Investigation of the function and regulation of the TRPM7 ion channel in the renal proximal tubule

The TRPM7 (Transient Receptor Potential Melastatin 7) ion channel is a unique member of the TRP channel family, possessing its own functional kinase domain at its COOH-terminus. As a Mg2+-permeable ion channel, TRPM7 has frequently been linked to the regulation of magnesium reabsorption at both the cellular and whole-body level. Mg2+ plays a pivotal role in human health and disease, and therefore, its level in the body has to be tightly regulated via ion channels and transporters in the functional unit of the kidney, the nephron. TRPM6, the close homolog of TRPM7, has been identified to be the major player regulating Mg2+ reabsorption in the distal convoluted tubule of the nephron. A major gap in our knowledge of TRPM7 is whether the channel is involved in regulating magnesium homeostasis in the proximal tubule of the nephron, where TRPM7 is highly expressed. To gain insight into the function of TRPM7 in the proximal tubule, we generated two conditional strains of proximal tubule-specific trpm7 KO mice, using PEPCK-Cre and gGT-Cre mice. The Mg2+ status of the proximal tubule trpm7 knockout mice was assessed but we did not obtain any evidence that the Mg2+ homeostasis was disrupted in the animals, indicating TRPM7 does not play a major role in proximal tubule to regulate whole-body magnesium homeostasis. However, large cavities and reduced cortical layers in the kidney anatomy of some female gGT-Cre KO trpm7 mice were observed. TRPM7 has previously been implicated in the regulation of cell-cell adhesion, having recently been found to contribute to the intercellular junction formation in the bladder urothelium. We performed transmission electron microscopy (TEM) analysis of the tissue slides obtained from the cortex of the kidneys from gGT-Cre KO trpm7 mice and found that tubule epithelial cells from the trpm7 KO mice had more impaired intercellular junctions than that from the control mice. We next investigated the relationship between TRPM7 and cell-cell adhesion process, employing the proximal tubule epithelial cell line, opossum kidney (OK) cells, as a cellular model. Mass spectrometric analysis uncovered that TRPM7 interacted with a cell adhesion protein called plakoglobin. Using immunocytochemical assays, we discovered that TRPM7 co-localized with plakoglobin and another adherens junction protein called E-cadherin. Application of the TRPM7’s channel blocker NS8593 to OK cells reduced E-cadherin expression and localization to adherens junctions. Taken together, these data suggest that TRPM7 is involved in controlling cell-cell adhesion in proximal tubule epithelial cells. In this study, we also explored the mechanism(s) by which TRPM7’s cellular localization is regulated. Using biochemical and immunocytochemical approaches, we identified a regulatory site at the COOH-terminus of TRPM7, the channel’s PDZ-binding motif, through which the localization of TRPM7 in OK cells could be regulated. Deletion of the channel’s PDZ-binding motif shortened the retention time of the mutant TRPM7 (TRPM7ΔPDZ) at adherens junctions of OK cells. We also discovered that TRPM7’s localization to the adherens junctions in OK cells was potentiated by the activation of PKC, indicating that TRPM7 localization is potentially regulated by the PKC pathway. While our understanding of the relationship between TRPM7 and cell-cell adhesion is still in its infancy, the discoveries made in this study will guide future investigations into the physiological roles of the channel in the regulation of cell-cell adhesion.Ph.D.Includes bibliographical reference