STABLE EXPRESSION AND FUNCTIONAL-CHARACTERIZATION OF A HUMAN CARDIAC NA-CELLS( CHANNEL GENE IN MAMMALIAN)

In order to develop mammalian cell lines expressing a functional human heart Na+ channel gene (hH1), Chinese hamster ovary (CHO-K1) cells an d HeLa cells were transfected with the hH1 gene and the bacterial neom ycin (neo) resistance gene, In CHO-K1 cells, direct screening for hH1- positive, G418-resistant colonies by functional patch clamp analysis w as 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 hH1 DNA by PCR, (2) specific hH1 mRNA expression by RT-PCR, (3) hH1 p rotein production by immunoprecipitation with hH1-specific antisera, a nd (4) hH1 Na+ channel function by patch-clamp analysis. Using this st rategy we obtained two CHO-K1 cell lines which express functional huma n heart Na+ channels. However, using the same strategy, we were unsucc essful in obtaining functional, hH1-positive HeLa cell lines, even tho ugh hH1 mRNA and protein was produced in these cells. The two CHO-K1 c ell lines stably express human cardiac Na+ channels which retain norma l electrophysiological characteristics with respect to activation and inactivation. In addition, the Na+ channels expressed in these cells a re blocked by tetrodotoxin with an IC50 value of 2.5 mu M; consistent with known cardiac Na+ channel pharmacology. The density of channels i s high enough to permit recording of pseudomacroscopic currents in exc ised outside-out patches of membrane. Stable expression of the human h eart Na+ channel gene in non-cardiac mammalian cells further indicates many of the distinguishing properties of these channels are encoded b y this gene, In addition, the CHO-K1 cell system should prove useful i n the further molecular, biochemical and biophysical characterization of human cardiac Na+ channels.