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