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Transient receptor potential melastatin-like 4 (TRPM4) is a Ca(2+)-activated non-selective cation channel expressed in a wide range of human tissues. TRPM4 participates in a variety of physiological processes such as T cell activation, myogenic vasoconstriction, and allergic reactions. TRPM4 Ca(2+) sensitivity is enhanced by calmodulin (CaM) and phosphathydilinositol 4, 5-bisphosphate (PI(4,5)P2) binding, as well as, under certain conditions, PKC activation. However, information as to the mechanisms of modulation of this channel remains unknown, including direct identification of phosphorylation sites on TRPM4 and their role in channel features. Here, we use mass-spectrometric-based proteomic approaches (immunoprecipitation and tandem mass spectrometry) to unambiguously identify S839 as a phosphorylation site present on human TRPM4 expressed in a human cell line. Site-directed mutagenesis employing a serine to alanine mutation to eliminate phosphorylation, and a phospho-mimetic aspartate mutation, as well as biochemical and immunocytochemical experiments, revealed a role for S839 phosphorylation in the basolateral expression of TRPM4 channels in epithelial cells. Moreover, we demonstrated that casein kinase 1 (CK1) phosphorylates S839 and is responsible for the basolateral localization of TRPM4.

Introduction: A comprehensive pathophysiological mechanism to explain the relationship between high-salt intake and hypertension remains undefined. Evidence suggests that chloride, as the accompanying anion of sodium in dietary salt, is necessary to develop hypertension. We evaluated whether reducing dietary Cl− while keeping a standard Na+ intake modified blood pressure, cardiac hypertrophy, renal function, and vascular contractility after angiotensin II (AngII) infusion. Methods: C56BL/6J mice fed with standard Cl− diet or a low-Cl− diet (equimolar substitution of Cl− by a mixture of Na+ salts, both diets with standard Na+ content) received AngII (infusion of 1.5 mg/kg/day) or vehicle for 14 days. We measured systolic blood pressure (SBP), glomerular filtration rate (GFR), natriuretic response to acute saline load, and contractility of aortic rings from mice infused with vehicle and AngII, in standard and low-Cl− diet. Results: The mice fed the standard diet presented increased SBP and cardiac hypertrophy after AngII infusion. In contrast, low-Cl− diet prevented the increase of SBP and cardiac hypertrophy. AngII-infused mice fed a standard diet presented hampered natriuretic response to saline load, meanwhile the low-Cl− diet preserved natriuretic response in AngII-infused mice, without change in GFR. Aortic rings from mice fed with standard diet or low-Cl− diet and infused with AngII presented a similar contractile response. Conclusion: We conclude that the reduction in dietary Cl− as the accompanying anion of sodium in salt is protective from AngII pro-hypertensive actions due to a beneficial effect on kidney function and preserved natriuresis. [ABSTRACT FROM AUTHOR]

Transient Receptor Potential Melastatin 4 (TRPM4) is a Ca2+‐activated and voltage‐dependent monovalent cation channel, which depolarizes the plasma cell membrane, thereby modulating Ca2+ influx across Ca2+‐permeable pathways. TRPM4 is involved in different physiological processes such as T cell activation and the migration of endothelial and certain immune cells. Overexpression of this channel has been reported in various types of tumors including prostate cancer. In this study, a significant overexpression of TRPM4 was found only in samples from cancer with a Gleason score higher than 7, which are more likely to spread. To evaluate whether TRPM4 overexpression was related to the spreading capability of tumors, TRPM4 was knockdown by using shRNAs in PC3 prostate cancer cells and the effect on cellular migration and invasion was analyzed. PC3 cells with reduced levels of TRPM4 (shTRPM4) display a decrease of the migration/invasion capability. A reduction in the expression of Snail1, a canonical epithelial to mesenchymal transition (EMT) transcription factor, was also observed. Consistently, these cells showed a significant change in the expression of key EMT markers such as MMP9, E‐cadherin/N‐cadherin, and vimentin, indicating a partial reversion of the EMT process. Whereas, the overexpression of TRPM4 in LnCaP cells resulted in increased levels of Snail1, reduction in the expression of E‐cadherin and increase in their migration potential. This study suggests a new and indirect mechanism of regulation of migration/invasion process by TRPM4 in prostate cancer cells, by inducing the expression of Snail1 gene and consequently, increasing the EMT. TRPM4 expression and function modulate the inhibitory phosphorylation of GSK‐3β promoting the β‐Catenin stability and its transcriptional activity on several genes involved in proliferation as Myc and CCND1 and EMT markers, such as Zeb, Vimentin, and Snail1. Also, this effect could regulate the stability of Snail1, promoting the invasive phenotype thought the regulation of the EMT markers as E‐Cadherin, N‐Cadherin, MMPs, and others. Interestingly, TRPM4 it is involved directly in the focal adhesion turnover promoting the cellular migration. These finding highlight this ion channel as a new potential target for future cancer therapies. [ABSTRACT FROM AUTHOR]

Formylated peptides are chemotactic agents generated by pathogens. The most relevant peptide is fMLF (formyl-Met-Leu-Phe) which participates in several immune functions, such as chemotaxis, phagocytosis, cytokine release and generation of reactive oxygen species. In macrophages fMLF-dependent responses are dependent on both, an increase in intracellular calcium concentration and on a hyperpolarization of the membrane potential. However, the molecular entity underlying this hyperpolarization remains unknown and it is not clear whether changes in membrane potential are linked to the increase in intracellular Ca2+. In this study, differentiated U937 cells, as a macrophage-like cell model, was used to characterize the fMLF response using electrophysiological and Ca2+ imaging techniques. We demonstrate by means of pharmacological and molecular biology tools that fMLF induces a Ca2+-dependent hyperpolarization via activation of the K+ channel KCa3.1 and thus, enhancing fMLF-induced intracellular Ca2+ increase through an amplification of the driving force for Ca2+ entry. Consequently, enhanced Ca2+ influx would in turn lengthen the hyperpolarization, operating as a positive feedback mechanism for fMLF-induced Ca2+ signaling. [ABSTRACT FROM AUTHOR]

Altered expression of some members of the TRP ion channel superfamily has been associated with the development of pathologies like cancer. In particular, TRPM4 levels are reportedly elevated in diffuse large B-cell non-Hodgkin lymphoma, prostate, and cervical cancer. However, whether such changes in TRPM4 expression may be relevant to genesis or progression of cancer remains unknown. Here we show that reducing TRPM4 expression decreases proliferation of HeLa cells, a cervical cancer-derived cell line. In this cell line, constitutive TRPM4 silencing promoted GSK-3β-dependent degradation of β-catenin and reduced β-catenin/Tcf/Lef-dependent transcription. Conversely, overexpression of TRPM4 in T-REx 293 cells (a HEK293-derived cell line) increased cell proliferation and β-catenin levels. Our results identify TRPM4 as an important, unanticipated regulator of the β-catenin pathway, where aberrant signaling is frequently associated with cancer. J. Cell. Physiol. 226: 103-109, 2010. © 2010 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Volume-sensitive outwardly rectifying (VSOR) Cl- channels participate in several physiological processes such as regulatory volume decrease, cell cycle regulation, proliferation and apoptosis. Recent evidence points to a significant role of hydrogen peroxide (H2O2) in VSOR Cl- channel activation. The aim of this study was to determine the signalling pathways responsible for H2O2-induced VSOR Cl- channel activation. In rat hepatoma (HTC) cells, H2O2 elicited a transient increase in tyrosine phosphorylation of phospholipase Cγ1 (PLCγ1) that was blocked by PP2, a Src-family protein kinases inhibitor. Also, H2O2 triggered an increase in cytosolic [Ca2+] that paralleled the time course of PLCγ1 phosphorylation. The H2O2-induced [Ca2+]i rise was prevented by the generic phospholipase C (PLC) inhibitor U73122 and the inositol 1,4,5-trisphosphate-receptor (IP3R) blocker 2-APB. In line with these results, manoeuvres that prevented PLCγ1 activation and/or [Ca2+]i rise, abolished H2O2-induced VSOR Cl- currents. Furthermore, in cells that overexpress a phosphorylation-defective dominant mutant of PLCγ1, H2O2 did not induce activation of VSOR Cl- currents. All these H2O2-induced effects were independent of extracellular Ca2+. Our findings suggest that activation of PLCγ1 and subsequent Ca2+i mobilisation mediate H2O2-induced VSOR Cl- currents, indicating that H2O2 operates via redox-sensitive signalling pathways akin to those activated by osmotic challenges. Copyright © 2007 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

In a previous work the involvement of a fenamate-sensitive Ca2+activated nonselective cation channel (NSCC) in free radical-induced rat liver cell necrosis was demonstrated (5). Therefore, we studied the effect of radical oxygen species and oxidizing agents on the gating behavior of a NSCC in a liver-derived epithelial cell line (HTC). Single-channel currents were recorded in HTC cells by the excised inside-out configuration of the patch-clamp technique. In this cell line, we characterize a 19-pS Ca2+-activated, ATP- and fenamate-sensitive NSCC nearly equally permeable to monovalent cations. In the presence of Fe2+, exposure of the intracellular side of NSCC to H2O2 increased their open probability (Po) by ∼40% without affecting the unitary conductance. Desferrioxamine as well as the hydroxyl radical (·OH) scavenger MCI-186 inhibited the effect of H2O2, indicating that the increase in Po was mediated by ·OH. Exposure of the patch membrane to the oxidizing agent 5,5'-dithio-bis-2-nitrobenzoic acid (DTNB) had a similar effect to ·OH. The increase in Po induced by ·OH or DTNB was not reverted by preventing formation or by DTNB washout, respectively. However, the reducing agent dithiothreitol completely reversed the effects on Po of both ·OH and DTNB. A similar increase in Po was observed by applying the physiological oxidizing molecule GSSG. Moreover, GSSG-oxidized channels showed enhanced sensitivity to Ca2+. The effect of GSSG was fully reversed by GSH. These results suggest an intracellular site(s) of action of oxidizing agents on cysteine targets on the fenamatesensitive NSCC protein implicated in epithelial cell necrosis. [ABSTRACT FROM AUTHOR]

Cell swelling following hypoosmotic stress leads to the activation of volume-sensitive ion channels that allow a K+ and Cl− efflux accompanied by water loss. A Ca2+-insensitive K+ channel (IK,vol) has been described in Ehrlich cells that can be activated by hypotonicity and leukotriene D4 and is inhibited by clofilium. We have studied the activation and deactivation by osmotic stimuli of this channel. A G-protein appears to be involved in these processes since GTP-γ-S accelerates deactivation, while GDP-β-S blocks the channel in the open state, a result mimicked by pertussis toxin (PTX). In addition, PTX accelerates the onset of IK,vol. We propose that IK,vol is tonically inhibited by a PTX-sensitive G-protein. [Copyright &y& Elsevier]

The patch-clamp technique was used to study the effect of extracellular and intracellular iodide on the properties of the volume-activated anion current in HeLa cells. Upon hypotonic challenge, HeLa cells responded by activating an outwardly rectifying Cl– current. Replacement of extracellular Cl– by I–, a more permeable anion, increased the peak outward and inward current, reduced the magnitude of deactivation observed at depolarized potentials and shifted the half-maximal (V0.5) deactivation voltage towards more positive values. On the other hand, when internal Cl– was replaced by I– the volume-activated current was not observed in normal, Cl–-rich hypotonic extracellular solution. However, switching to a hypotonic extracellular solution containing a mixture of Cl– and I– resulted in the activation of the volume-sensitive current. Furthermore, once the current was activated, I– could be excluded from the external solution without significantly affecting the current properties. These results suggest that the permeant anion plays a crucial role in the gating mechanism of the volume-activated Cl– current, influencing the swelling-dependent activation and the voltage-dependent deactivation processes. [ABSTRACT FROM AUTHOR]

The chloride conductance (GCl,swell) that participates in the regulatory volume decrease process triggered by osmotic swelling in HeLa cells was impaired by removal of extracellular Ca2+, depletion of intracellular Ca2 stores with thapsigargin, or by preloading the cells with BAPTA-AM (1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid). Furthermore, overnight exposure to the phorbol ester tetradecanoyl phorbol acetate and acute incubation with inhibitors of the conventional protein kinase C (PKC) isoforms bisindolylmaleimide I and Gö6976 inhibited GC1,swell. Treatment of HeLa cells with U73122, a phospholipase C inhibitor, also prevented GCl,swell. Hypotonicity induced selective PKCα accumulation in the membrane/cytoskeleton fraction in fractionation experiments and translocation of a green fluorescent protein-PKCα fusion protein to the plasma membrane of transiently transfected HeLa cells. To further explore the role of PKCs in hypotonicity-induced GC1,swell , HeLa clones stably expressing either a kinase-dead dominant negative variant of the Ca2+-dependent PKC isoform α (PKCα K386R) or of the atypical PKC isoform ζ (PKCζ K275W) were generated. GC1,swell was significantly reduced in HeLa cells expressing the dominant negative PKCα mutant but remained unaltered in cells expressing dominant negative PKCζ. These findings strongly implicate PKCα as a critical regulatory element that is required for efficient regulatory volume decrease in HeLa cells. [ABSTRACT FROM AUTHOR]

Cellular swelling triggers the activation of Cl- channels (volume-sensitive outwardly rectifying (VSOR) Cl- channels) in many cell types. Ensuing regulatory volume decrease has been considered the primary function of these channels. However, Cl- channels, which share functional properties with volume-sensitive Cl- channels, have been shown to be involved in other physiological processes, including cell proliferation and apoptosis, raising the question of their physiological roles and the signal transduction pathways involved in their activation. Here we report that exogenously applied H2O2 elicited VSOR Cl- channel activation. Furthermore, activation of these channels was found to be coupled to NAD(P)H oxidase activity. Also, epidermal growth factor, known to increase H2O2 production, activated Cl- channels with properties identical to swelling-sensitive Cl- channels. It is concluded that NAD(P)H oxidasederived H2O2 is the common signal transducing molecule that mediates the activation of these ubiquitously expressed anion channels under a variety of physiological conditions. [ABSTRACT FROM AUTHOR]

Transient receptor potential melastatin-like 4 (TRPM4) is a Ca(2+)-activated non-selective cation channel expressed in a wide range of human tissues. TRPM4 participates in a variety of physiological processes such as T cell activation, myogenic vasoconstriction, and allergic reactions. TRPM4 Ca(2+) sensitivity is enhanced by calmodulin (CaM) and phosphathydilinositol 4, 5-bisphosphate (PI(4,5)P2) binding, as well as, under certain conditions, PKC activation. However, information as to the mechanisms of modulation of this channel remains unknown, including direct identification of phosphorylation sites on TRPM4 and their role in channel features. Here, we use mass-spectrometric-based proteomic approaches (immunoprecipitation and tandem mass spectrometry) to unambiguously identify S839 as a phosphorylation site present on human TRPM4 expressed in a human cell line. Site-directed mutagenesis employing a serine to alanine mutation to eliminate phosphorylation, and a phospho-mimetic aspartate mutation, as well as biochemical and immunocytochemical experiments, revealed a role for S839 phosphorylation in the basolateral expression of TRPM4 channels in epithelial cells. Moreover, we demonstrated that casein kinase 1 (CK1) phosphorylates S839 and is responsible for the basolateral localization of TRPM4.