Localization and interaction of epitope-tagged GIRK1 and CIR inward rectifier K+ channel subunits.
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.
Kennedy
M E
ME
Mayo Foundation, Rochester, MN 55901, USA.
Nemec
J
J
Clapham
D E
DE
D000818:Animals; D019556:COS Cells; D000939:Epitopes / Q000032:analysis*; D005455:Fluorescent Antibody Technique; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; D009865:Oocytes / Q000378:metabolism; D015221:Potassium Channels / Q000032:analysis / Q000276:immunology*; D024661:Potassium Channels, Inwardly Rectifying; D011233:Precipitin Tests; D014162:Transfection; D014982:Xenopus laevis
D000939:Epitopes; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; C484274:KCNJ5 protein, human; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying