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 mammalian cells. Immunoblot analysis of transfected human embryonic kidney 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 abundant 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 cells expressing GIRK1-cp protein, appropriate K+ currents could not be detected. In contrast, whole cell recordings made directly from transfected beta TC3 cells expressing GIRK1-cp revealed inwardly rectifying, pertussis toxin-sensitive currents activated by norepinephrine and galanin. Single channel recordings in excised patches of beta TC3 cells expressing GIRK1-cp showed rectifying K+ currents when activated by 50 microM 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 demonstrates the utility of this system for further biochemical and biophysical analyses of G protein-K+ channel interactions.
Philipson
L H
LH
Department of Medicine, University of Chicago, Illinois 60637, USA.
Kuznetsov
A
A
Toth
P T
PT
Murphy
J F
JF
Szabo
G
G
Ma
G H
GH
Miller
R J
RJ
D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D017931:DNA Primers; D000939:Epitopes / Q000737:chemistry*; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; D006801:Humans; D008969:Molecular Sequence Data; D015221:Potassium Channels / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D024661:Potassium Channels, Inwardly Rectifying; D051381:Rats; D015398:Signal Transduction; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D000939:Epitopes; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying