TRPM7 activation potentiates SOCE in enamel cells but requires ORAI

Abstract

Calcium (Ca 2+) release-activated Ca 2+ (CRAC) channels mediated by STIM1/2 and ORAI (ORAI1-3) proteins form the dominant store-operated Ca 2+ entry (SOCE) pathway in a variety of cells. Among these, the enamel-forming cells known as ameloblasts rely on CRAC channel function to enable Ca 2+ influx, which is important for enamel mineralization. This key role of the CRAC channel is supported by human mutations and animal models lacking STIM1 and ORAI1, which results in enamel defects and hypomineralization. A number of recent reports have highlighted the role of the chanzyme TRPM7 (transient receptor potential melastanin 7), a transmembrane protein containing an ion channel permeable to divalent cations (Mg 2+ , Ca 2+), as a modulator of SOCE. This raises the question as to whether TRPM7 should be considered an alternative route for Ca 2+ influx, or if TRPM7 modifies CRAC channel activity in enamel cells. To address these questions, we monitored Ca 2+ influx mediated by SOCE using the pharmacological TRPM7 activator naltriben and the inhibitor NS8593 in rat primary enamel cells and in the murine ameloblast cell line LS8 cells stimulated with thapsigargin. We also measured Ca 2+ dynamics in ORAI1/2-deficient (shOrai1/2) LS8 cells and in cells with siRNA knock-down of Trpm7. We found that primary enamel cells stimulated with theTRPM7 activator potentiated Ca 2+ influx via SOCE compared to control cells. However, blockade of TRPM7 with NS8593 did not decrease the SOCE peak. Furthermore, activation of TRPM7 in shOrai1/2 LS8 cells lacking SOCE failed to elicit Ca 2+ influx, and Trpm7 knock-down had no effect on SOCE. Taken together, our data suggest that TRPM7 is a positive modulator of SOCE potentiating Ca 2+ influx in enamel cells, but its function is fully dependent on the prior activation of the ORAI channels.

TRPM7-Mediated Calcium Transport in HAT-7 Ameloblasts

“…However, the SOCE inhibitor BTP2 strongly blocked the naltriben-induced Ca 2+ entry, suggesting a pivotal role for Orai channels in these processes. This observation is in agreement with a previous report by Souza Bomfim et al [ 34 ].…”

“…Although the electrophysiology of TRPM7 channels is usually investigated, as here, by using Cs + as the conducting ion, these channels are highly permeable to divalent cations. We therefore next examined whether they might contribute directly to Ca 2+ uptake into HAT-7 cells, in addition to their role in modulating the SOCE pathway [ 34 ].…”

“…To investigate the contribution of SOCE to the Ca 2+ influx evoked by stimulation of TRPM7 (as proposed by Souza Bonfim et al [ 34 ]), responses to the two TRPM7 activators were also measured in the presence of a known SOCE blocker, BTP2 [ 35 ]. First, HAT-7 cells were pretreated with thapsigargin in the absence of extracellular Ca 2+ to deplete the ER stores, and the effect of Ca 2+ readdition was then tested in the presence and absence of BTP2.…”

Mapping TRPM7 Function by NS8593

“…Using DT40 cells expressing a kinase-deficient mutant of TRPM7, Faouzi et al [ 25 ] showed that TRPM7 regulates SOCE through its kinase domain. In line with these findings, NS8593 in combination with naltriben and siRNA approach was instrumental in demonstrating that in rat primary enamel cells and murine ameloblast LS8 cells TRPM7 acts as a positive regulator of SOCE and that this function of TRPM7 is dependent on ORAI1/2 channels, known molecular correlates of SOCE [ 117 ].…”

Hypo-Osmotic Loading Induces Expression of IL-6 in Nucleus Pulposus Cells of the Intervertebral Disc Independent of TRPV4 and TRPM7

“…This is partially in line with an earlier study on human articular chondrocytes, where TRPM7 was highly expressed in non-passaged cells, but contrary to our study, its expression greatly diminished with passaging ( Gavenis et al., 2009 ). Naltriben, with an EC 50 20 µM (HEK 293-mTRPM7 ( Hofmann et al., 2014 )), 24 µM (rat ventricular myocytes ( Tashiro et al., 2019 )), and 45 µM (ameloblast cell line LS8 cells ( Souza Bomfim et al., 2020 )), and NS8593, with an IC 50 1.6 µM (HEK 293 cells ( Chubanov et al., 2012 )) and 2 µM (rat ventricular myocytes ( Tashiro et al., 2014 )), are compounds that respectively activate and block TRPM7 channel. Naltriben was initially characterized as an antagonist of δ-opioid receptors ( June et al., 1999 ) and may compete with the inhibitory effect of NS8598, but does not stimulate other TRP channels including TRPM2, TRPM3, TRPM8, and TRPV1 ( Hofmann et al., 2014 ).…”