Tanshinone II-A sodium sulfonate (DS-201) enhances human BKCa channel activity by selectively targeting the pore-forming α subunit
Abstract
Xiao-qiu TAN1, 2, #, Xiu-li CHENG1, #, Yan YANG2, Li YAN1, Jing-li GU1, Hui LI1, Xiao-rong ZENG2, *, Ji-min CAO1, *
1Department of Physiology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine, Peking Union Medical College, Beijing 100005, China; 2Key Laboratory of Medical Electrophysiology, Ministry of Education, Collaborative Innovation Center for Prevention and Treatment of Cardiovascular Disease/Institute of Cardiovascular Research, Luzhou Medical College, Luzhou 646000, China
Aim: Tanshinone II-A sodium sulfonate (DS-201), a water-soluble derivative of Tanshinone II-A, has been found to induce vascular relaxation and activate BKCa channels. The aim of this study was to explore the mechanisms underlying the action of DS-201 on BKCa channels.
Methods: Human BKCa channels containing α subunit alone or α plus β1 subunits were expressed in HEK293 cells. BKCa currents were recorded from the cells using patch-clamp technique. The expression and trafficking of BKCa subunits in HEK293 cells or vascular smooth muscle cells (VSMCs) were detected by Western blotting, flow cytometry and confocal microscopy.
Results: DS-201 (40–160 μmol/L) concentration-dependently increased the total open probability of BKCa channels in HEK293 cells, associated with enhancements of Ca2+ and voltage dependence as well as a delay in deactivation. Coexpression of β1 subunit did not affect the action of DS-201: the values of EC50 for BKCa channels containing α subunit alone and α plus β1 subunit were 66.6±1.5 and 62.0±1.1 μmol/L, respectively. In both HEK293 cells and VSMCs, DS-201 (80 μmol/L) markedly increased the expression of α subunit without affecting β1 subunit. In HEK293 cells, DS-201 enriched the membranous level of α subunit, likely by accelerating the trafficking and suppressing the internalization of α subunit. In both HEK293 cells and VSMCs, DS-201 (≥320 μmol/L) induced significant cytotoxicity.
Conclusion: DS-201 selectively targets the pore-forming α subunit of human BKCa channels, thus enhancing the channel activities and increasing the subunit expression and trafficking, whereas the β1 subunit does not contribute to the action of DS-201.
Keywords: Danshen; tanshinone; BKCa channel; vascular relaxation; protein trafficking; vascular smooth muscle cell
This work was supported by grants from the National Natural Science Foundation of China (31300948 to Xiao-qiu TAN, 81173661 to Yan YANG, 81300139 to Jing-li GU, and 31171088 to Ji-min CAO). We thank Prof Isao INOUE for his helpful comments during the manuscript preparation.
# These authors contributed equally to this work.
* To whom correspondence should be addressed.
E-mail caojimin@126.com (Ji-min CAO); lyxjd7151@163.com (Xiao-rong ZENG)
Received 2014-04-04 Accepted 2014-07-18
Keywords:
1Department of Physiology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine, Peking Union Medical College, Beijing 100005, China; 2Key Laboratory of Medical Electrophysiology, Ministry of Education, Collaborative Innovation Center for Prevention and Treatment of Cardiovascular Disease/Institute of Cardiovascular Research, Luzhou Medical College, Luzhou 646000, China
Aim: Tanshinone II-A sodium sulfonate (DS-201), a water-soluble derivative of Tanshinone II-A, has been found to induce vascular relaxation and activate BKCa channels. The aim of this study was to explore the mechanisms underlying the action of DS-201 on BKCa channels.
Methods: Human BKCa channels containing α subunit alone or α plus β1 subunits were expressed in HEK293 cells. BKCa currents were recorded from the cells using patch-clamp technique. The expression and trafficking of BKCa subunits in HEK293 cells or vascular smooth muscle cells (VSMCs) were detected by Western blotting, flow cytometry and confocal microscopy.
Results: DS-201 (40–160 μmol/L) concentration-dependently increased the total open probability of BKCa channels in HEK293 cells, associated with enhancements of Ca2+ and voltage dependence as well as a delay in deactivation. Coexpression of β1 subunit did not affect the action of DS-201: the values of EC50 for BKCa channels containing α subunit alone and α plus β1 subunit were 66.6±1.5 and 62.0±1.1 μmol/L, respectively. In both HEK293 cells and VSMCs, DS-201 (80 μmol/L) markedly increased the expression of α subunit without affecting β1 subunit. In HEK293 cells, DS-201 enriched the membranous level of α subunit, likely by accelerating the trafficking and suppressing the internalization of α subunit. In both HEK293 cells and VSMCs, DS-201 (≥320 μmol/L) induced significant cytotoxicity.
Conclusion: DS-201 selectively targets the pore-forming α subunit of human BKCa channels, thus enhancing the channel activities and increasing the subunit expression and trafficking, whereas the β1 subunit does not contribute to the action of DS-201.
Keywords: Danshen; tanshinone; BKCa channel; vascular relaxation; protein trafficking; vascular smooth muscle cell
This work was supported by grants from the National Natural Science Foundation of China (31300948 to Xiao-qiu TAN, 81173661 to Yan YANG, 81300139 to Jing-li GU, and 31171088 to Ji-min CAO). We thank Prof Isao INOUE for his helpful comments during the manuscript preparation.
# These authors contributed equally to this work.
* To whom correspondence should be addressed.
E-mail caojimin@126.com (Ji-min CAO); lyxjd7151@163.com (Xiao-rong ZENG)
Received 2014-04-04 Accepted 2014-07-18