2+ and Ca2+, and turned this blockade from a voltage-independent t o a -dependent process. The sensitivity of the mutant channels to Mg2 was enhanced at higher pH and by an increased ratio of mutant:WT mRNA , suggesting that the charge on the Arg site controlled the sensitivit y. The blocking rate of open channel blockers, such as Cs+ and Ba2+, w as facilitated by coexpression without significant change in the stead y state block. Evaluation of the electrical distance to the binding. s ite for Mg2+ or Ca?+ and that to the barrier peak for block by Cs+ or Ba2+ suggest that Arg(148) is located between the external blocking si te for Mg2+ or Ca2+ and the deeper blocking site for Cs+ or Ba2+ in th e IRK1 channel. It is concluded that Arg(148) serves as a barrier to c ationic blockers, keeping Mg2+ and Ca2+ out from the electric field of the membrane.