Functional expression of an epitope-tagged G protein-coupled K+ channel (GIRK1)

Louis H. Philipson*, Andrey Kuznetsov, Peter T. Toth, Joseph F. Murphy, Gabor Szabo, Gloria H. Ma, Richard J. Miller

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

23 Scopus citations

Abstract

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 (βTC3) 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 βTC3 cells expressing GIRK1-cp revealed inwardly rectifying, pertussis toxin-sensitive currents activated by norepinephrine and galanin. Single channel recordings in excised patches of βTC3 cells expressing GIRK1-cp showed rectifying K+ currents when activated by 50 μM 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.

Original languageEnglish (US)
Pages (from-to)14604-14610
Number of pages7
JournalJournal of Biological Chemistry
Volume270
Issue number24
DOIs
StatePublished - Jun 16 1995

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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