Transient Receptor Potential Melastatin 7 Kinase Research Articles

Abstract 13087: TRPM7 Kinase Regulates Inflammation in Hypomagnesemia-Induced Cardiac Diastolic Dysfunction

Introduction: Hypomagnesemia (HypoMg) is commonly observed in heart failure and associated with inflammation. We have reported that inflammation contributes to mitochondrial oxidative stress and causes cardiac diastolic dysfunction (DD) in diabetic mice and that Mg supplementation shows therapeutic benefits. Meanwhile, diet-induced HypoMg alone can lead to DD. Transient receptor potential melastatin 7 (TRPM7), a Mg transporter with a kinase domain, regulates Mg homeostasis in different types of cells. Object: We aim to assess the role of TRPM7 in HypoMg-associated inflammation and DD. Methods: Wild type (WT) C57BL/6J and transgenic kinase dead TRPM7 K1646R mice (with a global homozygous K1646R mutation in the kinase domain) were fed with a normal (2 g/kg Mg) or a HypoMg diet (15-30 mg/kg Mg) for 6 weeks. Echocardiography was performed to monitor E/e’ (an DD indicator when increased). Hearts were collected for protein levels of TRPM7, inflammatory markers IL-1β and NLRP3, and total protein oxidation. Isolated cardiac macrophages (Mϕs) were used for phenotyping by flow cytometry. Raw264.7 cells (a Mϕ cell line) and RL-14 cells (a cardiomyocyte cell line) were treated with a normal (0.81 mM Mg) or a HypoMg medium (0.04 mM Mg) for 48 h. TRPM7 protein and IL-1β collected from the culture media or from cell lysates were tested. Results: In the WT-HypoMg mouse group, E/e’, protein levels of TRPM7, IL-1β and NLRP3, and total protein oxidation were all significantly elevated, which were not observed in the TRPM7 K1646R -HypoMg group. Cardiac Mϕs phenotype showed increased recruited pro-inflammatory Mϕs and decreased resident pro-resolving Mϕs, while the former was prevented by TRPM7 K1646R . TRPM7 protein levels in Raw264.7 and RL-14 cells were also significantly increased by HypoMg treatment, as well as Mϕ-secreted IL-1β (2.7±0.6 pg/mL vs. undetectable in control, P=0.012) and IL-1β in RL-14 cell lysates (2.6±0.2-fold of control, P=0.006). Conclusions: TRPM7 kinase function is upregulated under HypoMg in both Mϕs and cardiomyocytes. It contributes to inflammatory Mϕ activation, elevated IL-1β production, increased oxidative stress, and cardiac DD, suggesting inflammation as a potential mediator of HypoMg-induced DD.

TRPM7 kinase inactivation results in macrophage alkalinization and increased phagocytic activity

Transient receptor potential melastatin 7 (TRPM7) is a unique protein that is both an ion channel and a cytosolic protein kinase. Normally, TRPM7 channels are tonically inhibited by cytoplasmic Mg2+, polyamines and acidic pH. Channel basal activity can be increased by alkalinization or Mg2+ depletion. We have used the TRPM7 kinase-dead (KD K1646R) mouse to investigate the consequences of kinase inactivation in immune cell function. It also serves as a convenient system to elucidate kinase-channel interactions at a functional level.

Phagocytic activity of splenic macrophages is enhanced and accompanied by cytosolic alkalinization in TRPM7 kinase-dead mice.

Transient receptor potential melastatin 7 (TRPM7) is a unique protein functioning as a cation channel as well as a serine/threonine kinase and is highly expressed in immune cells such as lymphocytes and macrophages. TRPM7 kinase-dead (KD) mouse model has been used to investigate the role of this protein in immune cells; these animals display moderate splenomegaly and ectopic hemopoiesis. The basal TRPM7 current magnitudes in peritoneal macrophages isolated from KD mice were higher; however, the maximum currents, achieved after cytoplasmic Mg2+ washout, were not different. In the present study, we investigated the consequences of TRPM7 kinase inactivation in splenic and peritoneal macrophages. We measured the basal phagocytic activity of splenic macrophages using fluorescent latex beads, pHrodo zymosan bioparticles, and opsonized red blood cells. KD macrophages phagocytized more efficiently and had slightly higher baseline calcium levels compared to WT cells. We found no obvious differences in store-operated Ca2+ entry between WT and KD macrophages. By contrast, the resting cytosolic pH in KD macrophages was significantly more alkaline than in WT. Pharmacological blockade of sodium hydrogen exchanger 1 (NHE1) reversed the cytosolic alkalinization and reduced phagocytosis in KD macrophages. Basal TRPM7 channel activity in KD macrophages was also reduced after NHE1 blockade. Cytosolic Mg2+ sensitivity of TRPM7 channels measured in peritoneal macrophages was similar in WT and KD mice. The higher basal TRPM7 channel activity in KD macrophages is likely due to alkalinization. Our results identify a novel role for TRPM7 kinase as a suppressor of basal phagocytosis and a regulator of cellular pH.

The kinase activity of the channel-kinase protein TRPM7 regulates stability and localization of the TRPM7 channel in polarized epithelial cells.

The channel-kinase transient receptor potential melastatin 7 (TRPM7) is a bifunctional protein with ion channel and kinase domains. The kinase activity of TRPM7 has been linked to the regulation of a broad range of cellular activities, but little is understood as to how the channel itself is regulated by its own kinase activity. Here, using several mammalian cell lines expressing WT TRPM7 or kinase-inactive variants, we discovered that compared with the cells expressing WT TRPM7, cells in which TRPM7's kinase activity was inactivated had faster degradation, elevated ubiquitination, and increased intracellular retention of the channel. Mutational analysis of TRPM7 autophosphorylation sites further revealed a role for Ser-1360 of TRPM7 as a key residue mediating both TRPM7 stability and intracellular trafficking. Additional trafficking roles were uncovered for Ser-1403 and Ser-1567, whose phosphorylation by TRPM7's kinase activity mediated the interaction of the channel with the signaling protein 14-3-3θ. In summary, our results point to a critical role for TRPM7's kinase activity in regulating proteasome-mediated turnover of the TRPM7 channel and controlling its cellular localization in polarized epithelial cells. Overall, these findings improve our understanding of the significance of TRPM7's kinase activity for functional regulation of its channel activity.

The crucial role of the TRPM7 kinase domain in the early stage of amelogenesis

Transient receptor potential melastatin-7 (TRPM7) is a bi-functional protein containing a kinase domain fused to an ion channel. TRPM7 is highly expressed in ameloblasts during tooth development. Here we show that TRPM7 kinase-inactive knock-in mutant mice (TRPM7 KR mice) exhibited small enamel volume with opaque white-colored incisors. The TRPM7 channel function of ameloblast-lineage cells from TRPM7 KR mice was normal. Interestingly, phosphorylation of intracellular molecules including Smad1/5/9, p38 and cAMP response element binding protein (CREB) was inhibited in ameloblasts from TRPM7 KR mice at the pre-secretory stage. An immunoprecipitation assay showed that CREB was bound to TRPM7, suggesting that direct phosphorylation of CREB by TRPM7 was inhibited in ameloblast-lineage cells from TRPM7 KR mice. These results indicate that the function of the TRPM7 kinase domain plays an important role in ameloblast differentiation, independent of TRPM7 channel activity, via phosphorylation of CREB.

TRPM7 kinase domain rather than the channel regulates breast cancer cell migration and tumor metastasis

The channel‐kinase TRPM7 (transient receptor potential melastatin 7) is a bifunctional protein consisting of a cation channel that is permeable to Mg2+, Ca2+ and Mn2+ ions fused to a C‐terminal kinase domain. A growing number of studies with clinical significance suggest that TRPM7 is linked to adhesion and migration of breast cancer cells and promotes breast tumor metastasis. While the channel properties of TRPM7 have been studied extensively, little is known about its kinase activity and how it is linked to the control of actomyosin contractility. To understand the functions of the kinase domain we identified the first cell‐permeable inhibitor of the kinase domain (TRPM7‐IN‐1) and developed MDA‐MB‐231 cell lines in which TRPM7 is knocked out by CRISPR/Cas9 (KO), and in which various forms of TRPM7 were stably re‐expressed. These were wild type TRPM7 (WT), a kinase‐inactive mutant of TRPM7 (KD), and TRPM7 containing a truncated kinase domain (KT). TRPM7 KO significantly inhibited MDA‐MB‐231 cells migration. Only expression of the wild type TRPM7 (WT) rescued the migration phenotype, supporting a role for the kinase domain and not the channel in the regulation of cell migration. TRPM7‐IN‐1 decreased the binding of Myosin IIB to TRPM7 in HEK293 and MDA‐MB‐231 cells. And when MDA‐MB‐231 cells were treated with increasing doses of TRPM7‐IN‐1, TRPM7 phosphorylation at Ser‐1569 and its downstream Myosin IIa phosphorylation at Ser1943 were completely abrogated at a concentration of 5 μM. TRPM7‐IN‐1 inhibited MDA‐MB‐231 and BT 549 cell migration and invasion, while treatment of the KO cells with TRPM7‐IN‐1 showed no further inhibition of migration. Electrophysiological assessment of the TRPM7 channel revealed that the inhibitor did not affect the channel function in MD‐MB‐231 cells, supporting the notion that the inhibitor affects the migration exclusively through the inhibition of the TRPM7 kinase domain. Finally, in an experimental metastasis model, TRPM7‐IN‐1 significantly impeded metastasis to the lung. In conclusion, inhibition of TRPM7 kinase activity may reduce or block breast tumor progression and/or metastasis.Support or Funding InformationFinancial support by grants from the National Institute of General Medical Sciences (R01GM059802), the Welch Foundation (F‐1390) and Cancer Prevention and Research Institute of Texas to KND.

The TRPM7 channel kinase regulates store-operated calcium entry.

Pharmacological and molecular inhibition of transient receptor potential melastatin 7 (TRPM7) reduces store-operated calcium entry (SOCE). Overexpression of TRPM7 in TRPM7-/- cells restores SOCE. TRPM7 is not a store-operated calcium channel. TRPM7 kinase rather than channel modulates SOCE. TRPM7 channel activity contributes to the maintenance of store Ca2+ levels at rest. The transient receptor potential melastatin 7 (TRPM7) is a protein that combines an ion channel with an intrinsic kinase domain, enabling it to modulate cellular functions either by conducting ions through the pore or by phosphorylating downstream proteins via its kinase domain. In the present study, we report store-operated calcium entry (SOCE) as a novel target of TRPM7 kinase activity. TRPM7-deficient chicken DT40 B lymphocytes exhibit a strongly impaired SOCE compared to wild-type cells as a result of reduced calcium release activated calcium currents, and independently of potassium channel regulation, membrane potential changes or changes in cell-cycle distribution. Pharmacological blockade of TRPM7 with NS8593 or waixenicin A in wild-type B lymphocytes results in a significant decrease in SOCE, confirming that TRPM7 activity is acutely linked to SOCE, without TRPM7 representing a store-operated channel itself. Using kinase-deficient mutants, we find that TRPM7 regulates SOCE through its kinase domain. Furthermore, Ca2+ influx through TRPM7 is essential for the maintenance of endoplasmic reticulum Ca2+ concentration in resting cells, and for the refilling of Ca2+ stores after a Ca2+ signalling event. We conclude that the channel kinase TRPM7 and SOCE are synergistic mechanisms regulating intracellular Ca2+ homeostasis.

Transient Receptor Potential Melastatin 7 Cation Channel Kinase: New Player in Angiotensin II-Induced Hypertension.

Transient receptor potential melastatin 7 (TRPM7) is a bifunctional protein comprising a magnesium (Mg(2+))/cation channel and a kinase domain. We previously demonstrated that vasoactive agents regulate vascular TRPM7. Whether TRPM7 plays a role in the pathophysiology of hypertension and associated cardiovascular dysfunction is unknown. We studied TRPM7 kinase-deficient mice (TRPM7Δkinase; heterozygous for TRPM7 kinase) and wild-type (WT) mice infused with angiotensin II (Ang II; 400 ng/kg per minute, 4 weeks). TRPM7 kinase expression was lower in heart and aorta from TRPM7Δkinase versus WT mice, effects that were further reduced by Ang II infusion. Plasma Mg(2+) was lower in TRPM7Δkinase versus WT mice in basal and stimulated conditions. Ang II increased blood pressure in both strains with exaggerated responses in TRPM7Δkinase versus WT groups (P<0.05). Acetylcholine-induced vasorelaxation was reduced in Ang II-infused TRPM7Δkinase mice, an effect associated with Akt and endothelial nitric oxide synthase downregulation. Vascular cell adhesion molecule-1 expression was increased in Ang II-infused TRPM7 kinase-deficient mice. TRPM7 kinase targets, calpain, and annexin-1, were activated by Ang II in WT but not in TRPM7Δkinase mice. Echocardiographic and histopathologic analysis demonstrated cardiac hypertrophy and left ventricular dysfunction in Ang II-treated groups. In TRPM7 kinase-deficient mice, Ang II-induced cardiac functional and structural effects were amplified compared with WT counterparts. Our data demonstrate that in TRPM7Δkinase mice, Ang II-induced hypertension is exaggerated, cardiac remodeling and left ventricular dysfunction are amplified, and endothelial function is impaired. These processes are associated with hypomagnesemia, blunted TRPM7 kinase expression/signaling, endothelial nitric oxide synthase downregulation, and proinflammatory vascular responses. Our findings identify TRPM7 kinase as a novel player in Ang II-induced hypertension and associated vascular and target organ damage.

Abstract A17: TRPM7 kinase domain is involved in breast tumor cell metastasis

Abstract Introduction: TRPM7 (transient receptor potential melastatin 7) is a non-selective cation channel fused to protein kinase domain at the C-terminal whose activity is linked to the control of actomyosin contractility. TRPM7 mediates adhesion and migration of breast cancer cells and promotes breast tumor metastasis. The lack of cell-permeable inhibitors of the kinase domain represents a barrier to understand the kinase function. Methods: Herein, we discover the first small molecule (KD-1) that targets TRPM7 kinase activity and characterize its mechanism of action in-vitro, in-cells and in-vivo. By using this inhibitor, we are investigated the involvement of TRPM7 kinase domain in breast tumor cell metastasis. Results: Mg2+ starvation, which promotes TRPM7 kinase activity, induces phosphorylation of eEF2. Treatment of Mg2+-starved HEK293 cells with KD-1 decreased eEF2 phosphorylation, consistent with TRPM7 kinase activity suppression in-cells. KD-1 decreased the binding of Myosin IIB to TRPM7 in HEK293 and MDA-MB-231 cells. And when MDA-MB-231 and BT 549 cells were treated with increasing doses of KD-1, no change in cell viability was seen. Interestingly, KD-1 inhibited migration and invasion in the MDA-MB-231 and BT 549 cells that is reportedly regulated by TRPM7 kinase activity. Finally, the bioluminescent signals (to assess lung metastasis) were significantly lower in KD-1-treated mice (25 and 50 mg/kg/day) than in mice treated with vehicle control (P ≤0.05, 2-sided t-test.). Conclusion: The discovered compound represents the first inhibitor that targets the kinase activity of transient receptor potential melastatin 7 (TRPM7). Inhibition of TRPM7 kinase activity may reduce or block breast tumor progression and/or metastasis. Citation Format: Tamer S. Kaoud, Xuemei Xie, Jihyun Park, Clint D.J. Tavares, Shreya Mitra, Micael Cano, Mohamed F. Radwan, Chandra Bartholomeusz, Kevin N. Dalby. TRPM7 kinase domain is involved in breast tumor cell metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr A17.

TRPM7 kinase activity regulates murine mast cell degranulation.

The Mg(2+) and Ca(2+) conducting transient receptor potential melastatin 7 (TRPM7) channel-enzyme (chanzyme) has been implicated in immune cell function. Mice heterozygous for a TRPM7 kinase deletion are hyperallergic, while mice with a single point mutation at amino acid 1648, silencing kinase activity, are not. As mast cell mediators trigger allergic reactions, we here determine the function of TRPM7 in mast cell degranulation and histamine release. Our data establish that TRPM7 kinase activity regulates mast cell degranulation and release of histamine independently of TRPM7 channel function. Our findings suggest a regulatory role of TRPM7 kinase activity on intracellular Ca(2+) and extracellular Mg(2+) sensitivity of mast cell degranulation. Transient receptor potential melastatin 7 (TRPM7) is a divalent ion channel with a C-terminally located α-kinase. Mice heterozygous for a TRPM7 kinase deletion (TRPM7(+/∆K) ) are hypomagnesaemic and hyperallergic. In contrast, mice carrying a single point mutation at amino acid 1648, which silences TRPM7 kinase activity (TRPM7(KR) ), are not hyperallergic and are resistant to systemic magnesium (Mg(2+) ) deprivation. Since allergic reactions are triggered by mast cell-mediated histamine release, we investigated the function of TRPM7 on mast cell degranulation and histamine release using wild-type (TRPM7(+/+) ), TRPM7(+/∆K) and TRPM7(KR) mice. We found that degranulation and histamine release proceeded independently of TRPM7 channel function. Furthermore, extracellular Mg(2+) assured unperturbed IgE-DNP-dependent exocytosis, independently of TRPM7. However, impairment of TRPM7 kinase function suppressed IgE-DNP-dependent exocytosis, slowed the cellular degranulation rate, and diminished the sensitivity to intracellular calcium (Ca(2+) ) in G protein-induced exocytosis. In addition, G protein-coupled receptor (GPCR) stimulation revealed strong suppression of histamine release, whereas removal of extracellular Mg(2+) caused the phenotype to revert. We conclude that the TRPM7 kinase activity regulates murine mast cell degranulation by changing its sensitivity to intracellular Ca(2+) and affecting granular mobility and/or histamine contents.

Targeting the transient receptor potential‐melastatin‐like 7 (Trpm7) kinase domain with the first inhibitor, inhibited breast cancer cell migration, invasion and tumor metastasis.

TRPM7 (transient receptor potential melastatin 7) is a non‐selective cation channel fused to protein kinase domain at the C‐terminal whose activity is linked to the control of actomyosin contractility. TRPM7 mediates adhesion and migration of breast cancer cells and promotes breast tumor metastasis. The lack of cell‐permeable inhibitors of the kinase domain represents a barrier to understand the kinase function. Herein, we discovered the first small molecule (KD‐1) that targets TRPM7 kinase activity. Mg2+ starvation, which promotes TRPM7 kinase activity, induces phosphorylation of eEF2. Treatment of Mg2+‐starved HEK293 cells with KD‐1 decreased eEF2 phosphorylation, consistent with TRPM7 kinase activity suppression in‐cells. KD‐1 decreased the binding of Myosin IIB to TRPM7 in HEK293 and MDA‐MB‐231 cells. And when MDA‐MB‐231 cells were treated with increasing doses of KD‐1, no change in cell viability was seen. Interestingly, KD‐1 inhibited MDA‐MB‐231 cells migration and invasion that have been reported to be regulated by TRPM7 kinase activity. Finally, the bioluminescent signals (to assess metastasis) were significantly lower in KD‐1‐treated mice (25 and 50 mg/kg/day) than in mice treated with vehicle control (P 蠄 0.05, 2‐sided t‐test.). As a conclusion, inhibition of TRPM7 kinase activity can be a valid approach to reduce or block breast tumor progression and/or metastasis.

Abstract 4: Transient Receptor Potential Melastatin 7 Cation Channel (TRPM7) Kinase Domain - a New Player in Angiotensin II-induced Hypertension and Cardiac Hypertrophy

Transient receptor potential melastatin 7 (TRPM7) cation channel is a unique protein that has the dual ability to act as a channel to regulate transmembrane Mg 2+ transport and also as a kinase to promote cellular signaling. Despite increasing awareness of the importance of Mg 2+ in cardiovascular biology nothing is known about TRPM7 and its kinase domain in the pathophysiology of hypertension. We previously demonstrated that Ang II regulates TRPM7 in vitro . Here we studied TRPM7 kinase-deficient mice to explore the role of the TRPM7 kinase domain in Ang II-induced hypertension. TRPM7 kinase deficient mice (TRPM7 +/- ) and wild type (WT) counterparts were infused with Ang II (400 ng/kg/min; minipumps) for 4 weeks. Blood pressure (BP) was measured by tail cuff. Vascular reactivity and structure studies were performed by myography in mesenteric arteries. Although baseline BP tended to be higher in TRPM7 +/- versus WT mice (127 ± 6.0 vs 119 ± 2.2 mmHg), significance was not achieved. TRPM7 +/- mice displayed earlier onset of BP increase by Ang II (2 weeks; WT-Ang II: 145 ± 5 vs TRPM7 +/- Ang II: 178 ± 9; mmHg). After 4 weeks, BP was significantly higher in TRPM7 +/- (174 ± 10 mmHg) than in WT mice (147 ± 8 mmHg). Ang II-induced hypertension was associated with cardiac hypertrophy, an effect that was exaggerated in TRPM7 +/- mice (WT: 4.4 ± 0.1; WT-Ang II: 5.0 ± 0.2; TRPM7 +/- : 4.5 ± 0.1; TRPM7 +/- Ang II: 5.7 ± 0.1 g/body weight). Mesenteric arteries from Ang II-infused TRPM7 +/- mice exhibited decreased sensitivity to acetylcholine (pD2; WT-Ang II: 7.6 ± 0.3 vs. TRPM7 +/- Ang II: 6.7 ± 0.4), and reduced maximal relaxation compared to WT mice (WT-Ang II: 88 ± 8% vs TRPM7 +/- Ang II: 59 ± 10%). Ang II induced a leftward shift in the stress-strain relationship for both WT and TRPM7 +/- mice in a similar fashion. Plasma analysis revealed that TRPM7 +/- mice were hypomagnesemic, and that Ang II increased Mg 2+ levels to a greater extent in WT than in TRPM7 +/- mice (WT: 0.65 ± 0.02; WT-Ang II: 0.74 ± 0.04; TRPM7 +/- : 0.60 ± 0.01; TRPM7 +/- Ang II: 0.64 ± 0.02; mmol/L). In conclusion, our findings demonstrate that hypertension, cardiac hypertrophy and endothelial dysfunction are exaggerated by Ang II in TRPM7 +/- hypomagnesemic mice, suggesting a novel role for TRPM7 kinase domain in cardiovascular pathophysiology.

Abstract 218: TRPM7 Kinase - A Novel Signaling Pathway for Ang II-induced MAPK Activation in Vascular Smooth Muscle Cells.

Transient receptor potential melastatin 7 (TRPM7) cation channel is a unique protein, which regulates transmembrane magnesium (Mg2+) transport through its channel domain, and intracellular signaling through its kinase domain. We previously demonstrated that TRPM7, is expressed on vascular smooth muscle cells (VSMC), controls Mg2+ influx, and is regulated by angiotensin II (Ang II). Low Mg2+ levels are among the many factors leading to hypertension, where intracellular Mg2+ deficiency and TRPM7 downregulation is observed. Despite the advancement in the understanding of Mg2+ and TRPM7 biology, the role of TRPM7, and its kinase, in hypertension remains elusive. In this study, we investigated whether deletion of TRPM7 kinase domain alters Ang II-induced Mg2+ influx and signaling in VSMCs. Cultured VSMCs from mesenteric arteries obtained from heterozygous mice for the TRPM7 kinase deletion (+/-) and WT mice were used. Mg2+ influx was assessed by fluorescence microscopy. Activation of MAP kinases was evaluated by immunoblotting. Ang II (10-7M) increase in Mg2+ influx was similar in VSMC from both, WT and TRPM7+/- mice (2.5-fold; p&lt;0.001). Ang II increased phosphorylation of JNK (10-fold), p38MAPK (4-fold) and ERK1/2 (6.4-fold) in VSMC from WT mice (p&lt;0.001). Ang II-induced MAPK activation was blunted in TRPM7+/- VSMC. Moreover, Ang II treatment induced PDGF-R (2-fold) and Akt phosphorylation (3-fold) in VSMC from WT mice (p&lt;0.05), an effect that was also blunted in TRPM+/- VSMC. Ang II induced SMAD2/3 activation in VSMC from WT (1.5-fold; p&lt;0.05) and, with a greater extent, TRPM7+/- mice (3.5-fold; p&lt;0.05). To understand whether the blunted effects of Ang II on intracellular signaling in VSMC from TRPM7+/- mice could be attributed to downregulation of Ang II receptors, AT1R and AT2R expression was assessed. No differences in receptor expression were observed between VSMC from WT and TRPM+/-. In conclusion, our findings identify that TRPM7 kinase domain is not necessary for Mg2+ influx, but it seems to be a novel and important player in Ang II-induced signaling in VSMCs.

Waixenicin A Inhibits Cell Proliferation through Magnesium-dependent Block of Transient Receptor Potential Melastatin 7 (TRPM7) Channels

Transient receptor potential melastatin 7 (TRPM7) channels represent the major magnesium-uptake mechanism in mammalian cells and are key regulators of cell growth and proliferation. They are expressed abundantly in a variety of human carcinoma cells controlling survival, growth, and migration. These characteristics are the basis for recent interest in the channel as a target for cancer therapeutics. We screened a chemical library of marine organism-derived extracts and identified waixenicin A from the soft coral Sarcothelia edmondsoni as a strong inhibitor of overexpressed and native TRPM7. Waixenicin A activity was cytosolic and potentiated by intracellular free magnesium (Mg(2+)) concentration. Mutating a Mg(2+) binding site on the TRPM7 kinase domain reduced the potency of the compound, whereas kinase deletion enhanced its efficacy independent of Mg(2+). Waixenicin A failed to inhibit the closely homologous TRPM6 channel and did not significantly affect TRPM2, TRPM4, and Ca(2+) release-activated Ca(2+) current channels. Therefore, waixenicin A represents the first potent and relatively specific inhibitor of TRPM7 ion channels. Consistent with TRPM7 inhibition, the compound blocked cell proliferation in human Jurkat T-cells and rat basophilic leukemia cells. Based on the ability of the compound to inhibit cell proliferation through Mg(2+)-dependent block of TRPM7, waixenicin A, or structural analogs may have cancer-specific therapeutic potential, particularly because certain cancers accumulate cytosolic Mg(2+).

9 - Lymphomas and Dysproteinaemias

The channel-kinase transient receptor potential melastatin 7 (TRPM7) is a bifunctional protein with ion channel and kinase domains. The kinase activity of TRPM7 has been linked to the regulation of a broad range of cellular activities, but little is understood as to how the channel itself is regulated by its own kinase activity. Here, using several mammalian cell lines expressing WT TRPM7 or kinase-inactive variants, we discovered that compared with the cells expressing WT TRPM7, cells in which TRPM7's kinase activity was inactivated had faster degradation, elevated ubiquitination, and increased intracellular retention of the channel. Mutational analysis of TRPM7 autophosphorylation sites further revealed a role for Ser-1360 of TRPM7 as a key residue mediating both TRPM7 stability and intracellular trafficking. Additional trafficking roles were uncovered for Ser-1403 and Ser-1567, whose phosphorylation by TRPM7's kinase activity mediated the interaction of the channel with the signaling protein 14-3-3θ. In summary, our results point to a critical role for TRPM7's kinase activity in regulating proteasome-mediated turnover of the TRPM7 channel and controlling its cellular localization in polarized epithelial cells. Overall, these findings improve our understanding of the significance of TRPM7's kinase activity for functional regulation of its channel activity.