IADR Abstract Archives
The Role of TRPM7 Channel in the Ca2+ and Mg2+ Homeostasis of Ameloblast Cells
Objectives: TRPM7 is a divalent cation-permeable channel, with serine/threonine protein kinase activity, which plays an important role in the regulation of Ca2+ and Mg2+ homeostasis in various cell types. TRPM7 channel activity is sensitive to intracellular Mg2+ and pH, and these channels are known to be expressed in ameloblasts. We have previously found that TRPM7 mRNA is expressed in rat HAT-7 cells, an established model for ameloblast epithelial transport. Thus, our objective was to functionally characterize the TRPM7 channel in HAT-7 cells.
Methods: Transmembrane ion currents were measured using patch-clamp electrophysiology in the whole-cell configuration (voltage-clamp mode) in the presence of different intracellular Mg2+ concentrations ([Mg2+]i; 0, 0.9, 3.6 mM) in response to the agonist mibefradil (50 µM) as well as to TRPM7 antagonists (20 µM NS8593 and 2 µM FTY720). Changes in intracellular Ca2+ concentration [Ca2+]i were measured by ratiometric Ca2+-imaging using the fluorescent dye Fura-2.
Results: Mg2+-free pipette solutions significantly increased, while elevated [Mg2+]i (3.6 mM) decreased the whole-cell currents measured at +80 mV. Both NS8593 and FTY720 inhibited the TRPM7 channel activity triggered by the Mg2+-free intracellular environment. Mibefradil evoked a characteristic outward-rectifying current that was significantly suppressed by elevated [Mg2+]i (3.6 mM). Mibefradil elicited an increase in [Ca2+]i in a dose-dependent manner which was largely dependent on the presence of extracellular Ca2+. Intracellular alkalinization induced by an NH4Cl pulse enhanced the Ca2+ influx from the extracellular space.
Conclusions: Both the characteristic mibefradil-induced and [Mg2+]i-sensitive transmembrane ion currents and the mibefradil- and intracellular alkalinization-evoked Ca2+ influx indicate the functional presence of TRPM7 channels in HAT-7 cells. These data suggest that HAT-7 cells could serve as suitable model for the investigation of epithelial Ca2+ transport in ameloblasts.
Supported by the EFOP-3.6.2.-16-2017-0006, NKFIH K-125161 and NIH-NIDCR 1R01DE027971 grants.
Methods: Transmembrane ion currents were measured using patch-clamp electrophysiology in the whole-cell configuration (voltage-clamp mode) in the presence of different intracellular Mg2+ concentrations ([Mg2+]i; 0, 0.9, 3.6 mM) in response to the agonist mibefradil (50 µM) as well as to TRPM7 antagonists (20 µM NS8593 and 2 µM FTY720). Changes in intracellular Ca2+ concentration [Ca2+]i were measured by ratiometric Ca2+-imaging using the fluorescent dye Fura-2.
Results: Mg2+-free pipette solutions significantly increased, while elevated [Mg2+]i (3.6 mM) decreased the whole-cell currents measured at +80 mV. Both NS8593 and FTY720 inhibited the TRPM7 channel activity triggered by the Mg2+-free intracellular environment. Mibefradil evoked a characteristic outward-rectifying current that was significantly suppressed by elevated [Mg2+]i (3.6 mM). Mibefradil elicited an increase in [Ca2+]i in a dose-dependent manner which was largely dependent on the presence of extracellular Ca2+. Intracellular alkalinization induced by an NH4Cl pulse enhanced the Ca2+ influx from the extracellular space.
Conclusions: Both the characteristic mibefradil-induced and [Mg2+]i-sensitive transmembrane ion currents and the mibefradil- and intracellular alkalinization-evoked Ca2+ influx indicate the functional presence of TRPM7 channels in HAT-7 cells. These data suggest that HAT-7 cells could serve as suitable model for the investigation of epithelial Ca2+ transport in ameloblasts.
Supported by the EFOP-3.6.2.-16-2017-0006, NKFIH K-125161 and NIH-NIDCR 1R01DE027971 grants.