Stable expression and functional characterization of a human cardiac Na+ channel gene in mammalian cells.
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 hH1 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 hH1 DNA by PCR, (2) specific hH1 mRNA 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 hH1 mRNA and protein was produced in these cells. 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 microM; 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Krafte
D S
DS
Department of Vascular and Biochemical Pharmacology, Sterling Winthrop Inc., Collegeville, PA 19426-0900, USA.
Volberg
W A
WA
Rapp
L
L
Kallen
R G
RG
Lalik
P H
PH
Ciccarelli
R B
RB
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D016466:CHO Cells; D006224:Cricetinae; D004594:Electrophysiology; D018014:Gene Transfer Techniques; D006801:Humans; D008322:Mammals; D008969:Molecular Sequence Data; D009206:Myocardium / Q000378:metabolism*; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology; D012333:RNA, Messenger / Q000032:analysis; D015222:Sodium Channels / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D000906:Antibodies; D010455:Peptides; D012333:RNA, Messenger; D015222:Sodium Channels