Activation of Inwardly Rectifying Potassium Channels by Immobilized G-Protein Coupled Receptors

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Inwardly rectifying potassium (GIRK) channels open after binding G-protein βγ subunits, which are made available when G-protein coupled receptors (GPCRs) catalyze nucleotide exchange on the G-protein α subunits of Gαβγ heterotrimers. The standard model of GPCR to G-protein to GIRK coupling involves the random collision of signaling components that diffuse laterally in the plasma membrane. More recently it has been suggested that some (or all) components of this signaling pathway exist as preformed complexes. In order to investigate the mechanism of GPCR-GIRK coupling we have developed a technique to immobilize GPCRs in living cells, and to test activation of GIRK channels by mobile and immobile receptors. Cerebellar granule neurons or HEK293 cells were transiently transfected with a plasmid vector encoding the μ-opioid receptor fused at its (extracellular) n-terminus to superecliptic pHluorin, a pH-sensitive variant of the green fluorescent protein. The expressed protein (pH-MOR) was oriented correctly in the plasma membrane, as indicated by rapid acid quenching of cell surface fluorescence, and was functional, as indicated by GIRK channel activation in the presence of the μ-opioid agonist DAMGO. Fluorescence recovery after photobleaching (FRAP) experiments indicated that pH-MORs were mobile on the surface of living cells, similar to previous FRAP studies of GPCR mobility. The mobility of pH-MORs was dramatically reduced after cell surface proteins were biotinylated and crosslinked with soluble avidin. However, immobile pH-MORs activated GIRK channels with the same agonist sensitivity and kinetics as mobile pH-MORs. This result indicates that lateral diffusion of GPCRs is not required for activation of GIRK channels. Experiments designed to immobilize other components of this signaling pathway will constrain possible models of GPCR-GIRK coupling. Supported by NIH grants NS36455 and NS41055