Molecular expression of Mg2+ regulator TRPM7 and CNNM4 in rat odontoblasts

Objective: Magnesium, the second most abundant cation in cellular fluid, is critical for mineralization of hard tissues. Among the molecules involved in cellular Mg2+ homeostasis, functional impairment of Mg2+ permeable ion channel TRPM7 or Mg2+ transporter CNNM4 have been found to result in severe hypomineralization of the enamel and dentin. However, molecular expressions of TRPM7, CNNM4 and their respective homologues have not been fully investigated in adult odontoblasts. Design: Expressions of TRPM6, TRPM7, CNNM1, CNNM2, CNNM3, CNNM4 were screened in acutely dissociated rat odontoblasts by single cell RT-PCR. Among these candidates, expression levels of TRPM7 and CNNM4 were compared along the odontoblast layer by immunohistochemical analysis. Finally, the coexpression pattern of TRPM7 and CNNM4 in subcellular regions was examined by immunocytochemical analysis. Results: ScRT-PCR revealed high expression rate of TRPM7 and CNNM4 in odontoblasts, with CNNM4 detected almost exclusively in TRPM7-positive odontoblasts. However, CNNM2 and CNNM3 were detected in only a small population of odontoblasts, and TRPM6 and CNNM1 were not detected even in the pulp tissue. Immunohistochemical analysis revealed higher CNNM4 expression in the apical odontoblast layer than the coronal area, in contrast to the ubiquitous expression of TRPM7. Lastly, immunocytochemical analysis revealed colocalization of CNNM4 with TRPM7 in the odontoblastic process. Conclusions: CNNM4 and TRPM7 may serve as main Mg2+ regulators in odontoblasts, possibly with selective involvement of CNNM4 in apical dentin formation or mineralization. Colocalization of TRPM7 and CNNM4 in the odontoblastic process suggest functional coupling of these two molecules to maintain Mg2+ homeostasis.