Kennedy M E, Nemec J, Clapham D E
Mayo Foundation, Rochester, MN 55901, USA.
Neuropharmacology. 1996;35(7):831-9. doi: 10.1016/0028-3908(96)00132-3.
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.
GIRK1和CIR是G蛋白激活的内向整流钾通道亚基，它们结合形成异源多聚体IKACh，即Gβγ激活的心房钾通道，负责迷走神经介导的心率减慢。通过将不同的六个氨基酸表位引入GIRK1和CIR的C末端或假定的细胞外结构域，构建了表位标记的通道亚基。当在Cos细胞中表达时，羧基末端标记的亚基在反转片膜片中被纯化的Gβγ亚基激活。有趣的是，在GIRK1假定的细胞外结构域中插入三个氨基酸会产生一个无活性的亚基，当它与野生型GIRK1和CIR在非洲爪蟾卵母细胞中共表达时，会作为显性负性亚基起作用。表位标记的CIR-AU1亚基从代谢标记的Cos细胞中共免疫沉淀GIRK1-AU5。Cos细胞的免疫荧光标记将GIRK1-AU5定位到内部细胞骨架结构，这些结构与针对中间丝蛋白波形蛋白的抗体共染色。CIR-AU1主要定位于质膜。双重免疫荧光标记显示，只有当与CIR-AU1共表达时，才能检测到GIRK1-AU5的质膜染色。
