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 subu nits that combine to form the heteromultimer I-KACh, the G(beta gamma) -activated atrial K channel responsible for the vagal slowing of heart rate. Epitope-tagged channel subunits were constructed by the introdu ction of distinct six amino acid epitopes into the C-termini or putati ve extracellular domains of GIRK1 and CIR. Carboxyl-terminal tagged su bunits were activated by purified G(beta gamma) subunits in inside-out patches when expressed in Cos cells. Interestingly, insertion of thre e 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 CLR in Xenopus oocytes. The epit ope-tagged CIR-AU1 subunit coimmunoprecipitated GIRK1-AU5 from metabol ically labeled Cos cells. Immunofluorescence labeling of Cos cells loc alized GIRK1-AU5 to internal cytoskeletal structures that co-stained w ith antibodies against the intermediate filament protein, vimentin. CI R-AU1 localized primarily to the plasma membrane. Double immunofluores cence labeling showed that GIRK1-AU5 plasma membrane staining was dete ctable only when coexpressed with CIR-AU1. Copyright (C) 1996 Elsevier Science Ltd