Molecular mechanism for sodium‐dependent activation of G protein‐gated K+ channels

Abstract

G protein-gated inwardly rectifying K+ (GIRK) channels are activated independently by Gβγ and internal Na+ via mechanisms requiring phosphatidylinositol phosphates. An aspartate (Asp) at position 226 in GIRK2 is crucial for Na+-dependent activation of GIRK1–GIRK2 heteromeric channels. We expressed wild-type and mutant GIRK1–GIRK2 channels in Xenopus oocytes and tested the effects of Na+ and neutralizing Asp226 on the functional interactions of the channels with phosphatidylinositol 4,5-bisphosphate (PIP2). The rate of inhibition of GIRK1–GIRK2 currents by application of anti-PIP2 antibody to inside-out membrane patches was slowed > 2-fold by the D226N mutation in GIRK2 and by increasing internal [Na+]. The reverse mutation in GIRK1 (N217D) increased the rate of inhibition. The dose–response relationship for activation by purified PIP2 was shifted to lower concentrations in the presence of 20 mM Na+. Three synthetic isoforms of PIP2, PI(4,5)P2, PI(3,4)P2 and PI(3,5)P2, activated GIRK channels with similar potencies. We conclude that Na+ directly interacts with Asp226 of GIRK2 to reduce the negative electrostatic potential and promote the functional interaction of the channels with PIP2.