Functional comparison of the reverse mode of Na+/Ca2+ exchangers NCX1.1 and NCX1.5 expressed in CHO cells
Abstract
Yan LONG, Wei-ping WANG, Hui YUAN, Shi-ping MA, Nan FENG, Ling WANG, Xiao-liang WANG*
State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China
Aim: To investigate the reverse mode function of Na+/Ca2+ exchangers NCX1.1 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 NCX1.1 and brain NCX1.5. The expression of NCX1.1 and NCX1.5 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 perfusion 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 NCX1.1 and NCX1.5 expressed in CHO cells had no significant difference. The reverse modes of NCX1.1 and NCX1.5 in CHO cells exhibited a transient increase of [Ca2+]i, which was followed by a Ca2+ level plateau at higher external Ca2+ concentrations. In contrast, the wild type CHO cells showed a steady increase of [Ca2+]i at higher external Ca2+ concentrations. The PKC activator PMA (0.3–10 μmol/L) and PKA activator 8-Br-cAMP (10–100 μmol/L) significantly enhanced the reverse mode activity of NCX1.1 and NCX1.5 in CHO cells. NCX1.1 was 2.4-fold more sensitive to PKC activation than NCX1.5, whereas the sensitivity of the two NCX isoforms to PKA activation had no difference. Both PKC- and PKA-enhanced NCX reverse mode activities in CHO cells were suppressed by NCX inhibitor KB-R7943 (30 μmol/L).
Conclusion: Both NCX1.1 and NCX1.5 are functional in regulating and maintaining stable [Ca2+]i in CHO cells and differentially regulated by PKA and PKC. The two NCX isoforms might be useful drug targets for heart and brain protection.
Keywords: Na+/Ca2+ exchanger; NCX1.1; NCX1.5; intracellular Ca2+ level; PKC; PKA; PMA; 8-Br-cAMP; KB-R7943
We thank Dr Jonathan LYTTON for generously providing NCX1.1 and NCX1.5 plasmids and Dr Nai-hong CHEN for technical assistance on calcium imaging. This work was supported by the National 973 Fundamental Project of China (2011CB504103) and the National Major Special Project on New Drug Innovation of China (No 2012ZX09301002-004).
* To whom correspondence should be addressed.
E-mail wangxl@imm.ac.cn
Received 2012-11-18 Accepted 2013-01-11
Keywords:
State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China
Aim: To investigate the reverse mode function of Na+/Ca2+ exchangers NCX1.1 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 NCX1.1 and brain NCX1.5. The expression of NCX1.1 and NCX1.5 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 perfusion 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 NCX1.1 and NCX1.5 expressed in CHO cells had no significant difference. The reverse modes of NCX1.1 and NCX1.5 in CHO cells exhibited a transient increase of [Ca2+]i, which was followed by a Ca2+ level plateau at higher external Ca2+ concentrations. In contrast, the wild type CHO cells showed a steady increase of [Ca2+]i at higher external Ca2+ concentrations. The PKC activator PMA (0.3–10 μmol/L) and PKA activator 8-Br-cAMP (10–100 μmol/L) significantly enhanced the reverse mode activity of NCX1.1 and NCX1.5 in CHO cells. NCX1.1 was 2.4-fold more sensitive to PKC activation than NCX1.5, whereas the sensitivity of the two NCX isoforms to PKA activation had no difference. Both PKC- and PKA-enhanced NCX reverse mode activities in CHO cells were suppressed by NCX inhibitor KB-R7943 (30 μmol/L).
Conclusion: Both NCX1.1 and NCX1.5 are functional in regulating and maintaining stable [Ca2+]i in CHO cells and differentially regulated by PKA and PKC. The two NCX isoforms might be useful drug targets for heart and brain protection.
Keywords: Na+/Ca2+ exchanger; NCX1.1; NCX1.5; intracellular Ca2+ level; PKC; PKA; PMA; 8-Br-cAMP; KB-R7943
We thank Dr Jonathan LYTTON for generously providing NCX1.1 and NCX1.5 plasmids and Dr Nai-hong CHEN for technical assistance on calcium imaging. This work was supported by the National 973 Fundamental Project of China (2011CB504103) and the National Major Special Project on New Drug Innovation of China (No 2012ZX09301002-004).
* To whom correspondence should be addressed.
E-mail wangxl@imm.ac.cn
Received 2012-11-18 Accepted 2013-01-11