Stable expression and functional characterization of a human cardiac Na + channel gene in mammalian cells

Abstract

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 IC 50 value of 2.5 μ m; 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.