Cholesterol Stimulates the Transient Receptor Potential Melastatin 4 Channel in mpkCCDc14 Cells

Yong-Xu Cai; Bao-Long Zhang; Miao Yu; Yan-Chao Yang; Xue Ao; Di Zhu; Qiu-Shi Wang; Jie Lou; Chen Liang; Liang-Liang Tang; Ming-Ming Wu; Zhi-Ren Zhang; He-Ping Ma

doi:10.3389/fphar.2021.627875

Cholesterol Stimulates the Transient Receptor Potential Melastatin 4 Channel in mpkCCD_c14 Cells

Front Pharmacol. 2021 May 14:12:627875. doi: 10.3389/fphar.2021.627875. eCollection 2021.

Authors

Yong-Xu Cai¹, Bao-Long Zhang¹, Miao Yu¹, Yan-Chao Yang¹, Xue Ao¹, Di Zhu¹, Qiu-Shi Wang¹, Jie Lou¹, Chen Liang¹, Liang-Liang Tang¹, Ming-Ming Wu^{1

2}, Zhi-Ren Zhang^{1

3}, He-Ping Ma²

Affiliations

¹ 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.
² Department of Physiology, Emory University School of Medicine, Atlanta, GA, United States.
³ NHC Key Laboratory of Cell Transplantation, Harbin Medical University, Harbin, China.

Abstract

We have shown that cholesterol regulates the activity of ion channels in mouse cortical collecting duct (CCD) mpkCCD_c14 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 (Po) by regulating its sensitivity to Ca²⁺ in mpkCCD_c14 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 Po, 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_c14 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)P₂. Depletion of PI(4,5)P₂ with either wortmannin or PGE2 abrogated the stimulatory effects of exogenous cholesterol on TRPM4 activity, whereas exogenous PI(4,5)P₂ (diC8-PI(4,5)P₂, a water-soluble analog) increased the effects. These results suggest that cholesterol stimulates TRPM4 via a PI(4,5)P₂-dependent mechanism.

Keywords: Cholesterol; Lipid rafts; Lovastatin; PI(4,5)P2; TRPM4.