An Inter-intra Subunit Salt Bridge near the Selectivity Filter Stabilizes the Conducting State of Kir1.1

Abstract

ROMK (Kir1.1) potassium channels are normally closed by internal acidification with a pKa of 6.6. If this acidification occurs in the presence of low (1mM) external K, the channels also inactivate, such that channel activity is not recovered by realkalization until high K is returned to the external solution. Mutations in an inter-intra subunit salt bridge (E118-R128-E132-Kir1.1b) in the P-loop of the channel near the selectivity filter increased the K sensitivity of inactivation, such that the salt-bridge mutants E132Q-Kir1.1b and R128Y-Kir1.1b inactivated in 100mM K solutions after a transient acidification. However 300mM external K (but not 200mM Na +100mM K) protected E132Q and R128 from inactivation during this acidification, suggesting an altered K sensitivity in these mutants. External application of a modified honey bee toxin (TPNQ) protected wild-type ROMK from inactivation in 1mM K and protected E132Q or R128Y from inactivation in 100mM K, suggesting that TPNQ binding to the outer mouth of the channel stabilizes the conducting state. Nonetheless, TPNQ was unable to protect either E132Q or R128Y from inactivation in 1mM external K. However, both E132Q and R128Y were protected from inactivation in 1mM K either by a mutation that disrupted transmembrane helix H-bonding (K61M-Kir1.1b) or by a mutation that stabilized a selectivity-filter to helix-pore linkage (V121T-Kir1.1b). Our results are consistent with an inter-intra subunit salt bridge near the outer end of the selectivity filter that stabilizes the conductive state of the channel.