The Intersubunit Interaction Interface in the Kir 1.1 (ROMK) Channel Controls pH‐Dependent Gating

Abstract

Our previous studies on Bartter’s disease mutations in Kir 1.1 suggested a cytoplasmic C-terminal tail domain, involving residues 331–361, folds onto the cytoplasmic N-terminus of an adjacent subunit to maintain the local structure around the pH-trigger, K80, in an open state. Here, we critically test this hypothesis. A series of strategic mutations in the cytoplasmic N-and C-terminal domains were created and pH-dependent channel gating was examined. Alanine scanning mutagensis in the relevant C-terminal region pinpointed the pH-gating determinant to short track of amino-acids (335–343). This region precisely encompasses the beta-M stand, a structure predicted to form the chief C-terminal intersubunit interaction site. Point mutations in the beta-M strand (V339A, D340A, F341A) uniquely altered pH-dependent channel gating and these effects were specifically rescued with a suppressor mutation in the pH trigger (K80M). Channels bearing N-terminal mutations in the parallel beta-A strand exhibited identical gating phenotypes as channels bearing the C-terminal mutations. In summary, the beta-A/ beta-M intersubunit interaction interface in Kir 1.1 controls pH-dependent gating. Such an interaction may provide a mechanism to translate physiological relevant chemical modification of cytoplasmic COOH, such as phosphorylation or PIP2-binding, to the pH-dependent gating process.