TRPM7 Channel Activity Research Articles

Mechanism of TRPM7 Channel Inhibition by 2-Aminoethyl Diphenyl Borinate (2-APB)

2-APB is a widely used chemical compound in ion channel research. It inhibits numerous channels that include inositol 1,4,5-trisphosphate receptors, store-operated calcium channels, connexins and TRP family cation channels TRPC3, TRPC5, TRPC6, TRPM2, TRPM3 and TRPM7. Native and overexpressed TRPM7 channels are inhibited by 2-APB with IC50-s in the 70-170 μM range. A characteristic of TRPM7 channels is their inhibition by intracellular Mg2+ and acidic pH. using patch-clamp electrophysiology, we recorded native TRPM7 channel activity in Jurkat T lymphocytes and tested 2-APB at 10-300 μM for its ability to inhibit TRPM7 currents. When internal HEPES buffer concentration was low (1 mM), 100-300 μM 2-APB inhibited 60-70% of TRPM7 current with a slow time course. This inhibition was voltage-independent and reversible. Increasing the pH buffering capacity of internal solution to 140 mM HEPES abolished the inhibitory 2-APB effect. Simply making the internal recording solution alkaline, greatly diminished 2-APB inhibition. using single-cell pHi imaging with the fluorescent pH indicator BCECF, we found that at concentrations of 100 μM and higher, 2-APB potently acidifies the cytoplasm. A similar 2-APB-induced acidification was also observed in HEK293 cells, often used for TRPM7 channel overexpression. In contrast to 2-APB, its analog 2,2-diphenyltetrahydrofuran (DPTHF) did not produce cytosolic acidification, when applied at concentrations of 150-300 μM. We also found that Kv1.3 channels expressed in Jurkat T cells are modulated by 2-APB: the apparent voltage dependence of these channels was reversibly altered by the compound. Interestingly, this 2-APB effect on Kv1.3 channels did not depend on cytosolic pH. In summary, these experiments suggest that inhibition of TRPM7 channels by 2-APB is not direct but rather, can be explained by cytoplasmic acidification and resulting channel inhibition.

Phbylation Sites of the TRPM8 Protein: Implications to the Channel Structure and Function

The TRPM8 ion channel is expressed in sensory neurons and responsible for sensing environmental cold temperatures and a number of chemical compounds, such as menthol and icilin. The functional activity of the channel is regulated by various physical and chemical factors, and is likely to be pre-conditioned by its molecular composition. Our studies uncovered that TRPM8 channel forms a structural- functional complex with the polyester - poly-(R)-3-hydroxybutyrate (PHB). With mass spectrometric analysis, we identified a number of PHB-modified peptides in the N- terminus of the TRPM8 protein and in its extracellular S3-S4 linker. Removal of PHB by enzymatic hydrolysis, as well as site-directed mutagenesis of the covalent anchors of PHB - serine residues, and adjacent hydrophobic residues interacting with the methyl groups of the polymer, resulted in significant inhibition of TRPM8 channel activity. We conclude that TPRM8 channel undergoes posttranslational modification by PHB and that this modification is required for its normal functions.

Dual Effect of Phosphatidyl (4,5)-Bisphosphate PIP2 on Shaker K+ Channels

Dual Effect of Phosphatidyl (4,5)-Bisphosphate PIP2 on Shaker K+ Channels

Natural and synthetic modulators of SK (Kca2) potassium channels inhibit magnesium‐dependent activity of the kinase‐coupled cation channel TRPM7

Transient receptor potential cation channel subfamily M member 7 (TRPM7) is a bifunctional protein comprising a TRP ion channel segment linked to an α-type protein kinase domain. TRPM7 is essential for proliferation and cell growth. Up-regulation of TRPM7 function is involved in anoxic neuronal death, cardiac fibrosis and tumour cell proliferation. The goal of this work was to identify non-toxic inhibitors of the TRPM7 channel and to assess the effect of blocking endogenous TRPM7 currents on the phenotype of living cells. We developed an aequorin bioluminescence-based assay of TRPM7 channel activity and performed a hypothesis-driven screen for inhibitors of the channel. The candidates identified were further assessed electrophysiologically and in cell biological experiments. TRPM7 currents were inhibited by modulators of small conductance Ca²⁺ -activated K⁺ channels (K(Ca)2.1-2.3; SK) channels, including the antimalarial plant alkaloid quinine, CyPPA, dequalinium, NS8593, SKA31 and UCL 1684. The most potent compound NS8593 (IC₅₀ 1.6 µM) specifically targeted TRPM7 as compared with other TRP channels, interfered with Mg²⁺ -dependent regulation of TRPM7 channel and inhibited the motility of cultured cells. NS8593 exhibited full and reversible block of native TRPM7-like currents in HEK 293 cells, freshly isolated smooth muscle cells, primary podocytes and ventricular myocytes. This study reveals a tight overlap in the pharmacological profiles of TRPM7 and K(Ca)2.1-2.3 channels. NS8593 acts as a negative gating modulator of TRPM7 and is well-suited to study functional features and cellular roles of endogenous TRPM7.

Detailed examination of Mg2+ and pH sensitivity of human TRPM7 channels

TRPM7 channel kinase is a protein highly expressed in cells of hematopoietic lineage, such as lymphocytes. Studies performed in native and heterologous expression systems have shown that TRPM7 forms nonselective cation channels functional in the plasma membrane and activated on depletion of cellular Mg(2+). In addition to internal Mg(2+), cytosolic pH and the phospholipid phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P(2)] are potent physiological regulators of this channel: protons inhibit, while PI(4,5)P(2) is required for TRPM7 channel activity. These channels are also inhibited from inside by other metal cations and polyamines. While the regulation of TRPM7 channels by internal metal ions, acidic pH, and PI(4,5)P(2) is voltage independent, extracellular metal cations and polyamines block voltage dependently at micromolar concentrations and appear to occupy a distinct blocking site. In the present study we investigated intracellular Mg(2+) and pH dependence of native TRPM7 currents using whole cell patch-clamp electrophysiology in human Jurkat T lymphocytes and HEK293 cells. Our main findings are 1) Mg(2+) inhibition involves not one but two separate sites of high (∼10 μM) and low (∼165 μM) affinity; and 2) while sharing certain characteristics with Mg(2+) inhibition, protons most likely inhibit through one inhibitory site, corresponding to the low-affinity Mg(2+) site, with an estimated IC(50) of pH 6.3. Additionally, we present data on amplitude distribution of preactivated TRPM7 currents in Jurkat T lymphocytes in the absence of prior Mg(2+) or proton depletion.

TRPM2 Channel Membrane Currents in Primary Rat Megakaryocytes Were Activated by the Agonist ADP-Ribose but Not Oxidative Stress

Melastatin-like transient receptor potential 2 (TRPM2) channel activation/inhibition mechanisms in response to ADP-ribose (ADPR), oxidative stress, flufenamic acid (FFA) and 2-aminoethoxydiphenyl borate (2-APB) are not clear. We tested the effects of FFA and 2-APB on ADPR-induced TRPM2 cation channel currents in rat native bone marrow megakaryocytes. Megakaryocyte cells were freshly isolated from rat bone marrow and studied with the conventional whole-cell patch-clamp technique. Extracellular H2O2, FFA and 2-APB were added through the patch chamber, while intracellular ADPR was applied through the pipette. Nonselective cation currents were consistently induced by ADPR but not H2O2. Current density of ADPR in the cells was significantly (P<0.001) higher than in control. The time courses of ADPR effects in the megakaryocytes were characterized by a delay of 2.24±0.73. The ADPR-induced Ca2+ gate was not blocked by either the IP3 receptor inhibitor 2-APB or the PLC inhibitor FFA. In conclusion, TRPM2 channels were constitutively activated by intracellular ADPR, although the channel currents in rat native megakaryocytes were not affected by extracellular H2O2, 2-APB or FFA. Activation of TRPM2 channels in megakaryocytes seems to be intracellular and ADPR-dependent.

A novel role for TRPM8 in visceral afferent function

Transient receptor potential ion channel melastatin subtype 8 (TRPM8) is activated by cold temperatures and cooling agents, such as menthol and icilin. Compounds containing peppermint are reported to reduce symptoms of bowel hypersensitivity; however, the underlying mechanisms of action are unclear. Here we determined the role of TRPM8 in colonic sensory pathways. Laser capture microdissection, quantitative reverse transcription-polymerase chain reaction (RT-PCR), immunofluorescence, and retrograde tracing were used to localise TRPM8 to colonic primary afferent neurons. In vitro extracellular single-fibre afferent recordings were used to determine the effect of TRPM8 channel activation on the chemosensory and mechanosensory function of colonic high-threshold afferent fibres. TRPM8 mRNA was present in colonic DRG neurons, whereas TRPM8 protein was present on nerve fibres throughout the wall of the colon. A subpopulation (24%, n=58) of splanchnic serosal and mesenteric afferents tested responded directly to icilin (5μmol/L). Subsequently, icilin significantly desensitised afferents to mechanical stimulation (P<.0001; n=37). Of the splanchnic afferents responding to icilin, 21 (33%) also responded directly to the TRPV1 agonist capsaicin (3μmol/L), and icilin reduced the direct chemosensory response to capsaicin. Icilin also prevented mechanosensory desensitization and sensitization induced by capsaicin and the TRPA1 agonist AITC (40μmol/L), respectively. TRPM8 is present on a select population of colonic high threshold sensory neurons, which may also co-express TRPV1. TRPM8 couples to TRPV1 and TRPA1 to inhibit their downstream chemosensory and mechanosensory actions.TRPM8 was localised to high-threshold visceral afferent neurons. On visceral afferent peripheral endings, TRPM8 activation affected TRPV1 and TRPA1 downstream chemosensory and mechanosensory actions.

Endogenous Polyhydroxybutyrate (PHB) Regulates TRPM8 Channel

The transient receptor potential channel of the melastatin subfamily TRPM8 is the cold and menthol receptor. Our recent findings indicate that functional TRPM8 channel exists in a form of supramolecular complex of protein and two ubiquitous homopolymers inorganic polyphosphate (polyP) and polyhydroxybutyrate (PHB). We have demonstrated before that polyP is involved in regulation of TRPM8 voltage-gating properties and its removal from the complex completely prevented TRPM8 channel activity (Zakharian et al., 2009). In the current study we focused on the role of PHB in TRPM8 channel. To evaluate the role of PHB in the channel function we compared ion transporting properties of wild type TRPM8 complex with properties of the complex with enzymatically reduced PHB. Experiments were performed using HEK cells stably expressing TRPM8 protein and the calcium sensitive dye fura-2 as an indicator of channel activity. PHB was reduced by co-expression of PHB-depolymerase (Phaz7, courtesy of Dr. D. Jendrossek). To achieve PHB reduction, the bacterial Phaz7 DNA was cloned into mammalian expression system and transiently transfected into HEK cells stably expressing TRPM8 protein. Reduction of PHB associated with TRPM8 protein was confirmed by confocal microscopy, using the fluorescent dye Nile Red as an indicator of the level of PHB and by immunohistochemical analysis using PHB-IgG antibodies. We found that co-expression of the PHB depolymerase in HEK cells stably expressing TRPM8 channels significantly inhibited intracellular Ca2+ signals induced by menthol (∼60%, n=9, control n=8). This provides the first functional evidence of the role played by PHB in TRPM8 channel activity. Our findings suggest that association of PHB with TRPM8 complex is essential for normal channel function.Zakharian E, Thyagarajan B, French RJ, Pavlov E, Rohacs T (2009) Inorganic polyphosphate modulates TRPM8 channels. PLoS ONE 4:e5404.

Effects of opioids, cannabinoids, and vanilloids on body temperature

Cannabinoid and opioid drugs produce marked changes in body temperature. Recent findings have extended our knowledge about the thermoregulatory effects of cannabinoids and opioids, particularly as related to delta opioid receptors, endogenous systems, and transient receptor potential (TRP) channels. Although delta opioid receptors were originally thought to play only a minor role in thermoregulation compared to mu and kappa opioid receptors, their activation has been shown to produce hypothermia in multiple species. Endogenous opioids and cannabinoids also regulate body temperature. Mu and kappa opioid receptors are thought to be in tonic balance, with mu and kappa receptor activation producing hyperthermia and hypothermia, respectively. A particularly intense research focus is TRP channels, where TRPV1 channel activation produces hypothermia whereas TRPA1 and TRPM8 channel activation causes hyperthermia. The marked hyperthermia produced by TRPV1 channel antagonists suggests these warm channels tonically control body temperature. A better understanding of the roles of cannabinoid, opioid, and TRP systems in thermoregulation may have broad clinical implications and provide insights into interactions among neurotransmitter systems involved in thermoregulation.

What is the biological fate of mentholated cigarette smoking?

What is the biological fate of mentholated cigarette smoking?

Near-Membrane Dynamics and Capture of TRPM8 Channels within Transient Confinement Domains

BackgroundThe cold and menthol receptor, TRPM8, is a non-selective cation channel expressed in a subset of peripheral neurons that is responsible for neuronal detection of environmental cold stimuli. It was previously shown that members of the transient receptor potential (TRP) family of ion channels are translocated toward the plasma membrane (PM) in response to agonist stimulation. Because the spatial and temporal dynamics of cold receptor cell-surface residence may determine neuronal activity, we hypothesized that the movement of TRPM8 to and from the PM might be a regulated process. Single particle tracking (SPT) is a useful tool for probing the organization and dynamics of protein constituents in the plasma membrane.Methodology/Principal FindingsWe used SPT to study the receptor dynamics and describe membrane/near-membrane behavior of particles containing TRPM8-EGFP in transfected HEK-293T and F-11 cells. Cells were imaged using total internal reflection fluorescence (TIRF) microscopy and the 2D and 3D trajectories of TRPM8 molecules were calculated by analyzing mean-square particle displacement against time. Four characteristic types of motion were observed: stationary mode, simple Brownian diffusion, directed motion, and confined diffusion. In the absence of cold or menthol to activate the channel, most TRPM8 particles move in network covering the PM, periodically lingering for 2–8 s in confined microdomains of about 800 nm radius. Removing cholesterol with methyl-beta-cyclodextrin (MβCD) stabilizes TRPM8 motion in the PM and is correlated with larger TRPM8 current amplitude that results from an increase in the number of available channels without a change in open probability.Conclusions/SignificanceThese results reveal a novel mechanism for regulating TRPM8 channel activity, and suggest that PM dynamics may play an important role in controlling electrical activity in cold-sensitive neurons.

Contribution of the S5-Pore-S6 Domain to the Gating Characteristics of the Cation Channels TRPM2 and TRPM8

The closely related cation channels TRPM2 and TRPM8 show completely different requirements for stimulation and are regulated by Ca(2+) in an opposite manner. TRPM8 is basically gated in a voltage-dependent process enhanced by cold temperatures and cooling compounds such as menthol and icilin. The putative S4 voltage sensor of TRPM8 is closely similar to that of TRPM2, which, however, is mostly devoid of voltage sensitivity. To gain insight into principal interactions of critical channel domains during the gating process, we created chimeras in which the entire S5-pore-S6 domains were reciprocally exchanged. The chimera M2-M8P (i.e. TRPM2 with the pore of TRPM8) responded to ADP-ribose and hydrogen peroxide and was regulated by extracellular and intracellular Ca(2+) as was wild-type TRPM2. Single-channel recordings revealed the characteristic pattern of TRPM2 with extremely long open times. Only at far-negative membrane potentials (-120 to -140 mV) did differences become apparent because currents were reduced by hyperpolarization in M2-M8P but not in TRPM2. The reciprocal chimera, M8-M2P, showed currents after stimulation with high concentrations of menthol and icilin, but these currents were only slightly larger than in controls. The transfer of the NUDT9 domain to the C terminus of TRPM8 produced a channel sensitive to cold, menthol, or icilin but insensitive to ADP-ribose or hydrogen peroxide. We conclude that the gating processes in TRPM2 and TRPM8 differ in their requirements for specific structures within the pore. Moreover, the regulation by extracellular and intracellular Ca(2+) and the single-channel properties in TRPM2 are not determined by the S5-pore-S6 region.

Blockade of TRPM7 Channel Activity and Cell Death by Inhibitors of 5-Lipoxygenase

TRPM7 is a ubiquitous divalent-selective ion channel with its own kinase domain. Recent studies have shown that suppression of TRPM7 protein expression by RNA interference increases resistance to ischemia-induced neuronal cell death in vivo and in vitro, making the channel a potentially attractive pharmacological target for molecular intervention. Here, we report the identification of the 5-lipoxygenase inhibitors, NDGA, AA861, and MK886, as potent blockers of the TRPM7 channel. Using a cell-based assay, application of these compounds prevented cell rounding caused by overexpression of TRPM7 in HEK-293 cells, whereas inhibitors of 12-lipoxygenase and 15-lipoxygenase did not prevent the change in cell morphology. Application of the 5-lipoxygenase inhibitors blocked heterologously expressed TRPM7 whole-cell currents without affecting the protein's expression level or its cell surface concentration. All three inhibitors were also effective in blocking the native TRPM7 current in HEK-293 cells. However, two other 5-lipoxygenase specific inhibitors, 5,6-dehydro-arachidonic acid and zileuton, were ineffective in suppressing TRPM7 channel activity. Targeted knockdown of 5-lipoxygenase did not reduce TRPM7 whole-cell currents. In addition, application of 5-hydroperoxyeicosatetraenoic acid (5-HPETE), the product of 5-lipoxygenase, or 5-HPETE's downstream metabolites, leukotriene B4 and leukotriene D4, did not stimulate TRPM7 channel activity. These data suggested that NDGA, AA861, and MK886 reduced the TRPM7 channel activity independent of their effect on 5-lipoxygenase activity. Application of AA861 and NDGA reduced cell death for cells overexpressing TRPM7 cultured in low extracellular divalent cations. Moreover, treatment of HEK-293 cells with AA861 increased cell resistance to apoptotic stimuli to a level similar to that obtained for cells in which TRPM7 was knocked down by RNA interference. In conclusion, NDGA, AA861, and MK886 are potent blockers of the TRPM7 channel capable of attenuating TRPM7's function during cell stress, making them effective tools for the biophysical characterization and suppression of TRPM7 channel conductance in vivo.

Differential expression of TRPM2 and TRPV4 channels and their potential role in oxidative stress-induced cell death in organotypic hippocampal culture

TRPM2 and TPPV4 channels, two members of TRP channel family, are known to be widely expressed in the brain but their exact expression pattern and function are not well understood. Due to their high Ca 2+ permeability and gating by reactive oxygen species (TRPM2), or cell swelling, low pH and high temperature (TRPV4), they are likely to be involved in cell damage associated with various brain pathologies. The aim of this study was to investigate the expression of these channels and their potential role in oxidative stress-induced cell damage in organotypic hippocampal slice cultures, a model that retains the complex interaction between neurons and astrocytes. Channel expression was confirmed with RT-PCR and western blotting, while immunocytochemistry demonstrated TRPM2 in CA1–CA3 pyramidal neurons and TRPV4 in astrocytes. Oxidative stress induced by exogenous application of H 2O 2 (600 μM) caused preferential damage of pyramidal neurons, while oxidative stress evoked with mercaptosuccinate (MCS; 400 μM) or buthionine sulfoximine (BSO; 4 μM) mainly damaged astrocytes, as identified by propidium iodide fluorescence. Antioxidants (Trolox 500 μM; MitoE 2 μM) reduced both neuronal and astrocytic cell death. Blockers of TRPV4 channels (Gd 3+ 500 μM; Ruthenium red 1 μM) increased the viability of astrocytes following MCS or BSO treatments, consistent with the expression pattern of these channels. Blockers of TRPM2 channels clotrimazole (20 μM), N-( p-amylcinnomoyl)anthranilic acid (ACA, 25 μM) or flufenamic acid (FFA, 200 μM) failed to protect pyramidal neurons from damage caused by exogenous H 2O 2, and increased damage of these neurons caused by MCS and BSO. The differential expression of stress-sensitive TRPM2 and TRPV4 channels in hippocampal neurons and astrocytes that show distinct differences in vulnerability to different forms of oxidative stress suggests the specific involvement of these channels in oxidative stress-induced cell damage. However, the exact relationship between TRPM2 channel activation and cell death still remains to be determined due to the lack of protective effects of TRPM2 channel blockers.

TRPM7 Activates m-Calpain by Stress-Dependent Stimulation of p38 MAPK and c-Jun N-Terminal Kinase

TRPM7 is a Ca2+-permeant and Mg2+-permeant ion channel in possession of its own kinase domain. In a previous study, we showed that overexpression of the channel-kinase in HEK-293 cells produced cell rounding and loss of adhesion, which was dependent on the Ca2+-dependent protease m-calpain. The TRPM7-elicited change in cell morphology was channel-dependent and occurred without any significant increase in cytosolic Ca2+. Here we demonstrate that overexpression of TRPM7 increased levels of cellular reactive oxygen species (ROS) and nitric oxide, causing the activation of p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK). Application of inhibitors of p38 MAPK and JNK blocked TRPM7-induced cell rounding and activation of m-calpain, without affecting the phosphorylation state of the protease. Overexpression of TRPM7 increased intracellular Mg2+; however, when the concentration of either external Ca2+ or Mg2+ was increased to favor the permeation of one divalent cation over the other, a similar increase in cell rounding and calpain activity was detected, indicating that TRPM7-mediated activation of m-calpain is not dependent on the nature of the divalent conducted by the channel. Application of inhibitors of nitric oxide synthase and mitochondrial-derived ROS reduced TRPM7-induced increases in nitric oxide and ROS production, blocked the change in cell morphology, and reduced cellular calpain activity. Collectively, our data reveal that excessive TRPM7 channel activity causes oxidative and nitrosative stresses, producing cell rounding mediated by p38 MAPK/JNK-dependent activation of m-calpain.

Phosphoinositide Regulation of TRPM8 Channels in Planar Lipid Bilayers

The cold and menthol receptor TRPM8 is regulated by membrane phosphoinositides. To study the effects of lipids directly on the channel, we have reconstituted the purified TRPM8 in planar lipid bilayers. This system allows full control of the lipid composition in our experiments. The reconstituted channel was activated by menthol or cold, and its activity depended on the presence of specific phosphoinositides. In the presence of menthol, TRPM8 exhibited the highest probability of opening in the presence of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] with Po ∼ 0.89 at +100 mV and Po ∼ 0.4 at −100 mV. Less channel activity was induced by phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2] with Po ∼ 0.53 at +100 mV and Po ∼ 0.2 at −100 mV. Phosphatidylinositol 3,4,5-bisphosphate [PI(3,4,5) P2] resulted in irregular TRPM8 channel currents and lower open probability with Po ∼ 0.21 at +100 mV and Po ∼ 0.087 at −100 mV. Among the tested lipids the lowest TRPM8 channel activity was induced by phosphatidylinositol 4-phosphate [PI(4)P] with Po ∼ 0.12 at +100 mV and Po ∼ 0.019 at −100 mV. The lipid specificity profile in lipid bilayers is very similar to that observed in excised patches.We have also studied the activation of TRPM8 channels in lipid bilayer with cold. Cooling the system with reconstituted TRPM8 channels also required the presence of PI(4,5)P2. The main shift in the channel behavior was observed in the temperature range from 21°C to 18°C where the channel showed drastic changes in the open probability from 0.05 to 0.85 at +100 mV.These results demonstrate that the TRPM8 protein is directly activated by cold, menthol and phosphoinositides.

Transient receptor potential channels meet phosphoinositides

Transient receptor potential (TRP) cation channels are unique cellular sensors that are involved in multiple cellular functions, ranging from transduction of sensory signals to the regulation of Ca2+ and Mg2+ homoeostasis. Malfunctioning of TRP channels is now recognized as the cause of several hereditary and acquired human diseases. At the time of cloning of the first Drosophila TRP channel, a close connection between gating and phosphatidylinositol phosphates (PIPs) was already recognized. In this review, we summarize current knowledge about the mechanisms of interaction between TRP channels and PIPs, and discuss the possible functional implications of TRP–PIP interactions to human physiology and pathophysiology.

The Poly(ADP-ribose) Polymerase PARP-1 Is Required for Oxidative Stress-induced TRPM2 Activation in Lymphocytes

TRPM2 cation channels are widely expressed in the immune system and are thought to play a role in immune cell responses to oxidative stress. Patch clamp analyses suggest that TRPM2 channel activation can occur through a direct action of oxidants on TRPM2 channels or indirectly through the actions of a related group of adenine nucleotide 2nd messengers. However, the contribution of each gating mechanism to oxidative stress-induced TRPM2 activation in lymphocytes remains undefined. To better understand the molecular events leading to TRPM2 activation in lymphocytes, we analyzed oxidative stress-induced turnover of intracellular NAD, the metabolic precursor of adenine nucleotide 2nd messengers implicated in TRPM2 gating, and oxidative stress-induced TRPM2-mediated currents and Ca2+ transients in DT40 B cells. TRPM2-dependent Ca2+ entry did not influence the extent or time course of oxidative stress-induced turnover of NAD. Furthermore, expression of oxidative stress-activated poly(ADP-ribose) polymerases (PARPs) was required for oxidative stress-induced NAD turnover, TRPM2 currents, and TRPM2-dependent Ca2+ transients; no oxidant-induced activation of TRPM2 channels could be detected in PARP-deficient cells. Together, our results suggest that during conditions of oxidative stress in lymphocytes, TRPM2 acts as a downstream effector of the PARP/poly(ADP-ribose) glycohydrolase pathway through PARP-dependent formation of ADP-ribose.

AMTB, a TRPM8 channel blocker: evidence in rats for activity in overactive bladder and painful bladder syndrome

The activation of the TRPM8 channel, a member of the large class of TRP ion channels, has been reported to be involved in overactive bladder and painful bladder syndrome, although an endogenous activator has not been identified. In this study, N-(3-aminopropyl)-2-{[(3-methylphenyl) methyl]oxy}-N-(2-thienylmethyl)benzamide hydrochloride salt (AMTB) was evaluated as a TRPM8 channel blocker and used as a tool to evaluate the effects of this class of ion channel blocker on volume-induced bladder contraction and nociceptive reflex responses to noxious bladder distension in the rat. AMTB inhibits icilin-induced TRPM8 channel activation as measured in a Ca(2+) influx assay, with a pIC(50) of 6.23. In the anesthetized rat, intravenous administration of AMTB (3 mg/kg) decreased the frequency of volume-induced bladder contractions, without reducing the amplitude of contraction. The nociceptive response was measured by analyzing both visceromotor reflex (VMR) and cardiovascular (pressor) responses to urinary bladder distension (UBD) under 1% isoflurane. AMTB (10 mg/kg) significantly attenuated reflex responses to noxious UBD to 5.42 and 56.51% of the maximal VMR response and pressor response, respectively. The ID50 value on VMR response was 2.42 +/- 0.46 mg/kg. These results demonstrate that TRPM8 channel blocker can act on the bladder afferent pathway to attenuate the bladder micturition reflex and nociceptive reflex responses in the rat. Targeting TRPM8 channel may provide a new therapeutic opportunity for overactive bladder and painful bladder syndrome.

Recent developments in intestinal magnesium absorption

Recent identification and characterization of novel Mg transporters have clarified our understanding of intestinal magnesium absorption. The predominant Mg transporters include TRPM6 and TRPM7, members of the transient receptor potential melastatin family of cation channels. Mutations of TRPM6 result in a primary disorder termed hypomagnesemia with secondary hypocalcemia. Both TRPM6 and TRPM7 channels possess an atypical alpha-kinase domain. Recent studies have shown that TRPM7 channel activity is regulated by intracellular Mg and magnesium-nucleotides and modulated via this phosphotransferase kinase. TRPM6 channel function and intestinal magnesium absorption is altered by a variety of hormones and factors. Although it is apparent that TRPM6 and TRPM7 form heteromeric ion channels, controversy surrounds the nature of this interaction. Some studies show that TRPM6 may function on its own whereas other research concludes that TRPM7 is required for effective trafficking of TRPM6 to the plasma membrane. Finally, a number of other Mg transporters have been identified in intestinal epithelial cells but the role of these proteins is unclear. The recent developments in intestinal magnesium absorption and cellular magnesium homeostasis provide a basis for understanding magnesium deficiency disorders and provide a platform for future investigations.