The stoichiometry of G beta gamma binding to G-protein-regulated inwardly rectifying K+ channels (GIRKs)

G-protein-coupled inwardly rectifying K+ (GIRK; Kir3.x) channels are the pr imary effecters of numerous G-protein-coupled receptors, GIRK channels decr ease cellular excitability by hyperpolarizing the membrane potential in car diac cells, neurons, and secretory cells. Although direct regulation of GIR Ks by the heterotrimeric G-protein subunit G beta gamma has been extensivel y studied, little is known about the number of G beta gamma binding sites p er channel. Here we demonstrate that purified GIRK (Kir 3.x) tetramers can be chemically cross-linked to exogenously purified G beta gamma subunits. T he observed laddering pattern of G beta gamma attachment to GIRK4 homotetra mers was consistent with the binding of one, two, three, or four G beta gam ma molecules per channel tetramer. The fraction of channels chemically cros s-linked to four G beta gamma molecules increased with increasing G beta ga mma concentrations and approached saturation. These results suggest that GI RK tetrameric channels have four G beta gamma binding sites. Thus, GIRK (Ki r 3.x) channels, like the distantly related cyclic nucleotide-gated channel s, are tetramers and exhibit a 1:1 subunit/ligand binding stoichiometry.