FUNCTIONAL EXPRESSION OF AN EPITOPE-TAGGED G-PROTEIN-COUPLED K+ CHANNEL (GIRK1)

An epitope-tagged form of an inwardly rectifying and G protein-coupled K+ channel (GIRK1-cp) was expressed at high levels in transfected mam malian cells. Immunoblot analysis of transfected human embryonic kidne y cells (HEK293 and mouse insulinoma cells (beta TC3) revealed several GIRK1-cp polypeptides, including the major 59-kDa band, corresponding to the predicted mass of the GIRK1 polypeptide plus the epitope tag. Immunohistochemical staining using two anti-tag antibodies showed abun dant immunoreactive material, which was predominantly concentrated in the perinuclear area in both transfected cell types. While functional GIRK1-cp message was present in poly(A)+ RNA prepared from HEK293 cell s expressing GIRK1-cp protein, appropriate K+ currents could not be de tected. In contrast, whole cell recordings made directly from transfec ted beta TC3 cells expressing GIRK1-cp revealed inwardly rectifying, p ertussis toxin-sensitive currents activated by norepinephrine and gala nin. Single channel recordings in excised patches of beta TC3 cells ex pressing GIRK1-cp showed rectifying K+ currents when activated by 50 m u M guanosine 5'-O-(thiotriphosphate), with a slope conductance of 39. 1 +/- 1.0 picosiemens. This is the first report of stable heterologous expression of a functional G protein-coupled K+ channel in mammalian cells. The activity of an epitope-tagged channel in insulinoma cells d emonstrates the utility of this system for further biochemical and bio physical analyses of G protein-K+ channel interactions.