Receptor-mediated inhibition of G protein-coupled inwardly rectifying potassium channels involves Gαq family subunits, phospholipase C, and a readily diffusible messenger

G protein-coupled inwardly rectifying K+ (GIRK) channels can be activated or inhibited by distinct classes of receptor (G alpha (i/o)- and G alpha (q)-coupled), providing dynamic regulation of cellular excitability. Receptor-mediated activation involves direct effects of G betay subunits on GIRK channels, but mechanisms involved in GIRK channel inhibition have not been fully elucidated, An HEK293 cell line that stably expresses GIRK1/4 channels was used to test G protein mechanisms that mediate GIRK channel inhibition. In cells transiently or stably cotransfected with 5-HT1A (G alpha (i/o)-coupled) and TRH-R1 (G alpha (q)-coupled) receptors, 5-HT (5-hydroxytryptamine; serotonin) enhanced GIRK channel currents, whereas thyrotropin-releasing hormone (TRH) inhibited both basal and B-HT-activated GIRK channel currents. Inhibition of GIRK channel currents by TRH primarily involved signaling by G alpha (q) family subunits, rather than G beta gamma dimers: GIRK channel current inhibition was diminished by Pasteurella multocida toxin, mimicked by constitutively active members of the G alpha (q) family, and reduced by minigene constructs that disrupt G alpha (q) signaling, but was completely preserved in cells expressing constructs that interfere with signaling by G beta gamma subunits. Inhibition of GIRK channel currents by TRH and constitutively active G alpha (q) was reduced by U73122, an inhibitor of phospholipase C (PLC), Moreover, TRH-R1-mediated GIRK channel inhibition was diminished by minigene constructs that reduce membrane levels of the PLC substrate phosphatidylinositol bisphosphate, further implicating PLC. However, we found no evidence for involvement of protein kinase C, inositol trisphosphate, or intracellular calcium. Although these downstream signaling intermediaries did not contribute to receptor-mediated GIRK channel inhibition, bath application of TRH decreased GIRK channel activity in cell-attached patches. Together, these data indicate that receptor-mediated inhibition of GIRK channels involves PLC activation by G alpha subunits of the G alpha (q) family and suggest that inhibition may be communicated at a distance to GIRK channels via unbinding and diffusion of phosphatidylinositol bisphosphate away from the channel.