Me. Kennedy et al., LOCALIZATION AND INTERACTION OF EPITOPE-TAGGED GIRK1 AND CIR INWARD RECTIFIER K+ CHANNEL SUBUNITS, Neuropharmacology, 35(7), 1996, pp. 831-839
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