Receptor-mediated regulation of the TRPM7 channel through its endogenous protein kinase domain

Abstract

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.

The Mg2+ and Mg2+-Nucleotide-Regulated Channel-Kinase TRPM7

“…Another potential problem is the fact that the cells under investigation can have endogenous receptors that are activated in parallel with the overexpressed receptors, which is the case for muscarinic receptors, as some of them couple to PLC (M1, M3, M5) whereas others couple to Gi (M2, M4). Takezawa et al (2004) demonstrated that the muscarinic receptor-mediated inhibition of TRPM7 in HEK-293 cells by endogenous muscarinic receptors likely occurs via Gi-coupled pathways, since the effect was suppressed by pertussis toxin. They further demonstrated that TRPM7 currents are upregulated by isoproterenol (a beta receptor agonist that couples to Gs) or by perfusing cells with elevated cAMP.…”

“…Two conflicting reports have proposed regulatory mechanisms of TRPM7 by phosphatidylinositol bisphosphate (PIP 2 ) (Runnels et al 2002) and by cyclic AMP (cAMP) signaling (Takezawa et al 2004). Clapham and colleagues (2002) suggested that receptormediated PIP 2 is required to maintain TRPM7 activity, and its breakdown by receptormediated stimulation of phospholipase C (PLC) is responsible for the inhibition TRPM7 they observed when stimulating cells with agonists that couple to PLC.…”

“…Such overexpression is prone to significant pitfalls. Thus, the overexpression of TRPM7 leads to a complete suppression of PLC-mediated signaling and likely abolishes any PIP 2 breakdown, without affecting the receptor-mediated regulation of TRPM7 by muscarinic agonists (Takezawa et al 2004). This is presumably due to the fact that TRPM7 binds to and inhibits PLC (Runnels et al 2001).…”

“…Furthermore, these modulations were inhibited by protein kinase A inhibitors but not by the PLC antagonist U73122, suggesting that the receptor signaling cascade that regulates TRPM7 indeed involves Gi and/or Gscoupled receptors that elevate or decrease cAMP levels, which in turn regulate the activity of PKA. Interestingly, this PKA-dependent regulation additionally requires TRPM7's functional endogenous kinase, since phosphotransferase-deficient mutants lose their ability to be regulated via this mechanism (Takezawa et al 2004). It remains to be established whether the cAMP-dependent and the MgATP-mediated regulation of TRPM7 is caused by two separate and unrelated mechanisms or the two are somehow linked.…”

Emerging roles of TRPM6/TRPM7 channel kinase signal transduction complexes

“…According to Runnels et al phosphatidylinositol 4,5-biphosphate (PIP 2 ), the endogenous substrate of PLC, is required for TRPM7 channel activity and receptor-mediated activation of PLC decreases TRPM7 activity due to local hydrolysis of PIP 2 (Runnels et al 2002). On the contrary, Takezawa et al demonstrated that TRPM7, heterologously expressed in HEK293 cells, abolished activation of PLCβ via muscarinic and thrombin receptors coupled to G q proteins (Takezawa et al 2004). Moreover, TRPM7 channel activity was found to be positively modulated by receptors coupled to the G s /cAMP/ PKA signaling cascade.…”