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Localization and interaction of epitope-tagged GIRK1 and CIR inward rectifier K+ channel subunits.

M E Kennedy, J Nemec, D E Clapham

Neuropharmacology, 1996 , 35, 831-9

GIRK1 and CIR are G-protein activated inward rectifier K+ channel subunits that combine to form the heteromultimer IKACh, the G beta gamma-activated atrial K channel responsible for the vagal slowing of heart rate. Epitope-tagged channel subunits were constructed by the introduction of distinct six amino acid epitopes into the C-termini or putative extracellular domains of GIRK1 and CIR. Carboxyl-terminal tagged subunits were activated by purified G beta gamma subunits in inside-out patches when expressed in Cos cells. Interestingly, insertion of three amino acids into the putative extracellular domain of GIRK1 resulted in an inactive subunit that acted as a dominant negative subunit when coexpressed with wild type GIRK1 and CIR in Xenopus oocytes. The epitope-tagged CIR-AU1 subunit coimmunoprecipitated GIRK1-AU5 from metabolically labeled Cos cells. Immunofluorescence labeling of Cos cells localized GIRK1-AU5 to internal cytoskeletal structures that co-stained with antibodies against the intermediate filament protein, vimentin. CIR-AU1 localized primarily to the plasma membrane. Double immunofluorescence labeling showed that GIRK1-AU5 plasma membrane staining was detectable only when coexpressed with CIR-AU1.