A recombinant inwardly rectifying potassium channel coupled to GTP-binding proteins.
GTP-binding (G) proteins have been shown to mediate activation of inwardly rectifying potassium (K+) channels in cardiac, neuronal and neuroendocrine cells. Here, we report functional expression of a recombinant inwardly rectifying channel which we call KGP (or hpKir3.4), to signify that it is K+ selective, G-protein-gated and isolated from human pancreas. KGP expression in Xenopus oocytes resulted in sizeable basal (or agonist-independent) currents while coexpression with a G-protein-linked receptor, yielded additional agonist-induced currents. Coexpression of KGP and hGIRK1 (a human brain homolog of GIRK1/Kir3.1) produced much larger basal currents than those observed with KGP or hGIRK1 alone, and upon coexpression with receptor, similarly large agonist-induced currents could be obtained. Pertussis toxin treatment significantly diminished agonist-dependent currents due to either KGP or KGP/hGIRK1 expression. Interestingly, PTX also significantly reduced basal KGP or KGP/hGIRK1 currents, suggesting that basal activity is largely the result of G-protein gating as well. When the two channels were coexpressed with receptor, the relative increase in current elicited by agonist was similar whether KGP and hGIRK1 were expressed alone or together. When in vitro translated or when expressed in Xenopus oocytes or CHO mammalian cells, KGP gave rise to a nonglycosylated 45-kD protein. Antibodies directed against either KGP or hGIRK1 coprecipitated both proteins coexpressed in oocytes, providing evidence for the heteromeric assembly of the two channels and suggesting that the current potentiation seen with coexpression of the two channel subunits is due to specific interactions between them. An endogenous oocyte protein similar in size to KGP was also coprecipitated with hGIRK1.
Chan
K W
KW
Department of Physiology and Biophysics, City University of New York, New York 10029, USA.
Langan
M N
MN
Sui
J L
JL
Kozak
J A
JA
Pabon
A
A
Ladias
J A
JA
Logothetis
D E
DE
D000109:Acetylcholine / Q000502:physiology; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D016466:CHO Cells; D006224:Cricetinae; D015342:DNA Probes; D004594:Electrophysiology; D019204:GTP-Binding Proteins / Q000096:biosynthesis / Q000378:metabolism*; D006168:Guinea Pigs; D006801:Humans; D015151:Immunoblotting; D015640:Ion Channel Gating / Q000502:physiology; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism; D037342:Pertussis Toxin; D016133:Polymerase Chain Reaction; D015221:Potassium Channels / Q000096:biosynthesis / Q000378:metabolism*; D011233:Precipitin Tests; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D014158:Transcription, Genetic; D010566:Virulence Factors, Bordetella / Q000494:pharmacology; D014982:Xenopus laevis
D015342:DNA Probes; D015221:Potassium Channels; D011994:Recombinant Proteins; D010566:Virulence Factors, Bordetella; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D000109:Acetylcholine