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Immunological and Physical Characterization of the Brain G Protein-Gated Muscarinic Potassium Channel

https://doi.org/10.1006/bbrc.1995.2901Get rights and content

Abstract

The subunit composition of the G protein-gated inwardly rectifying K+ (KG) channel protein extracted from mouse forebrain membranes was examined. A polyclonal antibody (anti-GIRK1C1) was prepared against the carboxyl terminal region of GIRK1, the major subunit of the KG channel. The anti-GIRK1C1 IgG detected a single protein at ∼ 65 kDa in SDS-PAGE of brain membranes. This IgG immunoprecipitated a macroprotein complex composed of GIRK1 and several associated proteins whose molecular weights ranging from 50 to 62 kDa on SDS-polyacrylamide gel. Upon size fractionation by both sucrose density gradient centrifugation and gel filtration, the solubilized KG channel proteins migrated into a single peak, which suggests that the component subunits form a single macromolecule. On the basis of the values of size fractionation, the molecular mass of the KG channel macromolecule could be estimated at ∼ 231,000. These results suggest that the KG channel is most likely a tetrameric macroprotein composed of GIRK1 and co-immunoprecipitated proteins in the forebrain.

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