Journal
FRONTIERS IN PHARMACOLOGY
Volume 12, Issue -, Pages -Publisher
FRONTIERS MEDIA SA
DOI: 10.3389/fphar.2021.627875
Keywords
TRPM4; Cholesterol; PI(4; 5)P2; Lipid rafts; Lovastatin
Categories
Funding
- National Natural Science Foundation of China [81930009, 81870370, 91639202, 82070276]
- Department of Health and Human Services (National Institutes of Health) [5R01-DK067110, R01-DK100582]
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Cholesterol regulates TRPM4 channel activity by modulating its sensitivity to Ca2+, but does not affect the protein abundance of the channel on the plasma membrane. This regulation is likely mediated through a PI(4,5)P-2-dependent mechanism.
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-beta-cyclodextrin (M beta CD) 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)P-2. Depletion of PI(4,5)P-2 with either wortmannin or PGE2 abrogated the stimulatory effects of exogenous cholesterol on TRPM4 activity, whereas exogenous PI(4,5)P-2 (diC8-PI(4,5)P-2, a water-soluble analog) increased the effects. These results suggest that cholesterol stimulates TRPM4 via a PI(4,5)P-2-dependent mechanism.
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