Functional comparison of the reverse mode of Na＋/ Ca2＋ exchangers NCXI.1 and NCX1.5 expressed in CHO cells

To investigate the reverse mode function of Na＊/Ca2~ exchangers NCXI.1 and NCX1.5 expressed in CHO cells as well as their modulations by PKC and PKA. Methods： CHO-K1 cells were transfected with pcDNA3.1 （＋） plasmid carrying cDNA of rat cardiac NCXI.1 and brain NCXl.5. The expression of NCXI.1 and NC...

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Published in:中国药理学报：英文版 2013 (5), p.691-698
Main Author: Yan LONG Wei-ping WANG HuiYUAN Shi-ping MA Nan FENG Ling WANG Xiao-liang WANG
Format: Article
Language:English
Subjects:
CHO细胞
PKA
免疫印迹分析
功能比较
反向
热交换器
钙水平
钠
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Summary:To investigate the reverse mode function of Na＊/Ca2~ exchangers NCXI.1 and NCX1.5 expressed in CHO cells as well as their modulations by PKC and PKA. Methods： CHO-K1 cells were transfected with pcDNA3.1 （＋） plasmid carrying cDNA of rat cardiac NCXI.1 and brain NCXl.5. The expression of NCXI.1 and NCXI.5 was examined using Western blot analysis. The intracellular Ca2~ level （[Ca2＋]i） was measured using Ca2＋ imaging. Whole-cell NCX currents were recorded using patch-clamp technique. Reverse mode NCX activity was elicited by perfu- sion with Na＋-free medium. Ca2＋ paradox was induced by Ca2＋-free EBSS medium, followed by Ca2＋-containing solution （1.8 or 3.8 mmol/L CaCl2）. Results： The protein levels of NCXI.1 and NCXI.5 expressed in CliO cells had no significant difference. The reverse modes of NCXl.1 and NCXl.5 in CliO cells exhibited a transient increase of [Ca2＋]＋, which was followed by a Ca2＋ level plateau at higher external Ca2＋ concentrations. In contrast, the wild type CliO cells showed a steady increase of [Ca2i]＋ at higher external Ca2＋ concentrations. The PKC activator PMA （0.3-10 pmol/L） and PKA activator 8-Br-cAMP （10-100 pmol/L） significantly enhanced the reverse mode activity of NCXI.1 and NCXl.5 in CHO cells. NCXI.1 was 2.4-fold more sensitive to PKC activation than NCXI.5, whereas the sensitivity of the two NCX isoforms to PKA activation had no difference. Both PKC- and PKA-enhanced NCX reverse mode activities in CliO cells were suppressed by NCX inhibitor KB-R7943 （30 pmol/L）. Conclusion： Both NCXI.1 and NCX1.5 are functional in regulating and maintaining stable [Ca2＋]i in CliO cells and differentially regu- lated by PKA and PKC. The two NCX isoforms might be useful drug targets for heart and brain protection.
ISSN:1671-4083
1745-7254