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Cholesterol Stimulates the Transient Receptor Potential Melastatin 4 Channel in mpkCCDc14 Cells
Yong-Xu Cai1, Bao-Long Zhang1, Miao Yu1
1Departments 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.
Frontiers in Pharmacology
|May 31, 2021
Summary
Cholesterol regulates the activity of the TRPM4 ion channel in kidney cells. Cholesterol stimulates TRPM4 activity through a PI(4,5)P2-dependent pathway, impacting its function without altering protein levels.
Area of Science:
- Cell Biology
- Ion Channel Physiology
- Membrane Biophysics
Background:
- Cholesterol is known to regulate ion channel activity in kidney cells.
- The Transient Receptor Potential Melastatin 4 (TRPM4) channel is present in mouse cortical collecting duct (CCD) mpkCCDc14 cells.
- The specific role of cholesterol in regulating TRPM4 channel function remains undetermined.
Purpose of the Study:
- To investigate whether cholesterol regulates the activity of the TRPM4 channel in mpkCCDc14 cells.
- To elucidate the mechanism by which cholesterol influences TRPM4 channel function.
Main Methods:
- Inside-out patch-clamp electrophysiology to measure TRPM4 channel open probability (Po).
- Manipulation of cellular cholesterol levels using lovastatin and exogenous cholesterol.
- Cholesterol depletion using methyl-β-cyclodextrin (MβCD).
- Immunofluorescence, Western blot, and cell-surface biotinylation to assess TRPM4 protein abundance.
- Sucrose density gradient centrifugation to localize TRPM4 within lipid rafts.
- Lipid-protein overlay assays to identify direct lipid interactions.
Main Results:
- Inhibition of cholesterol synthesis decreased TRPM4 Po, while cholesterol enrichment increased it, by modulating Ca2+ sensitivity.
- Acute cholesterol depletion reduced TRPM4 Po, an effect reversible by exogenous cholesterol.
- Cholesterol modulation did not affect TRPM4 protein abundance in the plasma membrane.
- TRPM4 channels were localized in cholesterol-rich lipid rafts and directly interacted with PI(4,5)P2.
- PI(4,5)P2 depletion abrogated cholesterol's stimulatory effect on TRPM4, while exogenous PI(4,5)P2 enhanced it.
Conclusions:
- Cholesterol significantly regulates TRPM4 channel activity in mpkCCDc14 cells.
- Cholesterol stimulates TRPM4 channel function via a mechanism dependent on the phospholipid PI(4,5)P2.
- These findings highlight a novel cholesterol-PI(4,5)P2-TRPM4 signaling pathway in kidney cells.
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