The number, sign, and distribution of charged residues in the pore-forming H5 domain for inward-rectifying K channels (IRK1) are different from the otherwise homologous H5 domains of other voltage-gated K channels. We have mutated Arg148, which is perfectly conserved in all inward rectifiers, to His in the H5 of IRK1 (Kir2. 1). Channel activity was lost by the mutation, but coexpression of the mutant (R148H) along with the wild-type (WT) mRNA revealed populations of channels with reduced single-channel conductances. Long-lasting and flickery sublevels were 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 coexpression made the IRK1 channel more sensitive to extracellular block by Mg2+ and Ca2+, and turned this blockade from a voltage-independent to 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 sensitivity. The blocking rate of open channel blockers, such as Cs+ and Ba2+, was facilitated by coexpression without significant change in the steady state block. Evaluation of the electrical distance to the binding site for Mg2+ or Ca2+ and that to the barrier peak for block by Cs+ or Ba2+ suggest that Arg148 is located between the external blocking site for Mg2+ or Ca2+ and the deeper blocking site for Cs+ or Ba2+ in the IRK1 channel. It is concluded that Arg148 serves as a barrier to cationic blockers, keeping Mg2+ and Ca2+ out from the electric field of the membrane.
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