Abstract
The cardiac G protein-gated K+ channel, IKACh, is activated by application of purified and recombinant beta and gamma subunits (G beta gamma) of heterotrimeric G proteins to excised inside-out patches from atrial membranes (Logothetis, D. E., Kurachi, Y., Galper, J., Neer, E., and Clapham, D. E. (1987) Nature 325, 321-326; Wickman, K., Iniguez-Lluhi, J., Davenport, P., Taussig, R. A., Krapivinsky, G. B., Linder, M. E., Gilman, A., and Clapham, D.E. (1994) Nature 368, 255-257). Cardiac IKACh is composed of two inward rectifier K+ channel subunits, GIRK1 and CIR (Krapivinsky, G., Gordon, E., Wickman, K., Velimirovic, B., Krapivinsky, L., and Clapham, D. E. (1995) Nature 374, 135-141). We show that G beta gamma directly binds to immunoprecipitated cardiac IKACh as well as to recombinant CIR and GIRK1 subunits, with dissociation constants (Kd) of 55, 50, and 125 nM, respectively. In each case, binding appeared specific as judged by competition of unlabeled G beta gamma with radiolabeled G beta gamma and inhibition of binding by antigenic peptide or G alpha-GDP, but not G alpha-GTP gamma S (guanosine 5'-3-O-(thio)triphosphate). In contrast, G alpha (GTP gamma S- or GDP-bound) did not bind to the native channel. We conclude that G beta gamma binds directly and specifically to IKACh via interactions with both CIR and GIRK1 subunits to gate the channel.
Highlights
Cardiac IKACh is a heteromultimer of two homologous inward rectifier Kϩ channel subunits, GIRK1 [8, 9] and CIR [3]
Despite the widespread electrophysiological evidence for membrane-delimited G protein activation of ion channels [20], no biochemical evidence has been presented for a direct interaction between G protein subunits and channel proteins for two major reasons
In comparison to other G protein effectors such as adenylyl cyclase, cGMP phosphodiesterase, and phospholipase C, channel proteins are of lower abundance in cells
Summary
Cardiac IKACh is a heteromultimer of two homologous inward rectifier Kϩ channel subunits, GIRK1 [8, 9] and CIR [3]. Recent evidence suggests that a similar complex comprised of GIRK1 and GIRK2 [10], a structural and functional CIR homolog, forms a neuronal G protein-gated Kϩ channel [11]. We tested whether G␥ binds directly to cardiac IKACh and, if so, to which subunit(s). By immunoprecipitation with subunit-specific antibodies, we were able to effectively purify whole cardiac IKACh channel and individual recombinant subunits and study binding of 125I-labeled G␥ to these immunoprecipitated species. We show that G␥ binds directly and to the whole channel and to each IKACh subunit
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