Gβγ binding to GIRK4 subunit is critical for G protein-gated K+ channel activation

The cardiac G protein-gated K+ channel, I-KACh, is directly activated by G protein beta gamma subunits (G(beta gamma)). I-KACh is composed of two inward rectifier K+ channel subunits, GIRK1 and GIRK4. G(beta gamma) binds to both GIRK1 and GIRK4 subunits of the heteromultimeric I-KACh. Here we delineate the G(beta gamma) binding regions of I-KACh by studying direct G(beta gamma) interaction with native purified I-KACh, competition of this interaction with peptides derived from GIRK1 or GIRK4 amino acid sequences, mutational analysis of regions implicated in G(beta gamma) binding, and functional expression of mutated subunits in mammalian cells. Only two GIRK4 peptides, containing amino acids 209-225 or 226-245, effectively competed for G beta gamma binding. A single point mutation introduced into GIRK4 at position 216 (C216T) dramatically reduced the potency of the peptide in inhibiting G(beta gamma) binding and G(beta gamma) activation of expressed GIRK1/GIRK4(C216T) channels. Conversion of 5 amino acids in GIRK4 (226-245) to the corresponding amino acids found in the G protein-insensitive IRK1 channel, completely abolished peptide inhibition of G(beta gamma) binding to I-KACh and G(beta gamma) activation of GIRK1/mutant GIRK4 channels. We conclude from this data that G(beta gamma) binding to GIRK4 is critical for I-KACh activation.