Discrete Control of TRPV4 Channel Function in the Distal Nephron by Protein Kinases A and C

We have recently documented that the Ca²⁺-permeable TRPV4 channel, which is abundantly expressed in distal nephron cells, mediates cellular Ca²⁺ responses to elevated luminal flow. In this study, we combined Fura-2-based [Ca²⁺]_i imaging with immunofluorescence microscopy in isolated split-opened distal nephrons of C57BL/6 mice to probe the molecular determinants of TRPV4 activity and subcellular distribution. We found that activation of the PKC pathway with phorbol 12-myristate 13-acetate significantly increased [Ca²⁺]_i responses to flow without affecting the subcellular distribution of TRPV4. Inhibition of PKC with bisindolylmaleimide I diminished cellular responses to elevated flow. In contrast, activation of the PKA pathway with forskolin did not affect TRPV4-mediated [Ca²⁺]_i responses to flow but markedly shifted the subcellular distribution of the channel toward the apical membrane. These actions were blocked with the specific PKA inhibitor H-89. Concomitant activation of the PKA and PKC cascades additively enhanced the amplitude of flow-induced [Ca²⁺]_i responses and greatly increased basal [Ca²⁺]_i levels, indicating constitutive TRPV4 activation. This effect was precluded by the selective TRPV4 antagonist HC-067047. Therefore, the functional status of the TRPV4 channel in the distal nephron is regulated by two distinct signaling pathways. Although the PKA-dependent cascade promotes TRPV4 trafficking and translocation to the apical membrane, the PKC-dependent pathway increases the activity of the channel on the plasma membrane.     

Capsaicin inhibits intestinal Cl- secretion and promotes Na+ absorption by blocking TRPV4 channels in healthy and colitic mice

Although capsaicin has been studied extensively as an activator of the transient receptor potential vanilloid cation channel subtype 1 (TRPV1) channels in sensory neurons, little is known about its TRPV1-independent actions in gastrointestinal health and disease. Here, we aimed to investigate the pharmacological actions of capsaicin as a food additive and medication on intestinal ion transporters in mouse models of ulcerative colitis (UC). The short-circuit current (I_sc) of the intestine from WT, TRPV1-, and TRPV4-KO mice were measured in Ussing chambers, and Ca²⁺ imaging was performed on small intestinal epithelial cells. We also performed Western blots, immunohistochemistry, and immunofluorescence on intestinal epithelial cells and on intestinal tissues following UC induction with dextran sodium sulfate. We found that capsaicin did not affect basal intestinal I_sc but significantly inhibited carbachol- and caffeine-induced intestinal I_sc in WT mice. Capsaicin similarly inhibited the intestinal I_sc in TRPV1 KO mice, but this inhibition was absent in TRPV4 KO mice. We also determined that Ca²⁺ influx via TRPV4 was required for cholinergic signaling–mediated intestinal anion secretion, which was inhibited by capsaicin. Moreover, the glucose-induced jejunal I_scvia Na⁺/glucose cotransporter was suppressed by TRPV4 activation, which could be relieved by capsaicin. Capsaicin also stimulated ouabain- and amiloride-sensitive colonic I_sc. Finally, we found that dietary capsaicin ameliorated the UC phenotype, suppressed hyperaction of TRPV4 channels, and rescued the reduced ouabain- and amiloride-sensitive I_sc. We therefore conclude that capsaicin inhibits intestinal Cl^- secretion and promotes Na⁺ absorption predominantly by blocking TRPV4 channels to exert its beneficial anti-colitic action.     

Activation of TRPV4 channel in pancreatic INS-1E beta cells enhances glucose-stimulated insulin secretion via calcium-dependent mechanisms

Transient receptor potential channel vanilloid type 4 (TRPV4) is a Ca²⁺- and Mg²⁺-permeable cation channel that influences oxidative metabolism and insulin sensitivity. The role of TRPV4 in pancreatic beta cells is largely unknown. Here, we characterize the role of TRPV4 in controlling intracellular Ca²⁺ and insulin secretion in INS-1E beta cells. Osmotic, thermal or pharmacological activation of TRPV4 caused a rapid rise of intracellular Ca²⁺ and enhanced glucose-stimulated insulin secretion. In the presence of the TRPV channel blocker ruthenium red (RuR) or after suppression of TRPV4 protein production, TRPV4 activators failed to increase [Ca²⁺]_i and insulin secretion in INS-1E cells.     

Concerted actions of NHERF2 and WNK4 in regulating TRPV5

With-no-lysine (K) kinase 4 (WNK4) is a protein serine/threonine kinase associated with a Mendelian form of hypertension. WNK4 is an integrative regulator of renal transport of Na⁺, K⁺, and Cl⁻ as shown in Xenopus oocyte system. In addition, WNK4 enhances the surface expression of epithelial Ca²⁺ channel TRPV5, which plays a key role in the fine tuning of renal Ca²⁺ reabsorption. Variations in the magnitude of WNK4-mediated regulation on TRPV5 in Xenopus oocytes suggest additional cellular components with limited expression are required for the regulation. In this study, we identified the Na⁺/H⁺ exchanger regulating factor 2 (NHERF2) as a critical component for the positive regulation of TRPV5 by WNK4. NHERF2 augmented the positive effect of WNK4 on TRPV5, whereas its homolog NHERF1 had no effect when tested in the Xenopus oocyte system. The C-terminal PDZ binding motif of TRPV5 was required for the regulation by NHERF2. While NHERF2 interacted with TRPV5, no association between NHERF2 and WNK4 was detected using a GST pull-down assay. WNK4 increased the forward trafficking of TRPV5; however, it also caused an accelerated decline of the functional TRPV5 channels at later stage of co-expression. NHERF2 stabilized TRPV5 at the plasma membrane without interrupting the forward trafficking of TRPV5, thus prevented the decline of functional TRPV5 channel caused by WNK4 at later stage. The complementary and orderly regulations of WNK4 and NHERF2 allow TRPV5 functions at higher level for a longer period to maximize Ca²⁺ influx.     

A Highlights from MBoC Selection: Regulation of the epithelial Ca2+ channel TRPV5 by reversible histidine phosphorylation mediated by NDPK-B and PHPT1

The kidney, together with bone and intestine, plays a crucial role in maintaining whole-body calcium (Ca²⁺) homoeostasis, which is primarily mediated by altering the reabsorption of Ca²⁺ filtered by the glomerulus. The transient receptor potential-vanilloid-5 (TRPV5) channel protein forms a six- transmembrane Ca²⁺-permeable channel that regulates urinary Ca²⁺ excretion by mediating active Ca²⁺ reabsorption in the distal convoluted tubule of the kidney. Here we show that the histidine kinase, nucleoside diphosphate kinase B (NDPK-B), activates TRPV5 channel activity and Ca²⁺ flux, and this activation requires histidine 711 in the carboxy-terminal tail of TRPV5. In addition, the histidine phosphatase, protein histidine phosphatase 1, inhibits NDPK-B–activated TRPV5 in inside/out patch experiments. This is physiologically relevant to Ca²⁺ reabsorption in vivo, as short hairpin RNA knockdown of NDPK-B leads to decreased TRPV5 channel activity, and urinary Ca²⁺ excretion is increased in NDPK-B^−/− mice fed a high-Ca²⁺ diet. Thus these findings identify a novel mechanism by which TRPV5 and Ca²⁺ reabsorption is regulated by the kidney and support the idea that histidine phosphorylation plays other, yet-uncovered roles in mammalian biology.     

Modulation of TRPV4 gating by intra- and extracellular Ca

We have studied the modulation of gating properties of the Ca²⁺-permeable, cation channel TRPV4 transiently expressed in HEK293 cells. The phorbol ester 4αPDD transiently activated a current through TRPV4 in the presence of extracellular Ca²⁺. Increasing the concentration of extracellular Ca²⁺ ([Ca²⁺]_e) reduced the current amplitude and accelerated its decay. This decay was dramatically delayed in the absence of [Ca²⁺]_e. It was also much slower in the presence of [Ca²⁺]_e in a mutant channel, obtained by a point mutation in the 6th transmembrane domain, F707A. Mutant channels, containing a single mutation in the C-terminus of TRPV4 (E797), were constitutively open. In conclusion, gating of the 4αPDD-activated TRPV4 channel depends on both extra- and intracellular Ca²⁺, and is modulated by mutations of single amino acid residues in the 6th transmembrane domain and the C-terminus of the TRPV4 protein.     

TRPV1 stimulation triggers apoptotic cell death of rat cortical neurons

Transient receptor potential vanilloid 1 (TRPV1) functions as a polymodal nociceptor and is activated by several vanilloids, including capsaicin, protons and heat. Although TRPV1 channels are widely distributed in the brain, their roles remain unclear. Here, we investigated the roles of TRPV1 in cytotoxic processes using TRPV1-expressing cultured rat cortical neurons. Capsaicin induced severe neuronal death with apoptotic features, which was completely inhibited by the TRPV1 antagonist capsazepine and was dependent on extracellular Ca²⁺ influx. Interestingly, nifedipine, a specific L-type Ca²⁺ channel blocker, attenuated capsaicin cytotoxicity, even when applied 2-4 h after the capsaicin. ERK inhibitor PD98059 and several antioxidants, but not the JNK and p38 inhibitors, attenuated capsaicin cytotoxicity. Together, these data indicate that TRPV1 activation triggers apoptotic cell death of rat cortical cultures via L-type Ca²⁺ channel opening, Ca²⁺ influx, ERK phosphorylation, and reactive oxygen species production.     

P2Y1 Receptor Activation of the TRPV4 Ion Channel Enhances Purinergic Signaling in Satellite Glial Cells

Transient receptor potential (TRP) ion channels of peripheral sensory pathways are important mediators of pain, itch, and neurogenic inflammation. They are expressed by primary sensory neurons and by glial cells in the central nervous system, but their expression and function in satellite glial cells (SGCs) of sensory ganglia have not been explored. SGCs tightly ensheath neurons of sensory ganglia and can regulate neuronal excitability in pain and inflammatory states. Using a modified dissociation protocol, we isolated neurons with attached SGCs from dorsal root ganglia of mice. SGCs, which were identified by expression of immunoreactive Kir4.1 and glutamine synthetase, were closely associated with neurons, identified using the pan-neuronal marker NeuN. A subpopulation of SGCs expressed immunoreactive TRP vanilloid 4 (TRPV4) and responded to the TRPV4-selective agonist GSK1016790A by an influx of Ca²⁺ ions. SGCs did not express functional TRPV1, TRPV3, or TRP ankyrin 1 channels. Responses to GSK1016790A were abolished by the TRPV4 antagonist HC067047 and were absent in SGCs from Trpv4^−/− mice. The P2Y₁-selective agonist 2-methylthio-ADP increased [Ca²⁺]_i in SGCs, and responses were prevented by the P2Y1-selective antagonist MRS2500. P2Y₁ receptor-mediated responses were enhanced in TRPV4-expressing SGCs and HEK293 cells, suggesting that P2Y₁ couples to and activates TRPV4. PKC inhibitors prevented P2Y₁ receptor activation of TRPV4. Our results provide the first evidence for expression of TRPV4 in SGCs and demonstrate that TRPV4 is a purinergic receptor-operated channel in SGCs of sensory ganglia.     

Thermo-sensitive transient receptor potential vanilloid channel-1 regulates intracellular calcium and triggers chromogranin A secretion in pancreatic neuroendocrine BON-1 tumor cells

Transient receptor potential channels (TRPs) regulate tumor growth via calcium-dependent mechanisms. The (thermosensitive) capsaicin receptor TRPV1 is overexpressed in numerous highly aggressive cancers. TRPV1 has potent antiproliferative activity and is therefore potentially applicable in targeted therapy of malignancies. Recently, we characterized TRPM8 functions in pancreatic neuroendocrine tumors (NETs), however, the role of TRPV1 is unknown. Here, we studied the expression and the role of TRPV1 in regulating intracellular Ca²⁺ and chromogranin A (CgA) secretion in pancreatic NET BON-1 cell line and in primary NET cells (prNET). TRPV1 expression was detected by RT-PCR, Western blot and immunofluorescence. Intracellular free Ca²⁺ ([Ca²⁺]_i) was measured by fura-2; TRPV1 channel currents by the planar patch-clamp technique. Nonselective cation currents were analyzed by a color-coded plot method and CgA secretion by ELISA. Pancreatic BON-1 cells and NETs express TRPV1. Pharmacological blockade of TRPs by La³⁺ (100 μM) or by ruthenium-red (RuR) or by capsazepine (CPZ) (both at 10 μM) suppressed the capsaicin (CAP)- or heat-stimulated increase of [Ca²⁺]_i in NET cells. CAP (20 μM) also increased nonselective cation channel currents in BON-1 cells. Furthermore, CAP (10 μM) stimulated CgA secretion, which was inhibited by CPZ or by RuR (both 10 μM). La³⁺ potently reduced both stimulated and the basal CgA secretion. Our study shows for the first time that TRPV1 is expressed in pancreatic NETs. Activation of TRPV1 translates into changes of intracellular Ca²⁺, a known mechanism triggering the secretion of CgA. The clinical relevance of TRPV1 activation in NETs requires further investigations.     

The puzzle of TRPV4 channelopathies

Hereditary channelopathies, that is, mutations in channel genes that alter channel function and are causal for the pathogenesis of the disease, have been described for several members of the transient receptor potential channel family. Mutations in the TRPV4 gene, encoding a polymodal Ca²⁺ permeable channel, are causative for several human diseases, which affect the skeletal system and the peripheral nervous system, with highly variable phenotypes. In this review, we describe the phenotypes of TRPV4 channelopathies and overlapping symptoms. Putative mechanisms to explain the puzzle, and how mutations in the same region of the channel cause different diseases, are discussed and experimental approaches to tackle this surprising problem are suggested.     

Down-regulation of Intestinal Apical Calcium Entry Channel TRPV6 by Ubiquitin E3 Ligase Nedd4-2

Nedd4-2 is an archetypal HECT ubiquitin E3 ligase that disposes target proteins for degradation. Because of the proven roles of Nedd4-2 in degradation of membrane proteins, such as epithelial Na⁺ channel, we examined the effect of Nedd4-2 on the apical Ca²⁺ channel TRPV6, which is involved in transcellular Ca²⁺ transport in the intestine using the Xenopus laevis oocyte system. We demonstrated that a significant amount of Nedd4-2 protein was distributed to the absorptive epithelial cells in ileum, cecum, and colon along with TRPV6. When co-expressed in oocytes, Nedd4-2 and, to a lesser extent, Nedd4 down-regulated the protein abundance and Ca²⁺ influx of TRPV6 and TRPV5, respectively. TRPV6 ubiquitination was increased, and its stability was decreased by Nedd4-2. The Nedd4-2 inhibitory effects on TRPV6 were partially blocked by proteasome inhibitor MG132 but not by the lysosome inhibitor chloroquine. The rate of TRPV6 internalization was not significantly altered by Nedd4-2. The HECT domain was essential to the inhibitory effect of Nedd4-2 on TRPV6 and to their association. The WW1 and WW2 domains interacted with TRPV6 terminal regions, and a disruption of the interactions by D204H and D376H mutations in the WW1 and WW2 domains increased TRPV6 ubiquitination and degradation. Thus, WW1 and WW2 may serve as a molecular switch to limit the ubiquitination of TRPV6 by the HECT domain. In conclusion, Nedd4-2 may regulate TRPV6 protein abundance in intestinal epithelia by controlling TRPV6 ubiquitination.     

Modulation of TRPV4 and BKCa for treatment of brain diseases

As a member of transient receptor potential family, the transient receptor potential vanilloid 4 (TRPV4) is a kind of nonselective calcium-permeable cation channel, which belongs to non-voltage gated Ca²⁺ channel. Large-conductance Ca²⁺-activated K⁺ channel (BKCa) represents a unique superfamily of Ca²⁺-activated K⁺ channel (KCa) that is both voltage and intracellular Ca²⁺ dependent. Not surprisingly, aberrant function of either TRPV4 or BKCa in neurons has been associated with brain disorders, such as Alzheimer’s disease, cerebral ischemia, brain tumor, epilepsy, as well as headache. In these diseases, vascular dysfunction is a common characteristic. Notably, endothelial and smooth muscle TRPV4 can mediate BKCa to regulate cerebral blood flow and pressure. Therefore, in this review, we not only discuss the diverse functions of TRPV4 and BKCa in neurons to integrate relative signaling pathways in the context of cerebral physiological and pathological situations respectively, but also reveal the relationship between TRPV4 and BKCa in regulation of cerebral vascular tone as an etiologic factor. Based on these analyses, this review demonstrates the effective mechanisms of compounds targeting these two channels, which may be potential therapeutic strategies for diseases in the brain.     

A conserved gating element in TRPV6 channels

The Ca²⁺-selective tetrameric Transient Receptor Potential Vanilloid 6 (TRPV6) channel is an inwardly rectifying ion channel. The constitutive current endures Ca²⁺-induced inactivation as a result of the activation of phospholipase C followed depletion of phosphatidylinositol 4,5-bisphosphate, and calmodulin binding. Replacing a glycine residue within the cytosolic S4-S5 linker of the human TRPV6 protein, glycine 516, which is conserved in all TRP channel proteins, by a serine residue forces the channels into an open conformation thereby enhancing constitutive Ca²⁺ entry and preventing inactivation. Introduction of a second mutation (T621A) into TRPV6_G516S reduces constitutive activity and partially rescues the TRPV6 function. According to the recently revealed crystal structure of the rat TRPV6 the T621 is adjacent to the distal end of the transmembrane segment 6 (S6) within a short linker between S6 and the helix formed by the TRP domain. These results indicate that the S4-S5 linker and the S6-TRP-domain linker are critical constituents of TRPV6 channel gating and that disturbance of their sequences foster constitutive Ca²⁺ entry.     

The Identification of Histidine 712 as a Critical Residue for Constitutive TRPV5 Internalization

The epithelial Ca²⁺ channel TRPV5 constitutes the apical entry gate for Ca²⁺ transport in renal epithelial cells. Ablation of the trpv5 gene in mice leads to a reduced Ca²⁺ reabsorption. TRPV5 is tightly regulated by various calciotropic hormones, associated proteins, and other factors, which mainly affect channel activity via the C terminus. To further identify the role of the C terminus in TRPV5 regulation, we expressed channels harboring C-terminal deletions and studied channel activity by measuring intracellular Ca²⁺ concentration ([Ca²⁺]_i) using fura-2 analysis. Removal of amino acid His⁷¹² elevated the [Ca²⁺]_i, indicating enlarged TRPV5 activity. In addition, substitution of the positively charged His⁷¹² for a negative (H712D) or neutral (H712N) amino acid also stimulated TRPV5 activity. This critical role of His⁷¹² was confirmed by patch clamp analysis, which demonstrates increased Na⁺ and Ca²⁺ currents for TRPV5-H712D. Cell surface biotinylation studies revealed enhanced plasma membrane expression of TRPV5-H712D as compared with wild-type (WT) TRPV5. This elevated plasma membrane presence also was observed with the Ca²⁺-impermeable TRPV5-H712D and TRPV5-WT pore mutants, demonstrating that the elevation is not due to the increased [Ca²⁺]_i. Finally, using an internalization assay, we demonstrated a delayed cell surface retrieval for TRPV5-H712D, likely causing the increase in plasma membrane expression. Together, these results demonstrate that His⁷¹² plays an essential role in plasma membrane regulation of TRPV5 via a constitutive endocytotic mechanism.     

Loss of Function of Transient Receptor Potential Vanilloid 1 (TRPV1) Genetic Variant Is Associated with Lower Risk of Active Childhood Asthma

Transient receptor potential cation channels of the vanilloid subfamily (TRPV) participate in the generation of Ca²⁺ signals at different locations of the respiratory system, thereby controlling its correct functioning. TRPV1 expression and activity appear to be altered under pathophysiological conditions such as chronic cough and airway hypersensitivity, whereas TRPV4 single nucleotide polymorphisms (SNP) are associated with chronic obstructive pulmonary disease. However, to date, there is no information about the genetic impact of either TRPV1 or TRPV4 on asthma pathophysiology. We now report on the association of two functional SNPs, TRPV1-I585V and TRPV4-P19S, with childhood asthma. Both SNPs were genotyped in a population of 470 controls without respiratory symptoms and 301 asthmatics. Although none of the SNPs modified the risk of suffering from asthma, carriers of the TRPV1-I585V genetic variant showed a lower risk of current wheezing (odds ratio = 0.51; p = 0.01), a characteristic of active asthma, or cough (odds ratio = 0.57; p = 0.02). Functional analysis of TRPV1-I585V, using the Ca²⁺-sensitive dye fura-2 to measure intracellular [Ca²⁺] concentrations, revealed a decreased channel activity in response to two typical TRPV1 stimuli, heat and capsaicin. On the other hand, TRPV4-P19S, despite its loss-of-channel function, showed no significant association with asthma or the presence of wheezing. Our data suggest that genetically determined level of TRPV1 activity is relevant for asthma pathophysiology.     

Basal cells express functional TRPV4 channels in the mouse nasal epithelium

Basal cells in the nasal epithelium (olfactory and airway epithelia) are stem/progenitor cells that are capable of dividing, renewing and differentiating into specialized cells. These stem cells can sense their biophysical microenvironment, but the underlying mechanism of this process remains unknown. Here, we demonstrate the prominent expression of the transient receptor potential vanilloid type 4 (TRPV4) channel, a Ca²⁺-permeable channel that is known to act as a sensor for hypo-osmotic and mechanical stresses, in the basal cells of the mouse nasal epithelium. TRPV4 mRNA was expressed in the basal portions of the prenatal mouse nasal epithelium, and this expression continued into adult mice. The TRPV4 protein was also detected in the basal layers of the nasal epithelium in wild-type but not in TRPV4-knockout (TRPV4-KO) mice. The TRPV4-positive immunoreactions largely overlapped with those of keratin 14 (K14), a marker of basal cells, in the airway epithelium, and they partially overlapped with those of K14 in the olfactory epithelium. Ca²⁺ imaging analysis revealed that hypo-osmotic stimulation and 4α-phorbol 12,13 didecanoate (4α-PDD), both of which are TRPV4 agonists, caused an increase in the cytosolic Ca²⁺ concentration in a subset of primary epithelial cells cultured from the upper parts of the nasal epithelium of the wild-type mice. This response was barely noticeable in cells from similar parts of the epithelium in TRPV4-KO mice. Finally, there was no significant difference in BrdU-labeled proliferation between the olfactory epithelia of wild-type and TRPV4-KO mice under normal conditions. Thus, TRPV4 channels are functionally expressed in basal cells throughout the nasal epithelium and may act as sensors for the development and injury-induced regeneration of basal stem cells.     

The TRPV4 Channel Contributes to Intercellular Junction Formation in Keratinocytes

Transient receptor potential vanilloid 4 (TRPV4) channel is a physiological sensor for hypo-osmolarity, mechanical deformation, and warm temperature. The channel activation leads to various cellular effects involving Ca²⁺ dynamics. We found that TRPV4 interacts with β-catenin, a crucial component linking adherens junctions and the actin cytoskeleton, thereby enhancing cell-cell junction development and formation of the tight barrier between skin keratinocytes. TRPV4-deficient mice displayed impairment of the intercellular junction-dependent barrier function in the skin. In TRPV4-deficient keratinocytes, extracellular Ca²⁺-induced actin rearrangement and stratification were delayed following significant reduction in cytosolic Ca²⁺ increase and small GTPase Rho activation. TRPV4 protein located where the cell-cell junctions are formed, and the channel deficiency caused abnormal cell-cell junction structures, resulting in higher intercellular permeability in vitro. Our results suggest a novel role for TRPV4 in the development and maturation of cell-cell junctions in epithelia of the skin.     

Nerve Growth Factor Regulates Transient Receptor Potential Vanilloid 2 via Extracellular Signal-Regulated Kinase Signaling To Enhance Neurite Outgrowth in Developing Neurons

Neurite outgrowth is key to the formation of functional circuits during neuronal development. Neurotrophins, including nerve growth factor (NGF), increase neurite outgrowth in part by altering the function and expression of Ca²⁺-permeable cation channels. Here we report that transient receptor potential vanilloid 2 (TRPV2) is an intracellular Ca²⁺-permeable TRPV channel upregulated by NGF via the mitogen-activated protein kinase (MAPK) signaling pathway to augment neurite outgrowth. TRPV2 colocalized with Rab7, a late endosome protein, in addition to TrkA and activated extracellular signal-regulated kinase (ERK) in neurites, indicating that the channel is closely associated with signaling endosomes. In line with these results, we showed that TRPV2 acts as an ERK substrate and identified the motifs necessary for phosphorylation of TRPV2 by ERK. Furthermore, neurite length, TRPV2 expression, and TRPV2-mediated Ca²⁺ signals were reduced by mutagenesis of these key ERK phosphorylation sites. Based on these findings, we identified a previously uncharacterized mechanism by which ERK controls TRPV2-mediated Ca²⁺ signals in developing neurons and further establish TRPV2 as a critical intracellular ion channel in neuronal function.