A Conserved Arginine Near the Selectivity Filter of Kir1.1 Controls Rb/K Selectivity

Abstract

ROMK (Kir1.1) channels are important for K secretion and recycling in the collecting duct, connecting tubule and thick ascending limb of the mammalian nephron. We have identified a highly conserved Arg in the P loop of the channel near the selectivity filter that controls Rb/K selectivity. Mutation of this Arg to a Tyr (R128Y-Kir1.1b, R147Y-Kir1.1a) increased Rb/K selectivity by 11 ± 1 fold, NH4/K selectivity by 6 ±1 fold, and Cs/K selectivity by 32 ± 5 fold over wild-type (wt), with no significant change in K/Na selectivity. R128Y remained susceptible to block by both external Ba and the honey bee toxin, TPNQ, although it had a 100 fold lower affinity for TPNQ than wild-type. Single-channel R128Y-Kir1.1b conductance averaged 12 ± 0.5 pS in 100mM RbCl, 0 Mg, 0 Ca solutions, compared to 18 ± 2 pS for wt-Kir1.1b in the same Rb solutions. In excised, inside-out patches (polyamine-free, 0 Mg solutions), R128Y-Kir1.1b had a non-rectifying Rb conductance of 10 ± 0.6 pS, but no visible K current, consistent with its low K selectivity. The kinetics of Rb permeation through R128Y were similar to the kinetics of K permeation through wt-Kir1.1b, but with a longer open time (245 ms vs. 19ms for wt); and two closed states (2.8ms, 25ms) yielding an average open probability (Po) of 0.7 at −100mV, compared to a Po > 0.9 for wt-Kir1.1, which has a single closed state of 1.3ms. The observed 11 fold increase in Rb/K selectivity with no change in K/Na selectivity or rectification is consistent with R128Y-Kir1.1b causing a subtle change in the selectivity filter, perhaps by disruption of an intra-subunit salt bridge (R128-E118) near the filter.