Cation Channels In Plasma Membrane Research Articles

Gain-of-function variants in the ion channel gene TRPM3 underlie a spectrum of neurodevelopmental disorders.

TRPM3 is a temperature- and neurosteroid-sensitive plasma membrane cation channel expressed in a variety of neuronal and non-neuronal cells. Recently, rare de novo variants in TRPM3 were identified in individuals with developmental and epileptic encephalopathy, but the link between TRPM3 activity and neuronal disease remains poorly understood. We previously reported that two disease-associated variants in TRPM3 lead to a gain of channel function . Here, we report a further 10 patients carrying one of seven additional heterozygous TRPM3 missense variants. These patients present with a broad spectrum of neurodevelopmental symptoms, including global developmental delay, intellectual disability, epilepsy, musculo-skeletal anomalies, and altered pain perception. We describe a cerebellar phenotype with ataxia or severe hypotonia, nystagmus, and cerebellar atrophy in more than half of the patients. All disease-associated variants exhibited a robust gain-of-function phenotype, characterized by increased basal activity leading to cellular calcium overload and by enhanced responses to the neurosteroid ligand pregnenolone sulfate when co-expressed with wild-type TRPM3 in mammalian cells. The antiseizure medication primidone, a known TRPM3 antagonist, reduced the increased basal activity of all mutant channels. These findings establish gain-of-function of TRPM3 as the cause of a spectrum of autosomal dominant neurodevelopmental disorders with frequent cerebellar involvement in humans and provide support for the evaluation of TRPM3 antagonists as a potential therapy.

The Identification of a Novel Calcium-Dependent Link Between NAD+ and Glucose Deprivation-Induced Increases in Protein O-GlcNAcylation and ER Stress.

The modification of proteins by O-linked β-N-acetylglucosamine (O-GlcNAc) is associated with the regulation of numerous cellular processes. Despite the importance of O-GlcNAc in mediating cellular function our understanding of the mechanisms that regulate O-GlcNAc levels is limited. One factor known to regulate protein O-GlcNAc levels is nutrient availability; however, the fact that nutrient deficient states such as ischemia increase O-GlcNAc levels suggests that other factors also contribute to regulating O-GlcNAc levels. We have previously reported that in unstressed cardiomyocytes exogenous NAD+ resulted in a time and dose dependent decrease in O-GlcNAc levels. Therefore, we postulated that NAD+ and cellular O-GlcNAc levels may be coordinately regulated. Using glucose deprivation as a model system in an immortalized human ventricular cell line, we examined the influence of extracellular NAD+ on cellular O-GlcNAc levels and ER stress in the presence and absence of glucose. We found that NAD+ completely blocked the increase in O-GlcNAc induced by glucose deprivation and suppressed the activation of ER stress. The NAD+ metabolite cyclic ADP-ribose (cADPR) had similar effects on O-GlcNAc and ER stress suggesting a common underlying mechanism. cADPR is a ryanodine receptor (RyR) agonist and like caffeine, which also activates the RyR, both mimicked the effects of NAD+. SERCA inhibition, which also reduces ER/SR Ca2+ levels had similar effects to both NAD+ and cADPR on O-GlcNAc and ER stress responses to glucose deprivation. The observation that NAD+, cADPR, and caffeine all attenuated the increase in O-GlcNAc and ER stress in response to glucose deprivation, suggests a potential common mechanism, linked to ER/SR Ca2+ levels, underlying their activation. Moreover, we showed that TRPM2, a plasma membrane cation channel was necessary for the cellular responses to glucose deprivation. Collectively, these findings support a novel Ca2+-dependent mechanism underlying glucose deprivation induced increase in O-GlcNAc and ER stress.

Transient receptor potential polycystin-2 (TRPP2) regulates motility and intracellular calcium of porcine sperm.

Polycystin-2, also known as transient receptor potential polycystin-2 (TRPP2), is a membrane protein that regulates calcium homeostasis in renal epithelial cells. Mutations in PKD2, the gene encoding human TRPP2, cause enlarged cystic kidneys and contribute to polycystic kidney disease (PKD). Male Drosophila melanogaster with mutations in amo, the homolog of PKD2, display a mild decrease in sperm motility but have a drastic reduction in fertility due to failed sperm migration and storage within the female tract. Although TRPP2 has critical roles for Drosophila sperm function, the protein has not been described in mammalian sperm. Herein, we report the localization of TRPP2 in porcine sperm and identify functions of TRPP2 in regulating intracellular Ca2+ and motility. Porcine sperm treated with an antibody to TRPP2 in capacitating medium had reduced average path velocity and curvilinear velocity (p<.05). Blocking TRPP2 also increased sperm tail beat-cross frequency (p<.05). After 90min of capacitation, sperm incubated with TRPP2 antibody had decreased intracellular Ca2+ concentration compared to controls (p<.05), consistent with TRPP2 function as a plasma membrane cation channel. This is the first report that mammalian sperm contain TRPP2, which appears to regulate intracellular Ca2+ and motility patterns in porcine sperm.

Alveolar Stretch Activation of Endothelial Piezo1 Protects Adherens Junctions and Lung Vascular Barrier.

Disruption of alveolar-capillary barriers is a major complication of high-volume mechanical ventilation referred to as "ventilator-induced lung injury." The stretching force in alveoli is transmitted to endothelial cells, increasing the tension on underlying endothelial plasma membrane. The mechanosensor Piezo1, a plasma membrane cation channel, was inducibly deleted in endothelial cells of mice (Piezo1iEC-/-), which allowed us to study its role in regulating the endothelial barrier response to alveolar stretch. We observed significant increase in lung vascular permeability in Piezo1iEC-/- mice as compared with control Piezo1fl/fl mice in response to high-volume mechanical ventilation. We also observed that human lung endothelial monolayers depleted of Piezo1 and exposed to cyclic stretch had increased permeability. We identified the calcium-dependent cysteine protease calpain as a downstream target of Piezo1. Furthermore, we showed that calpain maintained stability of the endothelial barrier in response to mechanical stretch by cleaving Src kinase, which was responsible for disassembling endothelial adherens junctions. Pharmacological activation of calpain caused Src cleavage and thereby its inactivation, and it restored the disrupted lung endothelial barrier seen in Piezo1iEC-/- mice undergoing high-volume mechanical ventilation. Our data demonstrate that downregulation of Piezo1 signaling in endothelium is a critical factor in the pathogenesis of ventilator-induced lung injury, and thus augmenting Piezo1 expression or pharmacologically activating Piezo1 signaling may be an effective therapeutic strategy.

TRPC1 Channels Are Expressed in Pyramidal Neurons and in a Subset of Somatostatin Interneurons in the Rat Neocortex

Disturbances in calcium homeostasis due to canonical transient receptor potential (TRPC) and/or store-operated calcium (SOC) channels can play a key role in a large number of brain disorders. TRPC channels are plasma membrane cation channels included in the transient receptor potential (TRP) superfamily. The most widely distributed member of the TRPC subfamily in the brain is TRPC1, which is frequently linked to group I metabotropic glutamate receptors (mGluRs) and to the components of SOC channels. Proposing TRPC/SOC channels as a therapeutic target in neurological diseases previously requires a detailed knowledge of the distribution of such molecules in the brain. The aim of our study was to analyze the neuroanatomical distribution of TRPC1 in the rat neocortex. By double- and triple-labeling and confocal microscopy, we tested the presence of TRPC1 by using a series of specific neurochemical markers. TRPC1 was abundant in SMI 32-positive pyramidal neurons, and in some glutamic acid decarboxylase 67 (GAD67) interneurons, but was lacking in glial fibrillary acidic protein (GFAP)-positive glial cells. In neurons it colocalized with postsynaptic marker MAP2 in cell bodies and apical dendritic trunks and it was virtually absent in synaptophysin-immunoreactive terminals. By using a panel of antibodies to classify interneurons, we identified the GABAergic interneurons that contained TRPC1. TRPC1 was lacking in basket and chandelier parvalbumin (PVALB) cells, and a very low percentage of calretinin (CALR) or calbindin (CALB) interneurons expressed TRPC1. Moreover, 63% of somatostatin (SST) expressing-cells and 37% of reelin-positive cells expressed TRPC1. All the SST/TRPC1 double-labeled cells, many of which were presumptive Martinotti cells (MC), were positive for reelin. The presence of TRPC1 in the somata and apical dendritic trunks of neocortical pyramidal cells suggests a role for this channel in sensory processing and synaptic plasticity. Conversely in SST/reelin interneurons, TRPC1 could modulate GABAergic transmission, which is responsible for shaping the coordinated activity of the pyramidal cells in the cortical network. In future studies, it would be relevant to investigate whether TRPC1 could be involved in the expression or processing of reelin in SST inhibitory interneurons.

Abstract 1269: Enhanced cytotoxicity in primary human metastatic melanoma cells via inhibition of the transient receptor potential melastatin-2 (TRPM2) channel

Abstract The transient receptor potential melastatin-2 (TRPM2) cation channel was previously identified as a potential target in various cancers, where its pharmacologic inhibition caused increased DNA damage and the selective eradication of cancer cells. In this study, we utilized several human primary metastatic melanoma cell lines, in which we analyzed TRPM2 and its isoforms, and evaluated the ability of TRPM2 inhibition to modulate cell death. As TRPM2 is known to exist as a plasma membrane cation channel in noncancerous cells, we investigated the localization of TRPM2 in melanoma cells. In three lines of primary human metastatic melanoma cells, TRPM2 was localized in the nucleus, as compared to an extra-nuclear localization in noncancerous primary epidermal keratinocyes. Because TRPM2 exists in several isoforms in cancer cells, we investigated the cellular localization of these isoforms. Subcellular fractionations demonstrated that only full-length TRPM2 was localized to the nucleus in primary human metastatic melanoma cells, whereas two smaller isoforms were localized exclusively in the cytoplasmic fraction. Treatment with the TRPM2 inhibitor, clotrimazole, caused decreased proliferation in all three lines of primary human metastatic melanoma cells. Further, treatment with the DNA alkylating agent, temozolomide (TMZ), led to increased levels of cell death in melanoma cells pretreated with clotrimazole, but not in noncancerous primary epidermal keratinocyes after pretreatment. Similar increases in cell death were observed after RNAi silencing of TRPM2 followed by TMZ treatment. Taken together, this study demonstrated that TRPM2 inhibition selectively increases cell death in primary human metastatic melanoma cells. Further, the data suggests that, similar to our previous results in breast adenocarcinoma cells, full-length TRPM2 appears to have a novel role in melanoma cell growth and survival. The results therefore suggest that TRPM2 is a potential target in melanoma, where its inhibition may cause the selective eradication of metastatic melanoma. Citation Format: David W. Koh, Steven D. Blake, Daniel P. Powell. Enhanced cytotoxicity in primary human metastatic melanoma cells via inhibition of the transient receptor potential melastatin-2 (TRPM2) channel. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1269.

Modulation of Calcium Entry by the Endo-lysosomal System.

Endo-lysosomes are acidic organelles that besides the role in macromolecules degradation, act as intracellular Ca(2+) stores. Nicotinic acid adenine dinucleotide phosphate (NAADP), the most potent Ca(2+)-mobilizing second messenger, produced in response to agonist stimulation, activates Ca(2+)-releasing channels on endo-lysosomes and modulates a variety of cellular functions. NAADP-evoked signals are amplified by Ca(2+) release from endoplasmic reticulum, via the recruitment of inositol 1,4,5-trisphosphate and/or ryanodine receptors through a Ca(2+)-induced Ca(2+)- release (CICR) mechanism. The endo-lysosomal Ca(2+) channels activated by NAADP were recently identified as the two-pore channels (TPCs). In addition to TPCs, endo-lysosomes express another distinct family of Ca(2+)- permeable channels, namely the transient receptor potential mucolipin (TRPML) channels, functionally distinct from TPCs. TPCs belong to the voltage-gated channels, resembling voltage-gated Na(+) and Ca(2+) channels. TPCs have important roles in vesicular fusion and trafficking, in triggering a global Ca(2+) signal and in modulation of the membrane excitability. Depletion of acidic Ca(2+) stores has been shown to activate store-operated Ca(2+) entry in human platelets and mouse pancreatic β-cells. In human platelets, Ca(2+) influx in response to acidic stores depletion is facilitated by the tubulin-cytoskeleton and occurs through non-selective cation channels and transient receptor potential canonical (TRPC) channels. Emerging evidence indicates that activation of intracellular receptors, situated on endo-lysosomes, elicits canonical and non-canonical signaling mechanisms that involve CICR and activation of non-selective cation channels in plasma membrane. The ability of endo-lysosomal Ca(2+) stores to modulate the Ca(2+) release from other organelles and the Ca(2+) entry increases the diversity and complexity of cellular signaling mechanisms.

Abstract 1717: Selective induction of breast adenocarcinoma cell death via inhibition of the transient receptor potential melastatin-2 (TRPM2) cation channel

Abstract Transient receptor potential melastatin-2 (TRPM2) is a plasma membrane cation channel with important physiologic roles in noncancerous cells. However, in MCF-7 and MDA-MB-231 human breast adenocarcinoma cells, we previously demonstrated that TRPM2 was present in the nucleus, and pharmacologic inhibition or RNAi silencing of TRPM2 led to decreased proliferation. In this study, we evaluated the ability of TRPM2 inhibition to produce cell death in normal breast epithelial cells and breast adenocarcinoma cells. Further, we analyzed the cell death pathways induced. Treatment with the DNA alkylating agent, N-methyl-N’-nitro-N-nitrosoguanidine (MNNG), or doxorubicin (Dox) led to increased levels of cell death in MDA-MB-231 breast adenocarcinoma cells pretreated with TRPM2 inhibitors, but not in noncancerous mammary epithelial cells after pretreatment. Similar increases in cell death were observed after RNAi silencing of TRPM2 in both MDA-MB-231 and MCF-7 breast adenocarcinoma cells after Dox or tamoxifen treatment, respectively. Investigation of apoptosis after TRPM2 inhibition demonstrated decreased levels of caspase activity and no significant effect of Q-VD-OPh, a pan-caspase inhibitor, on cell death induced by TRPM2 inhibition. Further, pretreatment with 3-methyl adenine, an inhibitor of autophagy, also produced no significant effect on cell death induced by TRPM2 inhibition. These studies indicate that apoptosis or autophagy are not the primary cell death pathways induced by TRPM2 inhibition in breast cancer cells. Also, RNAi silencing of poly(ADP-ribose) glycohydrolase (PARG) and apoptosis-inducing factor (AIF), two proteins involved in initiating or facilitating alternative pathways of cell death, produced minimal changes in cell death induced by TRPM2 inhibition. Taken together, this study demonstrated that TRPM2 inhibition selectively increases cell death in breast adenocarcinoma cells. Further, the data suggests that apoptosis, autophagy, and PARG/AIF-mediated cell death are not the primarily cell death pathways involved in the cytotoxicity caused by TRPM2 inhibition. The results therefore suggest that TRPM2 is a potential target in breast cancer, where its inhibition may cause the selective eradication of breast adenocarcinoma cells. Citation Format: David W. Koh, Daniel P. Powell, Steven D. Blake, Joy L. Hoffman, Xiaoxing Feng. Selective induction of breast adenocarcinoma cell death via inhibition of the transient receptor potential melastatin-2 (TRPM2) cation channel. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1717. doi:10.1158/1538-7445.AM2015-1717

Inhibition of the transient receptor potential melastatin-2 channel causes increased DNA damage and decreased proliferation in breast adenocarcinoma cells.

Transient receptor potential, melastatin-2 (TRPM2) is a plasma membrane cation channel with important roles in sensory functions and promoting cell death. However, we demonstrated here that TRPM2 was present in the nuclei of MCF-7 and MDA-MB-231 human breast adenocarcinoma cells, and its pharmacologic inhibition or RNAi silencing caused decreased cell proliferation. Neither an effect on proliferation nor a localization of TRPM2 in the nucleus was observed in noncancerous HMEC and MCF-10A human mammary epithelial cells. Investigation of possible effects of TRPM2 function in the nucleus demonstrated that pharmacologic inhibition or RNAi silencing of TRPM2 in MCF-7 and MDA-MB-231 human breast adenocarcinoma cells caused up to 4-fold increases in DNA damage levels, as compared to noncancerous breast cells after equivalent treatments. These results indicate that TRPM2 has a novel nuclear function in human breast adenocarcinoma cells that facilitates the integrity of genomic DNA, a finding that is distinct from its previously reported role as a plasma membrane cation channel in noncancerous cells. In summary, we report here a novel effect promoted by TRPM2, where it functions to minimize DNA damage and thus may have a role in the protection of genomic DNA in breast cancer cells. Our study therefore provides compelling evidence that TRPM2 has a unique role in breast adenocarcinoma cells. Accordingly, these studies suggest that TRPM2 is a potential therapeutic target, where its pharmacologic inhibition may provide an innovative strategy to selectively increase DNA damage levels in breast cancer cells.

A Prominent Role for Leaf Calcium as a Yield and Quality Determinant in Upland Cotton (Gossypium hirsutum L.) Varieties Grown Under Irrigated Mediterranean Conditions

Seven cotton (Gossypium hirsutum L.) accessions were tested over 2 years under irrigated Mediterranean conditions on a loamy soil with nitrogen (N) as the only nutrient input. The study aimed to identify the critical nutritional and physiological factors determining seedcotton yield and fibre quality. A suite of leaf physiological traits [chlorophyll content (assessed by SPAD), carbon isotope discrimination (Δ), 15N natural abundance (δ15N), leaf water potential, N and C concentrations, C/N ratio, K, Na, Ca and Mg concentrations, their sum and ratios] was assessed, and their interrelationships then analysed. It was found that physiological indices such as SPAD, Δ and δ15N failed to discern genotypes for yield and did not relate with fibre quality traits. At the same time, leaf Ca concentration was the trait that showed the strongest correlation with both seedcotton (SY) and lint yield (LY). An increase of K/Na ratio up to 5.74 was beneficial for SY but higher ratios impacted yield adversely. In this line, exclusion of K in favour of Ca (lower K/Ca ratios) increased both SY and LY. The above results could be explained by Ca2+ control over activity of tonoplast and plasma membrane cation channels, resulting in redistribution of K+ between cell compartments. It is suggested that Ca2+-rich plants are more efficient in sequestering higher K+ quantities in leaf vacuoles, at the expense of cytosolic K+. Under K+-limiting conditions, such redistribution may trigger programmed cell death and enhance leaf senescence. This would remobilize and translocate nutrients (e.g. N) and organic substances to sinks (seedcotton), contributing to higher yields reported in the present work.

TRPC1 and metabotropic glutamate receptor expression in rat auditory midbrain neurons.

Canonical transient receptor potential (TRPC) channels are plasma membrane cation channels included in the TRP superfamily. TRPC1 is expressed widely in the central nervous system and is linked to group I metabotropic glutamate receptors (mGluRs). In the auditory brainstem, TRPC1 expression has never been described, although group I mGluRs are present. In the central nucleus of the inferior colliculus (CIC), activation of group I mGluRs induces an extracellular Ca(2+) influx after store depletion. Therefore, this study examines whether TRPC1 is expressed in this region to establish a correlation with mGluRs. By quantitative reverse transcription-polymerase chain reaction and Western blotting, this study assesses the presence of TRPC1 along with both group I mGluR subtypes mGluR1 and mGluR5 in the rat inferior colliculus (IC). All these molecules present a robust expression in the IC. By confocal double immunofluorescence, this study also demonstrates that TRPC1 colocalizes with parvalbumin, a CIC neuronal marker, in many cells. Conversely, TRPC1 was lacking in glial fibrillary acidic protein-positive glial cells. All the glutamate acid decarboxylase 67 (GAD67)-immunoreactive neurons and many GAD67-negative neurons were positive to TRPC1, which indicates the presence of TRPC1 in γ-aminobutyric acid (GABA)-ergic and non-GABAeregic neurons. With regard to subcellular distribution, TRPC1 was absent in synaptophysin-immunoreactive axonic terminals but colocalized with postsynaptic marker microtubule-associated protein 2 in cell bodies and dendrites. TRPC1 totally overlapped group I mGluRs, which supports the involvement of TRPC1 in the mGluR pathway and, likely, in auditory signal processing at the midbrain level. .

P2X4 Forms ATP-Activated Channels on Lysosomal Membranes Regulated by Luminal pH and SLC17A9 Proteins

P2X receptors are commonly known as plasma membrane cation channels involved in a wide variety of cell functions. Recently, it was shown that in addition to plasma membrane expression, mammalian P2X4 was also localized intracellularly in lysosomes. However, it was not clear whether the lysosomal P2X4 receptors function as channels and how they are regulated. In this study, we show that both P2X4 and its natural ligand, ATP, are enriched in lysosomes of mammalian cells. By directly recording membrane currents from enlarged lysosomal vacuoles, we demonstrate that lysosomal P2X4 proteins form channels activated by ATP from the luminal side in a pH dependent manner. While the acidic pH at the luminal side inhibits P2X4 activity, increasing the luminal pH in the presence of ATP causes P2X4 activation. We further show that SLC17A9 (solute carrier family 17 member 9), also known as VNUT (vesicular nucleotide transporter), functions as an ATP transporter in lysosomes where it transports ATP into lysosomes to activate P2X4 channels. Taken together, our data suggest that P2X4 acts as a lysosomal channel regulated by a number of factors including luminal ATP, luminal pH, and membrane SLC17A9. Such regulation may be important for lysosome physiology.

Dopaminergic neurotoxicants cause biphasic inhibition of purinergic calcium signaling in astrocytes.

Dopaminergic nuclei in the basal ganglia are highly sensitive to damage from oxidative stress, inflammation, and environmental neurotoxins. Disruption of adenosine triphosphate (ATP)-dependent calcium (Ca2+) transients in astrocytes may represent an important target of such stressors that contributes to neuronal injury by disrupting critical Ca2+-dependent trophic functions. We therefore postulated that plasma membrane cation channels might be a common site of inhibition by structurally distinct cationic neurotoxicants that could modulate ATP-induced Ca2+ signals in astrocytes. To test this, we examined the capacity of two dopaminergic neurotoxicants to alter ATP-dependent Ca2+ waves and transients in primary murine striatal astrocytes: MPP+, the active metabolite of 1-methyl 4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and 6-hydroxydopamine (6-OHDA). Both compounds acutely decreased ATP-induced Ca2+ transients and waves in astrocytes and blocked OAG-induced Ca2+ influx at micromolar concentrations, suggesting the transient receptor potential channel, TRPC3, as an acute target. MPP+ inhibited 1-oleoyl-2-acetyl-sn-glycerol (OAG)-induced Ca2+ transients similarly to the TRPC3 antagonist, pyrazole-3, whereas 6-OHDA only partly suppressed OAG-induced transients. RNAi directed against TRPC3 inhibited the ATP-induced transient as well as entry of extracellular Ca2+, which was augmented by MPP+. Whole-cell patch clamp experiments in primary astrocytes and TRPC3-overexpressing cells demonstrated that acute application of MPP+ completely blocked OAG-induced TRPC3 currents, whereas 6-OHDA only partially inhibited OAG currents. These findings indicate that MPP+ and 6-OHDA inhibit ATP-induced Ca2+ signals in astrocytes in part by interfering with purinergic receptor mediated activation of TRPC3, suggesting a novel pathway in glia that could contribute to neurotoxic injury.

P2X4 Forms Functional ATP-activated Cation Channels on Lysosomal Membranes Regulated by Luminal pH

P2X receptors are commonly known as plasma membrane cation channels involved in a wide variety of cell functions. The properties of these channels have been extensively studied on the plasma membrane. However, studies in amoeba suggest that P2X receptors are also present intracellularly and involved in vesicle fusion with the plasma membrane. Recently, it was shown that in addition to plasma membrane expression, mammalian P2X4 was also localized intracellularly in lysosomes. However, it was not clear whether the lysosomal P2X4 receptors function as channels and how they are activated and regulated. In this paper, we show that both P2X4 and its natural ligand, ATP, are enriched in lysosomes of COS1 and HEK293 cells. By directly recording membrane currents from enlarged lysosomal vacuoles, we demonstrated that lysosomal P2X4 formed channels activated by ATP from the luminal side in a pH-dependent manner. While the acidic pH at the luminal side inhibited P2X4 activity, increasing the luminal pH in the presence of ATP caused P2X4 activation. We further showed that, as for the plasma membrane P2X4, the lysosomal P2X4 was potentiated by ivermectin but insensitive to suramin and PPADS, and it permeated the large cation N-methyl-d-glucamine upon activation. Our data suggest that P2X4 forms functional ATP-activated cation channels on lysosomal membranes regulated by luminal pH. Together with the reported fusion effect of intracellular P2X in lower organisms, we speculate that the lysosome-localized P2X4 may play specific roles in membrane trafficking of acidic organelles in mammalian cells.

Drosophila TRPML Forms PI(3,5)P2-activated Cation Channels in Both Endolysosomes and Plasma Membrane

Transient Receptor Potential mucolipin (TRPML) channels are implicated in endolysosomal trafficking, lysosomal Ca(2+) and Fe(2+) release, lysosomal biogenesis, and autophagy. Mutations in human TRPML1 cause the lysosome storage disease, mucolipidosis type IV (MLIV). Unlike vertebrates, which express three TRPML genes, TRPML1-3, the Drosophila genome encodes a single trpml gene. Although the trpml-deficient flies exhibit cellular defects similar to those in mammalian TRPML1 mutants, the biophysical properties of Drosophila TRPML channel remained uncharacterized. Here, we show that transgenic expression of human TRPML1 in the neurons of Drosophila trpml mutants partially suppressed the pupal lethality phenotype. When expressed in HEK293 cells, Drosophila TRPML was localized in both endolysosomes and plasma membrane and was activated by phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) applied to the cytoplasmic side in whole lysosomes and inside-out patches excised from plasma membrane. The PI(3,5)P2-evoked currents were blocked by phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), but not other phosphoinositides. Using TRPML A487P, which mimics the varitint-waddler (Va) mutant of mouse TRPML3 with constitutive whole-cell currents, we show that TRPML is biphasically regulated by extracytosolic pH, with an optimal pH about 0.6 pH unit higher than that of human TRPML1. In addition to monovalent cations, TRPML exhibits high permeability to Ca(2+), Mn(2+), and Fe(2+), but not Fe(3+). The TRPML currents were inhibited by trivalent cations Fe(3+), La(3+), and Gd(3+). These features resemble more closely to mammalian TRPML1 than TRPML2 and TRPML3, but with some obvious differences. Together, our data support the use of Drosophila for assessing functional significance of TRPML1 in cell physiology.

Adenine Dinucleotide Second Messengers and T-lymphocyte Calcium Signaling

Calcium signaling is a universal signal transduction mechanism in animal and plant cells. In mammalian T-lymphocytes calcium signaling is essential for activation and re-activation and thus important for a functional immune response. Since many years it has been known that both calcium release from intracellular stores and calcium entry via plasma membrane calcium channels are involved in shaping spatio-temporal calcium signals. Second messengers derived from the adenine dinucleotides NAD and NADP have been implicated in T cell calcium signaling. Nicotinic acid adenine dinucleotide phosphate (NAADP) acts as a very early second messenger upon T cell receptor/CD3 engagement, while cyclic ADP-ribose (cADPR) is mainly involved in sustained partial depletion of the endoplasmic reticulum by stimulating calcium release via ryanodine receptors. Finally, adenosine diphosphoribose (ADPR) a breakdown product of both NAD and cADPR activates a plasma membrane cation channel termed TRPM2 thereby facilitating calcium (and sodium) entry into T cells. Receptor-mediated formation, metabolism, and mode of action of these novel second messengers in T-lymphocytes will be reviewed.

TRPV4 channels tune astrocytic endfoot Ca2+ to optimize neurovascular coupling

A rise in intracellular calcium ([Ca2+]i) in astrocytes is required to communicate the energy demands of neurons to the cerebral microcirculation to precisely match local cerebral blood flow to neuronal metabolism. This “neurovascular coupling” (NVC) is essential for neuronal viability and homeostasis. A moderate rise in [Ca2+]i in the perivascular “endfoot” terminals of astrocytes in response to neuronal activation produces vasodilation of the adjacent parenchymal arteriole, whereas high endfoot [Ca2+]i can produce vasoconstriction. Thus, precise regulation of astrocytic [Ca2+]i is necessary for proper function of the cerebral microcirculation. Transient receptor potential vanilloid 4 (TRPV4) channels are Ca2+‐permeant plasma membrane cation channels that participate in intracellular Ca2+ signaling in a variety of cell types. We previously reported that both the synthetic TRPV4 agonist, GSK1016790A, and the endogenous agonist, 11,12‐EET, increase the frequency and amplitude of perivascular astrocytic endfoot Ca2+ oscillations in brain slices. Here we provide evidence from multiphoton confocal microscopy in brain slices and cerebral blood flow (CBF) measurement by laser Doppler flowmetry in vivo, that Ca2+ entry through TRPV4 in astrocytic endfeet contributes to NVC. Incubation of brain slices with the selective TRPV4 antagonist, HC‐067047 (1 μM), attenuated the parenchymal arteriolar vasodilation to neuronal stimulation by 42% (n=5). This effect was accompanied by a reduction in pre‐stimulation endfoot [Ca2+]i and in the evoked increase in endfoot [Ca2+]i in response to stimulation. Superfusion of HC‐067047 over the somatosensory cortex in mice had no effect on resting CBF, but reduced the CBF response to contralateral whisker stimulation by 9% (n=7). These results indicate that TRPV4 channels tune perivascular astrocytic enfoot [Ca2+]i to optimize NVC. This study was supported by National Heart, Lung, and Blood Institute grants HL‐44455, T32 HL‐07944, and HL‐095489.

The over-expression of TRPC6 channels in HEK-293 cells favours the intracellular accumulation of zinc

TRPC6 are plasma membrane cation channels. By means of live-cell imaging and spectroscopic methods, we found that HEK cells expressing TRPC6 channels (HEK-TRPC6) are enriched in zinc and sulphur and have a reduced copper content when compared to HEK cells and HEK cells expressing TRPC3 channels (HEK-TRPC3). Hence, HEK-TRPC6 cells have larger pools of mobilizable Zn2+ and are more sensitive to an oxidative stress. Synchrotron X-ray fluorescence experiments showed a higher zinc content in the nuclear region indicating that the intracellular distribution of this metal was influenced by the over-expression of TRPC6 channels. Their properties were investigated with the diacylglycerol analogue SAG and the plant extract hyperforin. Electrophysiological recordings and imaging experiments with the fluorescent Zn2+ probe FluoZin-3 demonstrated that TRPC6 channels form Zn2+-conducting channels. In cortical neurons, hyperforin-sensitive channels co-exist with voltage-gated channels, AMPA and NMDA receptors, which are known to transport Zn2+. The ability of these channels to regulate the size of the mobilizable pools of Zn2+ was compared. The data collected indicate that the entry of Zn2+ through TRPC6 channels can up-regulate the size of the DTDP-sensitive pool of Zn2+. By showing that TRPC6 channels constitute a Zn2+ entry pathway, our study suggests that they could play a role in zinc homeostasis.

Functional evidence of TRPV4‐mediated Ca2+ signals in cortical astrocytes

A rise in intracellular calcium ([Ca2+]i) in astrocytes is required to communicate the energy demands of neurons to the cerebral microcirculation to precisely match local cerebral blood flow to neuronal metabolism. This “neurovascular coupling” is essential for neuronal viability and homeostasis. A moderate rise in [Ca2+]i in the perivascular “endfoot” terminals of astrocytes in response to neuronal activation produces vasodilation of the adjacent parenchymal arteriole, whereas high endfoot [Ca2+]i can produce vasoconstriction. Thus, precise regulation of astrocytic [Ca2+]i is necessary for proper function of the cerebral microcirculation. Transient receptor potential vanilloid 4 (TRPV4) channels are Ca2+‐permeant plasma membrane cation channels that participate in intracellular Ca2+ signaling in a variety of cell types. While there is some immunohistochemical evidence for TRPV4 expression, there is no evidence of functional TRPV4 channels in astrocytes. Here we provide evidence of TRPV4‐mediated Ca2+ oscillations in endfeet of cortical astrocytes obtained by multiphoton confocal microscopy in mouse brain slices. Activation of TRPV4 channels with the selective agonist GSK1016790A concentration‐dependently increased the amplitude of Ca2+ oscillations in astrocytic endfeet. Blocking neuron‐to‐astrocyte communication did not inhibit this effect, suggesting a direct effect of TRPV4 activation on astrocytic endfoot Ca2+. Additionally, the effect of TRPV4 activation on endfoot Ca2+ oscillations was blocked by the selective TRPV4 antagonist HC067047. These results provide evidence of functional TRPV4 channels in cortical astrocytes, and suggest that TRPV4 may play a role in regulating local cerebral blood flow through effects on astrocytic endfoot Ca2+. This study was supported by National Heart, Lung, and Blood Institute grant HL44455.

Endocannabinoids as Regulators of Transient Receptor Potential (TRP)Channels: a Further Opportunity to Develop New Endocannabinoid-Based Therapeutic Drugs

In the late 1990's, a series of experiments carried out independently in two laboratories led to establish an important connection between the function of the endocannabinoids, which, as exemplified in this special issue, is per se very complex and ubiquitous in animals, and that of the transient receptor potential (TRP) channels, a large family of plasma membrane cation channels involved in several mammalian and non-mammalian physiological and pathological conditions, overlapping only in part with those in which the cannabinoid receptors participate. These experiments were initially based on the observation that the endocannabinoid anandamide and the xenobiotic ligand of TRP channels of V1 type (TRPV1), capsaicin, are somehow chemically similar, both compounds being fatty acid amides, as are also synthetic activators of these channels and inhibitors of anandamide cellular re-uptake. As discussed in this article, the same type of "chemical thoughts" led to the discovery of N-arachidonoyl-dopamine, an endogenous ligand of TRPV1 channels that behaves also an endocannabinoid. The overlap between the ligand recognition properties of some TRP channels and proteins of the endocannabinoid system, namely the cannabinoid receptors and the proteins and enzymes catalyzing anandamide cellular re-uptake and hydrolysis, is being actively explored through the rational design and synthesis of new endocannabinoid-based drugs with multiple mechanisms of action. These aspects are discussed in this review article, together with the possible functional and pharmacological consequences of endocannabinoid-TRP channel interactions.