Receptor-mediated activation of heterotrimeric guanine nucleotide-binding proteins (G proteins) results in the dissociation of alpha from beta gamma subunits, thereby allowing both to regulate effectors. Little is known about the regions of effectors required for recognition of G beta gamma. A peptide encoding residues 956 to 982 of adenylyl cyclase 2 specifically blocked G beta gamma stimulation of adenylyl cyclase 2, phospholipase C-beta 3, potassium channels, and beta-adrenergic receptor kinase as well as inhibition of calmodulin-stimulated adenylyl cyclases, but had no effect on interactions between G beta gamma and G alpha o. Substitutions in this peptide identified a functionally important motif, Gln-X-X-Glu-Arg, that is also conserved in regions of potassium channels and beta-adrenergic receptor kinases that participate in G beta gamma interactions. Thus, the region defined by residues 956 to 982 of adenylyl cyclase 2 may contain determinants important for receiving signals from G beta gamma.

In heart, G-protein-activated channels are complexes of two homologous proteins, GIRK1 and GIRK4. Expression of either protein alone results in barely active or non-active channels, making it difficult to assess the individual contribution of each subunit to the channel complex. The residue Phe 137 , located within the H5 region of GIRK1, is critical to the synergy between GIRK1 and GIRK4 (Chan, K. W., Sui, J. L., Vivaudou, M., and Logothetis, D. E. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 14193-14198). By modifying this residue or the matching residue of GIRK4, Ser 143 , we have been able to generate mutant proteins that produced large inwardly rectifying, G-protein-modulated currents when expressed alone in Xenopus oocytes. The enhanced activity of the heterologous expression of each of two active mutants, GIRK1(F137S) and GIRK4(S143T), was not caused by association with an endogenous oocyte channel subunit, and these mutants did not display apparent differences in the ability to localize to the cell surface compared with their wild-type counterparts. When these functional mutant channels were compared individually with wild-type heteromeric channels, they responded with only small differences to a number of maneuvers involving coexpression with muscarinic receptors, G-protein ␤␥ subunits, wild-type or mutated G-protein ␣ subunits, and active protomers of pertussis toxin. These experiments, which confirmed the crucial, though not exclusive, role of G␤␥ in regulating channel activity, demonstrated that GIRK1(F137S) and GIRK4(S143T), and by extrapolation their wild-type counterparts, interact in a qualitatively similar way with G-protein subunits. These findings suggest that functionally important sites of interaction with G-proteins are likely to be located within the homologous regions of GIRK1 and GIRK4 rather than within the divergent terminal regions. They also raise the question of the functional advantage of a heteromeric over homomeric design for G-protein-gated channels.Inwardly rectifying potassium channels gated directly by GTP-binding proteins (G-proteins) 1 exist in many excitable cells, where they modulate membrane excitability in response to the stimulation of G-protein-coupled receptors. The best studied member of this family of channels is the cardiac K ACh channel, which is responsible for the negative chronotropic effect of acetylcholine (ACh) released by the vagus nerve. The binding of ACh to muscarinic type 2 (m2) receptors coupled to pertussis toxin (PTX)-sensitive G-proteins triggers the separation of the G␣ and G␤␥ subunits and activation of the K ACh channel by G␤␥ (1).K ACh channels appear to be a complex of two homologous subunits, GIRK1 (2-4) and GIRK4 (4 -6). Both of these subunits belong to the family of inward rectifier K ϩ channel proteins (7) characterized by cytoplasmic COOH and NH 2 termini, flanking two putative transmembrane helices linked by a hydrophilic loop that is thought to line the channel lumen. Other members of the GIRK family include the brain GIRK2 and GIRK3 (6...

G protein-gated inwardly rectifying K ؉ (GIRK) channels, which are important regulators of membrane excitability both in heart and brain, appear to function as heteromultimers. GIRK1 is unique in the GIRK channel family in that although it is by itself inactive, it can associate with the other family members (GIRK2-GIRK5) to enhance their activity and alter their single-channel characteristics. By generating a series of chimeras, we identified a phenylalanine residue, F137, in the pore region of GIRK1 that critically controls channel activity. F137 is found only in GIRK1, while the remaining GIRK channels possess a conserved serine residue in the analogous position. The single-point mutant GIRK4(S143F) behaved as a GIRK1 analog, forming multimers with GIRK2, GIRK4, or GIRK5 channels that exhibited prolonged single-channel open-time duration and enhanced activity compared with that of homomultimers. Expression of the corresponding GIRK1(F137S) mutant alone resulted in appreciable channel activity with novel characteristics that was further enhanced upon coexpression with other GIRK subunits. Thus, although the F137 residue renders the GIRK1 subunit inactive, when combined with other GIRK heteromeric partners it alters their gating and contributes to their enhanced activity.