Cai Yong-Xu, Zhang Bao-Long, Yu Miao, Yang Yan-Chao, Ao Xue, Zhu Di, Wang Qiu-Shi, Lou Jie, Liang Chen, Tang Liang-Liang, Wu Ming-Ming, Zhang Zhi-Ren, Ma He-Ping
Departments of Cardiology and Clinical Pharmacy, Harbin Medical University Cancer Hospital, Institute of Metabolic Disease, Heilongjiang Academy of Medical Science, Heilongjiang key laboratory for Metabolic disorder and cancer related cardiovascular diseases, and Key Laboratories of Education Ministry for Myocardial Ischemia Mechanism and Treatment, Harbin, China.
Department of Physiology, Emory University School of Medicine, Atlanta, GA, United States.
Front Pharmacol. 2021 May 14;12:627875. doi: 10.3389/fphar.2021.627875. eCollection 2021.
We have shown that cholesterol regulates the activity of ion channels in mouse cortical collecting duct (CCD) mpkCCD cells and that the transient receptor potential melastatin 4 (TRPM4) channel is expressed in these cells. However, whether TRPM4 channel is regulated by cholesterol remains unclear. Here, we performed inside-out patch-clamp experiments and found that inhibition of cholesterol biosynthesis by lovastatin significantly decreased, whereas enrichment of cholesterol with exogenous cholesterol significantly increased, TRPM4 channel open probability () by regulating its sensitivity to Ca in mpkCCD cells. In addition, inside-out patch-clamp data show that acute depletion of cholesterol in the membrane inner leaflet by methyl-β-cyclodextrin (MβCD) significantly reduced TRPM4 , which was reversed by exogenous cholesterol. Moreover, immunofluorescence microscopy, Western blot, cell-surface biotinylation, and patch clamp analysis show that neither inhibition of intracellular cholesterol biosynthesis with lovastatin nor application of exogenous cholesterol had effect on TRPM4 channel protein abundance in the plasma membrane of mpkCCD cells. Sucrose density gradient centrifugation studies demonstrate that TRPM4 was mainly located in cholesterol-rich lipid rafts. Lipid-protein overlay experiments show that TRPM4 directly interacted with several anionic phospholipids, including PI(4,5)P. Depletion of PI(4,5)P with either wortmannin or PGE2 abrogated the stimulatory effects of exogenous cholesterol on TRPM4 activity, whereas exogenous PI(4,5)P (diC8-PI(4,5)P, a water-soluble analog) increased the effects. These results suggest that cholesterol stimulates TRPM4 via a PI(4,5)P-dependent mechanism.
我们已经表明，胆固醇可调节小鼠皮质集合管（CCD）mpkCCD细胞中离子通道的活性，并且瞬时受体电位香草酸亚型4（TRPM4）通道在这些细胞中表达。然而，TRPM4通道是否受胆固醇调节仍不清楚。在此，我们进行了内面向外膜片钳实验，发现洛伐他汀抑制胆固醇生物合成会显著降低TRPM4通道开放概率（），而用外源性胆固醇富集胆固醇则会通过调节其对mpkCCD细胞中Ca的敏感性显著增加TRPM4通道开放概率。此外，内面向外膜片钳数据表明，甲基-β-环糊精（MβCD）急性耗尽膜内小叶中的胆固醇会显著降低TRPM4，而外源性胆固醇可使其逆转。此外，免疫荧光显微镜、蛋白质印迹、细胞表面生物素化和膜片钳分析表明，用洛伐他汀抑制细胞内胆固醇生物合成或应用外源性胆固醇均对mpkCCD细胞质膜中TRPM4通道蛋白丰度没有影响。蔗糖密度梯度离心研究表明，TRPM4主要位于富含胆固醇的脂筏中。脂质-蛋白质覆盖实验表明，TRPM4与几种阴离子磷脂直接相互作用，包括磷脂酰肌醇-4,5-二磷酸（PI(4,5)P）。用渥曼青霉素或前列腺素E2耗尽PI(4,5)P可消除外源性胆固醇对TRPM4活性的刺激作用，而外源性PI(4,5)P（二辛酰磷脂酰肌醇-4,5-二磷酸，一种水溶性类似物）则增强了这种作用。这些结果表明，胆固醇通过PI(4,5)P依赖性机制刺激TRPM4。
