Localization of a G-protein-coupled inwardly rectifying K+ channel, CIR, in the rat brain
Section snippets
Western blotting
Tissues were collected from freshly obtained rat brain, dissected, and homogenized in ice-cold phosphate-buffered saline (PBS) containing 2 mM phenylmethylsulfonyl fluoride, 20 μg/ml aprotinin, and 20 μg/ml leupeptin. Homogenates were centrifuged and membrane fractions were prepared. Aliquots of cell membranes (40 μg of protein) were subjected to electrophoresis on MULTIGEL 10/20 (Daiichi Pure Chemical Co., Ltd.) and transferred to GVHP filters (Millipore Ltd.) by electroblotting (200 mA, 2 h).
Immunoblotting analysis of CIR in rat brain
A specific polyclonal rabbit antibody was raised to the CIR protein by immunization with a synthetic peptide corresponding to the N-terminal region.[10]In a previous report, we demonstrated that the antibody could specifically recognize a single peptide in Western blot analysis of rat heart and in immunoprecipitation of recombinant CIR expressed in Xenopus oocytes.[10]The estimated size was comparable to the predicted molecular mass of ∼48,000 mol. wt. These results indicated that the antibody
Differential subcellular localization of CIR mRNA and its protein product
Parallel analyses of adjacent brain sections by immunocytochemistry and in situ hybridization revealed that CIR is present in at least a subset of nerve fibers and/or terminals within the rat brain. This is effectively demonstrated in the cerebellar cortex and the hippocampal formation. In the cerebellar cortex, CIR mRNA is expressed in the cell soma of both Purkinje and basket cells, and its product is present in the axons. Whereas, in the dentate gyrus of the hippocampal formation, the
Conclusions
We have demonstrated the cellular localization of CIR in the rat brain by CIR immunocytochemistry, in combination with in situ hybridization. CIR-containing neurons were found in the cerebellar cortex, hippocampal formation, olfactory system, cerebral cortex, basal ganglia and lower brain stem. The expression of CIR was also investigated in the choroid plexus in the lateral, third and fourth ventricles. CIR protein is present in a subset of nerve fibers and in other cases in axon terminals.
Acknowledgements
We thank Professors M. Tohyama and S. Shiosaka for their advice and stimulating discussion throughout this study. We thank Dr E. L. Barsoumian for his support, advice and encouragement throughout this project and for critical reading of the manuscript. We also thank Dr A. K. Kukula for critical reading and correction of the manuscript.
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