Probing the G-protein Regulation of GIRK1 and GIRK4, the Two Subunits of the KACh Channel, Using Functional Homomeric Mutants

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 Phe137, 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, Ser143, 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 betagamma subunits, wild-type or mutated G-protein alpha subunits, and active protomers of pertussis toxin. These experiments, which confirmed the crucial, though not exclusive, role of Gbetagamma 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.