Dominant-Negative Mutants Identify a Role for Girk Channels in D3 Dopamine Receptor-Mediated Regulation of Spontaneous Secretory Activity

The human D3 dopamine receptor can activate G-protein–coupled inward rectifier potassium channels (GIRKs), inhibit P/Q-type calcium channels, and inhibit spontaneous secretory activity in AtT-20 neuroendocrine cells (Kuzhikandathil, E.V., W. Yu, and G.S. Oxford. 1998. Mol. Cell. Neurosci. 12:390–402; Kuzhikandathil, E.V., and G.S. Oxford. 1999. J. Neurosci. 19:1698–1707). In this study, we evaluate the role of GIRKs in the D3 receptor-mediated inhibition of secretory activity in AtT-20 cells. The absence of selective blockers for GIRKs has precluded a direct test of the hypothesis that they play an important role in inhibiting secretory activity. However, the tetrameric structure of these channels provides a means of disrupting endogenous GIRK function using a dominant negative approach. To develop a dominant-negative GIRK mutant, the K⁺ selectivity amino acid sequence -GYG- in the putative pore domain of the human GIRK2 channels was mutated to -AAA-, -GLG-, or -GFG-. While the mutation of -GYG- to -GFG- did not affect channel function, both the -AAA- and -GLG- GIRK2 mutants were nonfunctional. This suggests that the aromatic ring of the tyrosine residue rather than its hydroxyl group is involved in maintaining the pore architecture of human GIRK2 channels. When expressed in AtT-20 cells, the nonfunctional AAA-GIRK2 and GLG-GIRK2 acted as effective dominant-negative mutants and significantly attenuated endogenous GIRK currents. Furthermore, these dominant-negative mutants interfered with the D3 receptor-mediated inhibition of secretion in AtT-20 cells, suggesting they are centrally involved in the signaling pathway of this secretory response. These results indicate that dominant-negative GIRK mutants are effective molecular tools to examine the role of GIRK channels in vivo.