Transient Receptor Potential Melastatin Member Research Articles

Abstract LB167: Acetalax (Oxyphenisatin acetate, NSC 59687) and bisacodyl (dulcolax) cause oncosis in triple negative breast cancer by poisoning the ion exchange membrane protein TRPM4

Abstract Triple negative breast cancer (TNBC) is generally unresponsive to current therapies, and the development of new anticancer agents is needed. Oxyphenisatin acetate, called Acetalax, which was used as a laxative in the 1950’s, has recently been reported to have anticancer activity in several murine cancer models. In this study we demonstrate that Acetalax and its structural analog laxative analog bisacodyl (Dulcolax) exhibit potent antiproliferative activity in TNBC cell lines. We show that both cause oncosis, a non-apoptotic cell death characterized by cellular and nuclear swelling and cell membrane blebbing that leads to mitochondrial dysfunction and ATP depletion. Acetalax was further shown to enhance immune and inflammatory responses and inhibit protein synthesis. Mechanistically, we provide evidence that TRPM4 (Transient Receptor Potential Melastatin member 4) is poisoned by Acetalax and bisacodyl. By blocking TRPM4, a monovalent cation channel, Acetalax and Bisacodyl cause oncosis, and TNBC cells without endogenous TRPM4 expression as well as TRPM 4 knockout TNBC cells were found to be Acetalax- and Bisacodyl resistant. TRPM4 was also lost in cells with acquired Acetalax resistance. Moreover TRPM4 is rapidly degraded the ubiquitin-proteasome system upon acute exposure to Acetalax and bisacodyl. Together, these results demonstrate that TRPM4 is a previously unknown target of Acetalax and Bisacodyl and that TRPM4 expression in cancer cells is a predictor of Acetalax and Bisacodyl efficacy, and could be used for the clinical development of these drugs as anticancer agents. Citation Format: Makito Mizunuma, Christophe Redon, Liton Saha, Naoko Takebe, Yves G. Pommier. Acetalax (Oxyphenisatin acetate, NSC 59687) and bisacodyl (dulcolax) cause oncosis in triple negative breast cancer by poisoning the ion exchange membrane protein TRPM4 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB167.

Abstract P6-10-01: The Executioner Protein for Immunogenic Anticancer Drug-induced Necrosis in ER Positive Breast Cancer is Transient Receptor Potential Melastatin Member 4

Abstract Immunotherapy has dramatically impacted cancer therapy, but it has been challenging to apply immunotherapy to estrogen receptor (ER) positive breast cancer and many other solid tumors that do not display neoantigens. One way to target these tumors is to induce necrosis, which robustly activates immune cells, inducing immunogenic cell death. However, anticancer therapy-induced necrosis was primarily characterized by morphological changes, and the molecular drivers of necrosis were largely obscure. To probe necrosis, we used our necrosis inducing anticancer agents, the small molecules BHPI and second-generation ErSO, which kill cancer cells by hyperactivating the anticipatory unfolded protein response (a-UPR). In orthotopic mouse xenografts, ErSO induces complete regression without recurrence of large, therapy-resistant primary ER positive breast tumors, of most lung, bone, and liver metastases, near complete regression of challenging breast cancer brain metastases and robust responses in PDX and patient derived organoids (PDOs) models. ErSO also induces complete or near complete regression in mouse xenograft models of ER positive ovarian and endometrial cancer. Using genome wide CRISPR-Cas9 screens with negative selection against our necrosis-inducing a-UPR hyperactivators, BHPI and ErSO, we identified the calcium-activated, ATP-inhibited, plasma membrane sodium channel, Transient Receptor Potential Melastatin Member 4 (TRPM4) as critical for anticancer therapy induced necrosis. TRPM4 knockout in multiple models abolished ErSO-induced ATP depletion, sustained UPR activation, cell swelling, necrotic cell death and increased migration of immune cells. Notably, knockout of TRPM4 completely abolished the ability of ErSO to induce regression of ER positive breast tumors in mice. Supporting a broad role for the TRPM4 pathway in anticancer therapy induced necrosis, rapid cancer cell death induced by four necrosis-inducing cancer therapies unrelated to ErSO, that range from FDA-approved to preclinical, is strongly reversed by TRPM4 knockout. ErSO treatment induces migration of macrophage into regressing tumors. Medium from cancer cells killed by necrosis-inducing ErSO, but not by an apoptosis inducer, dramatically increases macrophage migration and activation, as shown by induction of pro-inflammatory cytokines. This work identifies a protein that plays a pivotal role in the action of diverse anticancer therapies inducing immunogenic necrosis. Since increasing levels of TRPM4 increase sensitivity of breast cancer cells to killing by ErSO, TRPM4 is a novel biomarker whose levels can be used to identify patients most likely to benefit from ErSO and other necrosis-inducing cancer therapies. Citation Format: Santanu Ghosh, Rachel Yang, Darjan Duraki, Ji Eun Kim, Junyao Zhu, Mara Livezey, Matthew Boudreau, Ben H. Park, TImothy Fan, Erik R. Nelson, Paul J. Hergenrother, David J. Shapiro. The Executioner Protein for Immunogenic Anticancer Drug-induced Necrosis in ER Positive Breast Cancer is Transient Receptor Potential Melastatin Member 4 [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-10-01.

Association of TRPM5 Asn235Ser Polymorphism and Trace Elements/Minerals in Chronic Gastritis Patients: a Case-Control Study.

The link between chronic gastritis and chemosensory receptors is considered promising for disease prediction and treatment. The transient receptor potential melastatin member channel 5 (TRPM5) is an ion channel and may be a chemosensor in the gastrointestinal tract. Trace elements and minerals involved in many protein structures can change ion channel activity. Our study aimed to determine trace element and mineral levels according to TRPM5 Asn235Ser missense polymorphism distributions in patients with H. pylori (+) and (-) gastritis and to uncover their possible association with disease pathogenesis. In 109 volunteer patients diagnosed with gastritis [64 H. pylori (+), 45 H. pylori (-)], TRPM5 (Asn235Ser) polymorphism was detected by Kompetitive Allele-Specific PCR method (KASP), but no difference was found between the groups. There are differences between the serum trace element (Se, Zn, Cu, Mg, and Cu/Zn) levels of the two groups. High serum Se and Cu/Zn ratios were detected in H. pylori (+) patients. Cu/Zn ratio can be used as a marker of bacterial inflammation. The amount of Se was found to be statistically significant in the serums of H. pylori (+) patients carrying TT and CT genotype, while the amounts of Zn, Cu, and Mg were found as significant of H. pylori (-) patients. The relationship of Se with the H. pylori needs to be investigated further. In addition, H. pylori (+) patients have a significantly higher neutrophils/lymphocytes ratio (NLR) compared to those with H. pylori (-) NLR can be used as an indicator of inflammation.

Species-Specific Effects of Cation Channel TRPM4 Small-Molecule Inhibitors.

Background: The Transient Receptor Potential Melastatin member 4 (TRPM4) gene encodes a calcium-activated non-selective cation channel expressed in several tissues. Mutations in TRPM4 have been reported in patients with different types of cardiac conduction defects. It is also linked to immune response and cancers, but the associated molecular mechanisms are still unclear. Thus far, 9-phenanthrol is the most common pharmacological compound used to investigate TRPM4 function. We recently identified two promising aryloxyacyl-anthranilic acid compounds (abbreviated CBA and NBA) inhibiting TRPM4. However, all aforementioned compounds were screened using assays expressing human TRPM4, whereas the efficacy of mouse TRPM4 has not been assessed. Mouse models are essential to investigate ion channel physiology and chemical compound efficacy. Aim: In this study, we performed comparative electrophysiology experiments to assess the effect of these TRPM4 inhibitors on human and mouse TRPM4 channels heterologously expressed in TsA-201 cells. Methods and Results: We identified striking species-dependent differences in TRPM4 responses. NBA inhibited both human and mouse TRPM4 currents when applied intracellularly and extracellularly using excised membrane patches. CBA inhibited human TRPM4, both intracellularly and extracellularly. Unexpectedly, the application of CBA had no inhibiting effect on mouse TRPM4 current when perfused on the extracellular side. Instead, its increased mouse TRPM4 current at negative holding potentials. In addition, CBA on the intracellular side altered the outward rectification component of the mouse TRPM4 current. Application of 9-phenanthrol, both intracellularly and extracellularly, inhibited human TRPM4. For mouse TRPM4, 9-phenanthrol perfusion led to opposite effects depending on the site of application. With intracellular 9-phenanthrol, we observed a tendency towards potentiation of mouse TRPM4 outward current at positive holding potentials. Conclusion: Altogether, these results suggest that pharmacological compounds screened using “humanised assays” should be extensively characterised before application in vivo mouse models.

Deletion of Trpm4 Alters the Function of the Nav1.5 Channel in Murine Cardiac Myocytes.

Transient receptor potential melastatin member 4 (TRPM4) encodes a Ca2+-activated, non-selective cation channel that is functionally expressed in several tissues, including the heart. Pathogenic mutants in TRPM4 have been reported in patients with inherited cardiac diseases, including conduction blockage and Brugada syndrome. Heterologous expression of mutant channels in cell lines indicates that these mutations can lead to an increase or decrease in TRPM4 expression and function at the cell surface. While the expression and clinical variant studies further stress the importance of TRPM4 in cardiac function, the cardiac electrophysiological phenotypes in Trpm4 knockdown mouse models remain incompletely characterized. To study the functional consequences of Trpm4 deletion on cardiac electrical activity in mice, we performed perforated-patch clamp and immunoblotting studies on isolated atrial and ventricular cardiac myocytes and surfaces, as well as on pseudo- and intracardiac ECGs, either in vivo or in Langendorff-perfused explanted mouse hearts. We observed that TRPM4 is expressed in atrial and ventricular cardiac myocytes and that deletion of Trpm4 unexpectedly reduces the peak Na+ currents in myocytes. Hearts from Trpm4−/− mice presented increased sensitivity towards mexiletine, a Na+ channel blocker, and slower intraventricular conduction, consistent with the reduction of the peak Na+ current observed in the isolated cardiac myocytes. This study suggests that TRPM4 expression impacts the Na+ current in murine cardiac myocytes and points towards a novel function of TRPM4 regulating the Nav1.5 function in murine cardiac myocytes.

TRPM Channels in Human Diseases.

The transient receptor potential melastatin (TRPM) subfamily belongs to the TRP cation channels family. Since the first cloning of TRPM1 in 1989, tremendous progress has been made in identifying novel members of the TRPM subfamily and their functions. The TRPM subfamily is composed of eight members consisting of four six-transmembrane domain subunits, resulting in homomeric or heteromeric channels. From a structural point of view, based on the homology sequence of the coiled-coil in the C-terminus, the eight TRPM members are clustered into four groups: TRPM1/M3, M2/M8, M4/M5 and M6/M7. TRPM subfamily members have been involved in several physiological functions. However, they are also linked to diverse pathophysiological human processes. Alterations in the expression and function of TRPM subfamily ion channels might generate several human diseases including cardiovascular and neurodegenerative alterations, organ dysfunction, cancer and many other channelopathies. These effects position them as remarkable putative targets for novel diagnostic strategies, drug design and therapeutic approaches. Here, we review the current knowledge about the main characteristics of all members of the TRPM family, focusing on their actions in human diseases.

TRIM4 interacts with TRPM8 and regulates its channel function through K423-mediated ubiquitination.

Transient receptor potential melastatin member 8 (TRPM8), a Ca2+ -permeable nonselective cation channel activated by cold and cooling agents, mediates allodynia. Dysfunction or abnormal expression of TRPM8 has been found in several human cancers. The role of ubiquitination in the regulation of TRPM8 function remains poorly understood. Here, we identified the ubiquitin (Ub)-ligase E3, tripartite motif-containing 4 (TRIM4), as a novel interaction partner of TRPM8 and confirmed that the TRIM4-TRPM8 interaction was mediated through the SPRY domain of TRIM4. Patch-clamp assays showed that TRIM4 negatively regulates TRPM8-mediated currents in HEK293 cells. Moreover, TRIM4 reduced the expression of TRPM8 on the cell surface by promoting the K63-linked ubiquitination of TRPM8. Further analyses revealed that the TRPM8 N-terminal lysine residue at 423 was the major ubiquitination site that mediates its functional regulation by TRIM4. A Ub-activating enzyme E1, Ub-like modifier-activating enzyme 1 (UBA1), was also found to interact with TRPM8, thereby regulating its channel function and ubiquitination. In addition, knockdown of UBA1 impaired the regulation of TRPM8 ubiquitination and function by TRIM4. Thus, this study demonstrates that TRIM4 downregulates TRPM8 via K423-mediated TRPM8 ubiquitination and requires UBA1 to regulate TRPM8.

Testosterone-androgen receptor: The steroid link inhibiting TRPM8-mediated cold sensitivity.

Recent studies have revealed gender differences in cold perception, and pointed to a possible direct action of testosterone (TST) on the cold-activated TRPM8 (Transient Receptor Potential Melastatin Member 8) channel. However, the mechanisms by which TST influences TRPM8-mediated sensory functions remain elusive. Here, we show that TST inhibits TRPM8-mediated mild-cold perception through the noncanonical engagement of the Androgen Receptor (AR). Castration of both male rats and mice increases sensitivity to mild cold, and this effect depends on the presence of intact TRPM8 and AR. TST in nanomolar concentrations suppresses whole-cell TRPM8-mediated currents and single-channel activity in native dorsal root ganglion (DRG) neurons and HEK293 cells co-expressing recombinant TRPM8 and AR, but not TRPM8 alone. AR cloned from rat DRGs shows no difference from standard AR. However, biochemical assays and confocal imaging reveal the presence of AR on the cell surface and its interaction with TRPM8 in response to TST, leading to an inhibition of channel activity.

TRPM6 N-Terminal CaM- and S100A1-Binding Domains.

Transient receptor potential (TRPs) channels are crucial downstream targets of calcium signalling cascades. They can be modulated either by calcium itself and/or by calcium-binding proteins (CBPs). Intracellular messengers usually interact with binding domains present at the most variable TRP regions—N- and C-cytoplasmic termini. Calmodulin (CaM) is a calcium-dependent cytosolic protein serving as a modulator of most transmembrane receptors. Although CaM-binding domains are widespread within intracellular parts of TRPs, no such binding domain has been characterised at the TRP melastatin member—the transient receptor potential melastatin 6 (TRPM6) channel. Another CBP, the S100 calcium-binding protein A1 (S100A1), is also known for its modulatory activities towards receptors. S100A1 commonly shares a CaM-binding domain. Here, we present the first identified CaM and S100A1 binding sites at the N-terminal of TRPM6. We have confirmed the L520-R535 N-terminal TRPM6 domain as a shared binding site for CaM and S100A1 using biophysical and molecular modelling methods. A specific domain of basic amino acid residues (R526/R531/K532/R535) present at this TRPM6 domain has been identified as crucial to maintain non-covalent interactions with the ligands. Our data unambiguously confirm that CaM and S100A1 share the same binding domain at the TRPM6 N-terminus although the ligand-binding mechanism is different.

Ligands Exert Biased Activity to Regulate Sigma 1 Receptor Interactions With Cationic TRPA1, TRPV1, and TRPM8 Channels.

The sigma 1 receptor (σ1R) and the mu-opioid receptor (MOR) regulate the transient receptor potential (TRP) V1 calcium channel. A series of proteins are involved in the cross-regulation between MORs and calcium channels like the glutamate N-methyl-D-aspartate receptor (NMDAR), including the histidine triad nucleotide-binding protein 1 (HINT1), calmodulin (CaM), and the σ1R. Thus, we assessed whether similar mechanisms also apply to the neural TRP ankyrin member 1 (TRPA1), TRP vanilloid member 1 (TRPV1), and TRP melastatin member 8 (TRPM8). Our results indicate that σ1R and CaM bound directly to cytosolic regions of these TRPs, and this binding increased in the presence of calcium. By contrast, the association of HINT1 with these TRPs was moderately dependent on calcium. The σ1R always competed with CaM for binding to the TRPs, except for its binding to the TRPA1 C-terminal where σ1R binding cooperated with that of CaM. However, σ1R dampened HINT1 binding to the TRPA1 N-terminal. When the effect of σ1R ligands was addressed, the σ1R agonists PRE084 and pregnenolone sulfate enhanced the association of the σ1R with the TRPM8 N-terminal and TRPV1 C-terminal in the presence of physiological calcium, as seen for the σ1R–NMDAR interactions. However, these agonists dampened σ1R binding to the TRPA1 and TRPV1 N-terminal domains, and also to the TRPA1 C-terminal, as seen for σ1R–binding immunoglobulin protein (BiP) interactions in the endoplasmic reticulum (ER). By contrast, the σ1R antagonists progesterone and S1RA reduced the association of σ1R with TRPA1 and TRPV1 C-terminal regions, as seen for the σ1R–NMDAR interactions. Conversely, they enhanced the σ1R interaction with the TRPA1 N-terminal, as seen for σ1R–BiP interactions, whereas they barely affected the association of σ1R with the TRPV1 N-terminal. Thus, depending on the calcium channel and the cytosolic region examined, the σ1R agonists pregnenolone sulfate and PRE084 opposed or collaborated with the σ1R antagonists progesterone and S1RA to disrupt or promote such interactions. Through the use of cloned cytosolic regions of selected TRP calcium channels, we were able to demonstrate that σ1R ligands exhibit biased activity to regulate particular σ1R interactions with other proteins. Since σ1Rs are implicated in essential physiological processes, exploiting such ligand biases may represent a means to develop more selective and efficacious pharmacological interventions.

TRPM4 and TRPM5 Channels Share Crucial Amino Acid Residues for Ca2+ Sensitivity but Not Significance of PI(4,5)P2.

Transient receptor potential melastatin member 4 (TRPM4) and 5 (TRPM5) channels are Ca2+-activated nonselective cation channels. Intracellular Ca2+ is the most important regulator for them to open, though PI(4,5)P2, a membrane phosphoinositide, has been reported to regulate their Ca2+-sensitivities. We previously reported that negatively-charged amino acid residues near and in the TRP domain are necessary for the normal Ca2+ sensitivity of TRPM4. More recently, a cryo-electron microscopy structure of Ca2+-bound (but closed) TRPM4 was reported, proposing a Ca2+-binding site within an intracellular cavity formed by S2 and S3. Here, we examined the functional effects of mutations of the amino acid residues related to the proposed Ca2+-binding site on TRPM4 and also TRPM5 using mutagenesis and patch clamp techniques. The mutations of the amino acid residues of TRPM4 and TRPM5 reduced their Ca2+-sensitivities in a similar way. On the other hand, intracellular applications of PI(4,5)P2 recovered Ca2+-sensitivity of desensitized TRPM4, but its effect on TRPM5 was negligible. From these results, the Ca2+-binding sites of TRPM4 and TRPM5 were shown to be formed by the same amino acid residues by functional analyses, but the impact of PI(4,5)P2 on the regulation of TRPM5 seemed to be smaller than that on the regulation of TRPM4.

Cool mechanism for sensing cool

Structural Biology In humans, cold is primarily sensed by transient receptor potential melastatin member 8 (TRPM8), a calcium channel. Yin et al. present cryo–electron microscopy structures of TRPM8 with cooling agents, membrane lipid phosphatidylinositol-4,5-bisphosphate (PIP2), and calcium. Structural and functional analyses showed that the PIP2 binding site in TRPM8 is completely different from PIP2 sites in other TRP channels. The binding of PIP2 and cooling agents allosterically enhance each other and activate the channel opening. Thus, the activation mechanism of TRPM8 is distinct from that used by other TRP channels. Science , this issue p. [eaav9334][1] [1]: /lookup/doi/10.1126/science.aav9334

Structural basis of cooling agent and lipid sensing by the cold-activated TRPM8 channel.

Transient receptor potential melastatin member 8 (TRPM8) is a calcium ion (Ca2+)-permeable cation channel that serves as the primary cold and menthol sensor in humans. Activation of TRPM8 by cooling compounds relies on allosteric actions of agonist and membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2), but lack of structural information has thus far precluded a mechanistic understanding of ligand and lipid sensing by TRPM8. Using cryo-electron microscopy, we determined the structures of TRPM8 in complex with the synthetic cooling compound icilin, PIP2, and Ca2+, as well as in complex with the menthol analog WS-12 and PIP2 Our structures reveal the binding sites for cooling agonists and PIP2 in TRPM8. Notably, PIP2 binds to TRPM8 in two different modes, which illustrate the mechanism of allosteric coupling between PIP2 and agonists. This study provides a platform for understanding the molecular mechanism of TRPM8 activation by cooling agents.

Propagation Failure by TRPM4 Overexpression

Transient receptor potential melastatin member 4 (TRPM4) channels are nonselective monovalent cationic channels found in human atria and conduction system. Overexpression of TRPM4 channels has been found in families suffering from inherited cardiac arrhythmias, notably heart block. In this study, we integrate a mathematical formulation of the TRPM4 channel into a Purkinje cell model (Pan-Rudy model). Instead of simply adding the channel to the model, a combination of existing currents equivalent to the TRPM4 current was constructed, based on TRPM4 current dynamics. The equivalent current was then replaced by the TRPM4 current to preserve the model action potential. Single-cell behavior showed early afterdepolarizations for increases in TRPM4 channel expression above twofold. In a homogeneous strand of tissue, propagation conducted faithfully for lower expression levels but failed completely for more than a doubling of TRPM4 channel expression. Only with a heterogeneous distribution of channel expression was intermittent heart block seen. This study suggests that in Purkinje fibers, TRPM4 channels may account for sodium background current (INab), and that a heterogeneous expression of TRPM4 channels in the His/Purkinje system is required for type II heart block, as seen clinically.

The vesicular transfer of CLIC1 from glioblastoma to microvascular endothelial cells requires TRPM7.

Chloride intracellular channel 1 (CLIC1) is highly expressed and secreted by human glioblastoma cells and cell lines such as U87, initiating cell migration and tumor growth. Here, we examined whether CLIC1 could be transferred to human primary microvascular endothelial cells (HMEC). We previously reported that the oncogenic microRNA, miR-5096, increased the release of extracellular vesicles (EVs) by which it increased its own transfer from U87 to surrounding cells. Thus, we also examined its effect on the CLIC1 transfer. In homotypic cultures, miR-5096 did not increase the expression of CLIC1 in U87 nor in HMEC. However, the endothelial CLIC1 level increased after exposure to EVs released by U87, and even more by miR-5096-loaded U87. The EVs-transferred CLIC1 was active in HMEC, promoting endothelial sprouting in matrigel. Cell exposure to EVs induced cytosolic Ca2+ spikes which were dependent on the transient receptor potential melastatin member 7 (TRPM7). TRPM7 silencing prevented Ca2+ spikes and the subsequent CLIC1 delivery into HMEC. Our data suggest that the vesicular transfer of CLIC1 between cells requires TRMP7 expression in recipient endothelial cells. How the vesicular transfer of CLIC1 is modulated in cancer therapy is a future challenge.

The ion channel TRPM4 in murine experimental autoimmune encephalomyelitis and in a model of glutamate-induced neuronal degeneration

Transient receptor potential melastatin member 4 (TRPM4), a Ca2+-activated nonselective cation channel, has been found to mediate cell membrane depolarization in immune response, insulin secretion, cardiovascular diseases, and cancer. In murine experimental autoimmune encephalomyelitis (EAE), TRPM4 deletion and administration of glibenclamide were found to ameliorate clinical symptoms and attenuate disease progression. However, the exact role of TRPM4 in EAE, as well as the molecular mechanisms underlining TRPM4 contribution in EAE, remain largely unclear. In the present study, EAE was induced in WT C57BL/6 N mice using myelin oligodendrocyte glycoprotein 35–55 (MOG35–55) and TRPM4 protein and mRNA expression were examined in spinal cord membrane extracts. Our results showed that TRPM4 protein and mRNA are upregulated in EAE, and that their upregulation correlated with disease progression. Moreover, newly-developed TRPM4 inhibitors, named compound 5 and compound 6, were shown to exert a better neuroprotection compared to currently used TRPM4 inhibitors in an in vitro model of glutamate-induced neurodegeneration. These results support the hypothesis that TRPM4 is crucial from early stages of EAE, and suggest that these more potent TRPM4 inhibitors could be used as novel protective therapeutic tools in glutamate-induced neurodegeneration.

Aberrant Deactivation-Induced Gain of Function in TRPM4 Mutant Is Associated with Human Cardiac Conduction Block

A gain-of-function mutation in the Ca2+-activated transient receptor potential melastatin member 4 (TRPM4A432T) is linked to life-threatening cardiac conduction disturbance, but the underlying mechanism is unclear. For deeper insights, we used photolysis of caged Ca2+, quantitative Ca2+, and electrophysiological measurements. TRPM4A432T's 2-fold larger membrane current was associated with 50% decreased plasma membrane expression. Kinetic analysis unveiled 4-fold slower deactivation that was responsible for the augmented membrane current progressively rising during repetitive human cardiac action potentials. Rational mutagenesis of TRPM4 at position 432 revealed that the bulkiness of the amino acid was key to TRPM4A432T's aberrant gating. Charged amino acids rendered the channel non-functional. The slow deactivation caused by an amino acid substitution at position 432 from alanine to the bulkier threonine represents a key contributor to the gain of function in TRPM4A432T. Thus, our results add a mechanism in the etiology of TRP channel-linked human cardiac channelopathies.

Four TRPM4 Cation Channel Mutations Found in Cardiac Conduction Diseases Lead to Altered Protein Stability.

Transient receptor potential melastatin member 4 (TRPM4), a non-selective cation channel, mediates cell membrane depolarization in immune response, insulin secretion, neurological disorders, and cancer. Pathological variants in TRPM4 gene have been linked to several cardiac phenotypes such as complete heart block (CHB), ventricular tachycardia, and Brugada syndrome (BrS). Despite recent findings regarding the functional implications of TRPM4 in cardiac diseases, the molecular and cellular mechanisms leading to altered conduction are poorly understood. In the present study, we identify and characterize four novel TRPM4 variants found in patients with CHB or ventricular fibrillation. Three of them, p.A101T, p.S1044C and a double variant p.A101T/P1204L, led to a decreased expression and function of the channel. On the contrary, the variant p.Q854R showed an increase in TRPM4 current. Recent evidence indicates that altered degradation rate of mutant proteins represents a pathogenic mechanism underlying genetic diseases. In consequence, protein turnover of WT-TRPM4 and TRPM4 variants overexpressed in HEK293 cells was analyzed using cycloheximide, an inhibitor of protein biosynthesis. Upon addition of cycloheximide, WT-TRPM4 decayed with a half-life of ~20 h, while loss-of-expression variants showed a ~30% increase in degradation rate, with a half-life close to 12 h. Together, the gain-of-expression variant showed a higher stability and a doubled half-life compared to WT-TRPM4. In conclusion, decreased or increased protein expression of several TRPM4 variants linked to cardiac conduction disorders or ventricular arrhythmias were found to be caused by altered TRPM4 half-life compared to the WT form.

Down-regulation of transient receptor potential melastatin member 7 prevents migration and invasion of renal cell carcinoma cells via inactivation of the Src and Akt pathway.

PurposeTransient receptor potential melastatin member 7 (TRPM7), an ion channel and serine/threonine protein kinase, has been linked with distinct human malignancies. However, the role of TRPM7 in renal cell carcinoma (RCC) has not been investigated. The aim of this study is to determine whether TRPM7 regulates the migration and invasion of RCC cells. Its relationship with signal transduction pathways was also studied.Materials and MethodsThe human RCC cell lines ACHN and SN12C were chosen for this study. The molecular mechanisms of TRPM7 action were studied using Western blot analysis and small interfering RNA (siRNA)-based knockdown. The effect of TRPM7 knockdown on RCC cells was measured by using Transwell invasion and wound healing migration assays.ResultssiRNA-induced silencing of TRPM7 notably decreased the migration and invasion of ACHN and SN12C RCC cells. The phosphorylation levels of Src in both cells were obviously reduced after TRPM7 silencing compared with that of the control ACHN and SN12C cells. Furthermore, the phosphorylation levels of Akt were greatly decreased in ACHN cells after siRNA-induced knockdown of TRPM7. Additionally, the treatment of cells with Src and Akt inhibitors clearly limited the migration and invasion of RCC cells.ConclusionsOur data show that TRPM7 regulated ACHN and SN12C RCC cell invasion via the Src/Akt signaling pathway. Therefore, targeting the Src/Akt signaling pathway and/or the expression or function of TRPM7 could be a potential beneficial treatment for patients with RCC.

Inhibition of TRPM7 suppresses cell proliferation of colon adenocarcinoma in vitro and induces hypomagnesemia in vivo without affecting azoxymethane-induced early colon cancer in mice

BackgroundMagnesium (Mg2+) is an essential cation implicated in carcinogenesis, solid tumor progression and metastatic potential. The Transient Receptor Potential Melastatin Member 7 (TRPM7) is a divalent ion channel involved in cellular and systemic Mg2+ homeostasis. Abnormal expression of TRPM7 is found in numerous cancers, including colon, implicating TRPM7 in this process.MethodsTo establish a possible link between systemic magnesium (Mg2+) status, the Mg2+ conducting channel TRPM7 in colon epithelial cells, and colon carcinogenesis, in vitro whole-cell patch clamp electrophysiology, qPCR, and pharmacological tools were used probing human colorectal adenocarcinoma HT-29 as well as normal primary mouse colon epithelial cells. This was extended to and combined with aberrant crypt foci development in an azoxymethane-induced colorectal cancer mouse model under hypomagnesemia induced by diet or pharmacologic intervention.ResultsWe find that TRPM7 drives colon cancer cell proliferation in human HT-29 and expresses in normal primary mouse colon epithelia. This is linked to TRPM7’s dominant role as Mg2+ transporter, since high extracellular Mg2+ supplementation cannot rescue inhibition of cell proliferation caused by suppressing TRPM7 either genetically or pharmacologically. In vivo experiments in mice provide evidence that the specific TRPM7 inhibitor waixenicin A, given as a single bolus injection, induces transient hypomagnesemia and increases intestinal absorption of calcium. Repeated injections of waixenicin A over 3 weeks cause hypomagnesemia via insufficient Mg2+ absorption by the colon. However, neither waixenicin A, nor a diet low in Mg2+, affect aberrant crypt foci development in an azoxymethane-induced colorectal cancer mouse model.ConclusionEarly stage colon cancer proceeds independent of systemic Mg2+ status and TRPM7, and waixenicin A is a useful pharmacological tool to study of TRPM7 in vitro and in vivo.