Stable Expression and Functional Characterization of a Human Cardiac Na+ Channel Gene in Mammalian Cells
Journal of molecular and cellular cardiology/Journal of Molecular and Cellular Cardiology(1995)
摘要
D. S. Krafte, W. A. Volberg, L. Rapp, R. G. Kallen, P. H. Lalik and R. B. Ciccarelli. Stable Expression and Functional Characterization of a Human Cardiac Na+ Channel Gene in Mammalian Cells. Journal of Molecular and Cellular Cardiology (1995) 27, 823–830. In order to develop mammalian cell lines expressing a functional human heart Na+ channel gene (hH1), Chinese hamster ovary (CHO-K1) cells and HeLa cells were transfected with the hill gene and the bacterial neomycin (neo) resistance gene. In CHO-K1 cells, direct screening for hH1-positive, G418-resistant colonies by functional patch clamp analysis was complicated due to low-level endogenous expression of a brain-type Na+ channel. Therefore, we developed a stepwise strategy for isolation of cell lines expressing functional hH1 Na+ channels: G418-resistant colonies were sequentially analysed for (1) chromosomal integration of hill DNA by PCR, (2) specific hill rnRNA expression by RT-PCR, (3) hH1 protein production by immunoprecipitation with hH1-specific antisera, and (4) hH1 Na+ channel function by patch-clamp analysis. Using this strategy we obtained two CHO-K1 cell lines which express functional human heart Na+ channels. However, using the same strategy, we were unsuccessful in obtaining functional, hH1-positive HeLa cell lines, even though hill mRNA and protein was produced in these ceils. The two CHO-K1 cell lines stably express human cardiac Na+ channels which retain normal electrophysiological characteristics with respect to activation and inactivation. In addition, the Na+ channels expressed in these cells are blocked by tetrodotoxin with an IC50 value of 2.5 μm; consistent with known cardiac Na+ channel pharmacology. The density of channels is high enough to permit recording of pseudomacroscopic currents in excised outside-out patches of membrane. Stable expression of the human heart Na+ channel gene in non-cardiac mammalian cells further indicates many of the distinguishing properties of these channels are encoded by this gene. In addition, the CHO-K1 cell system should prove useful in the further molecular, biochemical and biophysical characterization of human cardiac Na+ channels.
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关键词
HUMAN CARDIAC SODIUM CHANNEL,NA+ CHANNEL,HH1,STABLE EXPRESSION,CHINESE HAMSTER OVARY CELL,HELA CELL
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