Krafte D S, Volberg W A, Rapp L, Kallen R G, Lalik P H, Ciccarelli R B
Department of Vascular and Biochemical Pharmacology, Sterling Winthrop Inc., Collegeville, PA 19426-0900, USA.
J Mol Cell Cardiol. 1995 Feb;27(2):823-30. doi: 10.1016/0022-2828(95)90089-6.
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)
为了构建表达功能性人心脏钠通道基因（hH1）的哺乳动物细胞系，将hH1基因和细菌新霉素（neo）抗性基因转染至中国仓鼠卵巢（CHO-K1）细胞和人宫颈癌（HeLa）细胞中。在CHO-K1细胞中，由于脑型钠通道的内源性低水平表达，通过功能性膜片钳分析直接筛选hH1阳性、G418抗性克隆变得复杂。因此，我们开发了一种逐步策略来分离表达功能性hH1钠通道的细胞系：依次对G418抗性克隆进行如下分析：（1）通过聚合酶链反应（PCR）检测hH1 DNA的染色体整合情况；（2）通过逆转录-聚合酶链反应（RT-PCR）检测特异性hH1信使核糖核酸（mRNA）的表达；（3）用hH1特异性抗血清进行免疫沉淀检测hH1蛋白的产生；（4）通过膜片钳分析检测hH1钠通道功能。使用该策略，我们获得了两个表达功能性人心脏钠通道的CHO-K1细胞系。然而，使用相同策略，我们未能成功获得功能性的、hH1阳性的HeLa细胞系，尽管这些细胞中产生了hH1 mRNA和蛋白。这两个CHO-K1细胞系稳定表达人心脏钠通道，其在激活和失活方面保持正常的电生理特性。此外，这些细胞中表达的钠通道被河豚毒素阻断，半数抑制浓度（IC50）值为2.5微摩尔；这与已知的心脏钠通道药理学一致。通道密度足够高，能够在切除的外侧向外膜片中记录到假宏观电流。（摘要截短至250字）
