TRPM7 is a Ca2+- and Mg2+-permeable cation channel that also contains a protein kinase domain. While there is general consensus that the channel is inhibited by free intracellular Mg2+, the functional roles of intracellular levels of Mg·ATP and the kinase domain in regulating TRPM7 channel activity have been discussed controversially. To obtain insight into these issues, we have determined the effect of purine and pyrimidine magnesium nucleotides on TRPM7 currents and investigated the possible involvement of the channel's kinase domain in mediating them. We report here that physiological Mg·ATP concentrations can inhibit TRPM7 channels and strongly enhance the channel blocking efficacy of free Mg2+. Mg·ADP, but not AMP, had similar, albeit smaller effects, indicating a double protection against possible Mg2+ and Ca2+ overflow during variations of cell energy levels. Furthermore, nearly all Mg-nucleotides were able to inhibit TRPM7 activity to varying degrees with the following rank in potency: ATP > TTP > CTP ≥ GTP ≥ UTP > ITP ≈ free Mg2+ alone. These nucleotides also enhanced TRPM7 inhibition by free Mg2+, suggesting the presence of two interacting binding sites that jointly regulate TRPM7 channel activity. Finally, the nucleotide-mediated inhibition was lost in phosphotransferase-deficient single-point mutants of TRPM7, while the Mg2+-dependent regulation was retained with reduced efficacy. Interestingly, truncated mutant channels with a complete deletion of the kinase domain regained Mg·NTP sensitivity; however, this inhibition did not discriminate between nucleotide species, suggesting that the COOH-terminal truncation exposes the previously inaccessible Mg2+ binding site to Mg-nucleotide binding without imparting nucleotide specificity. We conclude that the nucleotide-dependent regulation of TRPM7 is mediated by the nucleotide binding site on the channel's endogenous kinase domain and interacts synergistically with a Mg2+ binding site extrinsic to that domain.

TRPM7 is a ubiquitously expressed and constitutively active divalent cation-selective ion channel, whose basal activity is regulated by intracellular levels of Mg 2؉ and Mg⅐ATP. We have investigated receptor-mediated mechanisms that may actively regulate TRPM7 activity. We here report that TRPM7 currents are suppressed by intracellular GTP␥S, suggesting the involvement of heterotrimeric G proteins. TRPM7 currents are also inhibited by stimulating endogenous muscarinic receptors, which is mediated by Gi because the inhibitory effect is blunted by pertussis toxin. Conversely, stimulation of endogenous Gs-coupled ␤-adrenergic receptors potentiates TRPM7 currents, whereas Gq-coupled thrombin receptors have little effect. Consistent with the involvement of Gs͞Gi in controlling adenylyl cyclase activity, elevations of intracellular cAMP levels enhance TRPM7 activity and prevent receptor-mediated modulation of TRPM7 activity by muscarinic and adrenergic agonists. This cAMP-dependent effect requires the functional integrity of both protein kinase A (PKA) and the endogenous kinase domain of TRPM7 because cAMP-mediated effects are abolished when treating cells with the PKA inhibitors H89 or KT5720 as well as in cells expressing phosphotransferase-deficient TRPM7 constructs. These mutant channels are also much less susceptible to GTP␥S-mediated inhibition, suggesting that the main regulatory effect occurs through Gi-and Gs-mediated changes in cAMP. Taken together, our results demonstrate that TRPM7 activity is up-and down-regulated through its endogenous kinase in a cAMP-and PKA-dependent manner.B ased on sequence similarities, the mammalian transient receptor potential (TRP) channel family is divided into three subfamilies [TRP classical (formerly short TRP channel)], TRP vanilloid [(formerly osm TRP channel), and TRP melastatin (formerly long TRP channel)] (1-4). TRPM7 (long TRP channel 7, TRP-phospholipase C interacting kinase and channel kinase 1), a Ca 2ϩ -and Mg 2ϩ -permeable divalent cation channel of the TRP melastatin ion channel subfamily, is required for cellular viability and noted for its ubiquitous distribution profile (5, 6). We have previously demonstrated that TRPM7 is regulated by millimolar levels of intracellular Mg 2ϩ and Mg⅐ATP and its activity appears to be linked to cellular energy metabolism (5, 7). In resting cells, physiological levels of these molecules strongly suppress the activity of TRPM7 channels and only a small constitutive activity remains, sufficient to maintain basal divalent cation fluxes. In whole-cell patch-clamp experiments, intracellular solutions that lack added Mg⅐ATP lead to activation of TRPM7-mediated currents (Fig.