Age-related changes in the distribution of transient receptor potential vanilloid 4 channel (TRPV4) in the central nervous system of rats.

Transient receptor potential vanilloid type 4 (TRPV4) channel is expressed in the central nervous system and its role in development of Alzheimer’s disease (AD) is largely unknown. To identify AD-related changes in the TRPV4 channel distribution in the central nervous system, we investigated the distribution and level changes of TRPV4 in brains of AD model mice. The expressions of TRPV4 in the brain of control mice, early stage and late stage AD model mice were compared using immunohistochemistry with antibodies recognizing TRPV4 on free floating sections and in addition we performed western blotting to supplement our findings. TRPV4 immunoreactivity was significantly increased in the cerebral cortex, hippocampal formation, striatum and thalamus of AD model mice compared with control mice. In the cerebral cortex, TRPV4 immunoreactivity was significantly increased in pyramidal cells of early stage and late stage AD model mice. In addition, TRPV4 immunoreactivity was increased in the hippocampal formation, striatum and thalamus of late stage AD model mice. This is the first demonstration of AD-related increases in TRPV4 expression in the brain and it may provide useful data for investigating the pathogenesis of AD-related neurodegenerative diseases. The regulation of TRPV4 in AD mouse model and its functional significance require further investigation.

Currently, the role of transient receptor potential vanilloid type 4 (TRPV4), a nonselective cation channel in the pathology of spinal cord injury (SCI), is not recognized. Herein, we report the expression and contribution of TRPV4 in the pathology of scarring and endothelial and secondary damage after SCI. TRPV4 expression increased during the inflammatory phase in female rats after SCI and was expressed primarily by cells at endothelial-microglial junctions. Two-photon microscopy of intracellular-free Ca2+ levels revealed a biphasic increase at similar time points after SCI. Expression of TRPV4 at the injury epicenter, but not intracellular-free Ca2+, progressively increases with the severity of the injury. Activation of TRPV4 with specific agonist altered the organization of endothelial cells, affected tight junctions in the hCMEC/D3 BBB cell line in vitro, and increases the scarring in rat spinal cord as well as induced endothelial damage. By contrast, suppression of TRPV4 with a specific antagonist or in female Trpv4 KO mouse attenuated inflammatory cytokines and chemokines, prevented the degradation of tight junction proteins, and preserve blood-spinal cord barrier integrity, thereby attenuate the scarring after SCI. Likewise, secondary damage was reduced, and behavioral outcomes were improved in Trpv4 KO mice after SCI. These results suggest that increased TRPV4 expression disrupts endothelial cell organization during the early inflammatory phase of SCI, resulting in tissue damage, vascular destabilization, blood-spinal cord barrier breakdown, and scarring. Thus, TRPV4 inhibition/knockdown represents a promising therapeutic strategy to stabilize/protect endothelial cells, attenuate nociception and secondary damage, and reduce scarring after SCI.SIGNIFICANCE STATEMENT TRPV4, a calcium-permeable nonselective cation channel, is widely expressed in both excitable and nonexcitable cells. Spinal cord injury (SCI) majorly caused by trauma/accidents is associated with changes in osmolarity, mechanical injury, and shear stress. After SCI, TRPV4 was increased and were found to be linked with the severity of injury at the epicenter at the time points that were reported to be critical for repair/treatment. Activation of TRPV4 was damaging to endothelial cells that form the blood-spinal cord barrier and thus contributes to scarring (glial and fibrotic). Importantly, inhibition/knockdown of TRPV4 prevented these effects. Thus, the manipulation of TRPV4 signaling might lead to new therapeutic strategies or combinatorial therapies to protect endothelial cells and enhance repair after SCI.

Chapter 19 - The neuroscience of transient receptor potential vanilloid type 4 (TRPV4) and spinal cord injury

Bladder cancer (BLCA) is the second most common urologic cancer in the United States and worldwide and mostly affects the aging population. Despite several ongoing clinical trials, treatment paradigms for BLCA have not changed significantly. Here, we investigated the expression of transient receptor potential vanilloid type 4 (TRPV4) in patients with BLCA and its role in calcium influx, cell proliferation, and migration using normal human urothelial cells and BLCA cells. Bioinformatic analysis of the University of Alabama at Birmingham Cancer Data Analysis Portal and cBioPortal databases revealed that TRPV4 expression is significantly higher in human BLCA tissues than in normal adjacent tissues. Furthermore, TRPV4 expression was markedly elevated in early-stage BLCA and upregulated in muscle-invasive bladder cancer tissues. TRPV4 is expressed in both normal urothelial (SV-HUC-1) and BLCA (T-24) cells, and functional assays demonstrated enhanced TRPV4-mediated calcium influx in T-24 compared with SV-HUC-1 cells. T-24 cells exhibited higher spreading on extracellular matrix gels with increasing stiffness (0.2, 8, and 50 kPa) and exhibited a migratory phenotype compared to SV-HUC-1 cells. Pharmacological inhibition of TRPV4 significantly reduced proliferation and migration in T-24 cells but had minimal effects on normal cells. Finally, treatment with cisplatin significantly reduced TRPV4 protein levels and TRPV4-mediated calcium influx in chemosensitive UM-UC-3 cells but remained unchanged in chemoresistant T-24 cells, suggesting a potential role of TRPV4 in chemoresistance. In conclusion, TRPV4 may contribute to BLCA progression by regulating cell proliferation and migration and may impart resistance to chemotherapy. Targeting TRPV4 could present a novel therapeutic approach for managing BLCA progression and overcoming chemoresistance.Significance StatementThis study identified transient receptor potential vanilloid type 4 (TRPV4) as a critical driver of bladder cancer (BLCA) progression. TRPV4 gene expression is elevated in both early-stage and muscle-invasive BLCA tissues. Importantly, TRPV4 inhibition selectively reduces BLCA growth and motility. Furthermore, TRPV4 is downregulated by cisplatin in chemosensitive but not chemoresistant BLCA cells, underscoring its key role in bladder cancer chemoresistance. These findings position TRPV4 as a therapeutic target for enhancing BLCA treatment and overcoming drug resistance.

Functional change in the 5-HT presynaptic receptor in spinal cord of aged rats

Background: Right ventricular failure (RVF) is a common and fatal consequence of pulmonary hypertension (PH) and is a major determinant of morbidity and mortality. The transient receptor potential vanilloid type 4 (TRPV4) channel is a Ca2+ permeable, nonselective cation channel. The role of TRPV4 in PH-induced RVF (PH-RVF) is yet to be elucidated. Hypothesis: TRPV4 activation mediates PH-RVF and TRPV4 inhibition may rescue PH-RVF via reducing RV hypertrophy, fibrosis and inflammation. Methods: Male SD rats received s.c. Monocrotaline (MCT, 60mg/kg, n=9; 30-days), Sugen (20mg/kg, n=9; 3-wk hypoxia+2-wk normoxia; SuHx) or PBS (CTRL, n=9). Some MCT rats received TRPV4 agonist (GSK790A) or vehicle from day 0-30 while others received TRPV4 specific antagonist HC067047 or vehicle from day 14-30 (n=5). Echo, cath, RV-RNASeq, qPCR, WB, and IF validation were performed. RNASeq analysis was performed on human decompensated RVF vs Control (GSE198618), and human RVs were assessed for fibrosis and inflammation. Human cardiac fibroblasts (HCFs) were treated with TRPV4 agonist (under normoxia) and antagonist (under hypoxia+TGFβ1). Results: RNA-sequencing demonstrated an increase in hypertrophic, inflammatory, and fibrotic pathways and genes and an increase in TRPV4 expression in rat MCT, SuHx, PAB and human compensated and decompensated RVF compared to controls. RNA-sequencing also demonstrated increased downstream mediators of TRPV4 (collagens, ANP, BNP, inflammatory mediators) in rats and human RVF. Immunofluorescence imaging demonstrated increased colocalization of TRPV4 in RV cardiomyocytes, fibroblasts, and macrophages in PH-RVF. Interestingly, there was no significant increase in TRPV4 expression in the lungs of MCT and SuHx rats compared to controls. TRPV4 specific agonist GSK790A induced early PH-RVF in MCT rats in vivo and fibroblast to myofibroblast transition (FB-MFB) in vitro. Additionally, TRPV4-specific antagonist HC067047 rescued PH-RVF in MCT rats by improving RV function and reducing RV hypertrophy, fibrosis, and inflammation. Finally, TRPV4 specific antagonist GSK3874 inhibited FB-MFB transition in vitro and induced reversal of pro-fibrotic and inflammatory transcriptomic signatures in HCF. Conclusions: Pharmacological inhibition of TRPV4 rescues PH-RVF via reducing RV hypertrophy, fibrosis and inflammation.

Activation of the protease-activated receptor 2 (PAR2) or the transient receptor potential vanilloid type 1 (TRPV1) channels expressed in cardiac sensory afferents containing calcitonin gene-related peptide (CGRP) and/or substance P (SP) has been proposed to play a protective role in myocardial ischemia-reperfusion (I/R) injury. However, the interaction between PAR2 and TRPV1 is largely unknown. Using gene-targeted TRPV1-null mutant (TRPV1(-/-)) or wild-type (WT) mice, we test the hypothesis that TRPV1 contributes to PAR2-mediated cardiac protection via increasing the release of CGRP and SP. Immunofluorescence labeling showed that TRPV1 coexpressed with PAR2, PKC-epsilon, or PKAc in cardiomyocytes, cardiac blood vessels, and perivascular nerves in WT but not TRPV1(-/-) hearts. WT or TRPV1(-/-) hearts were Langendorff perfused with the selective PAR2 agonist, SLIGRL, in the presence or absence of various antagonists, followed by 35 min of global ischemia and 40 min of reperfusion (I/R). The recovery rate of coronary flow, the maximum rate of left ventricular pressure development, left ventricular end-diastolic pressure, and left ventricular developed pressure were evaluated after I/R. SLIGRL improved the recovery of hemodynamic parameters, decreased lactate dehydrogenase release, and reduced the infarct size in both WT and TRPV1(-/-) hearts (P < 0.05). The protection of SLIGRL was significantly surpassed for WT compared with TRPV1(-/-) hearts (P < 0.05). CGRP(8-37), a selective CGRP receptor antagonist, RP67580, a selective neurokinin-1 receptor antagonist, PKC-epsilon V1-2, a selective PKC-epsilon inhibitor, or H-89, a selective PKA inhibitor, abolished SLIGRL protection by inhibiting the recovery of the rate of coronary flow, maximum rate of left ventricular pressure development, and left ventricular developed pressure, and increasing left ventricular end-diastolic pressure in WT but not TRPV1(-/-) hearts. Radioimmunoassay showed that SLIGRL increased the release of CGRP and SP in WT but not TRPV1(-/-) hearts (P < 0.05), which were prevented by PKC-epsilon V1-2 and H-89. Thus our data show that PAR2 activation improves cardiac recovery after I/R injury in WT and TRPV1(-/-) hearts, with a greater effect in the former, suggesting that PAR2-mediated protection is TRPV1 dependent and independent, and that dysfunctional TRPV1 impairs PAR2 action. PAR2 activation of the PKC-epsilon or PKA pathway stimulates or sensitizes TRPV1 in WT hearts, leading to the release of CGRP and SP that contribute, at least in part, to PAR2-induced cardiac protection against I/R injury.

Na+ -K+ -2Cl- co-transporter (NKCC1) plays an important role in traumatic brain injury (TBI)-induced brain edema via the MAPK cascade. The transient receptor potential vanilloid type 4 (TRPV4) channel participates in neurogenic inflammation, pain transmission, and edema. In this study, we investigated the relationship between NKCC1 and TRPV4 and the related signaling pathways in TBI-induced brain edema and neuronal damage. TBI was induced by the calibrated weight-drop device. Adult male Wistar rats were randomly assigned into sham and experimental groups for time-course studies of TRPV4 expression after TBI. Hippocampal TRPV4, NKCC1, MAPK, and PI-3K cascades were analyzed by western blot, and brain edema was also evaluated among the different groups. Expression of hippocampal TRPV4 peaked at 8h after TBI, and phosphorylation of the MAPK cascade and Akt was significantly elevated. Administration of either the TRPV4 antagonist, RN1734, or NKCC1 antagonist, bumetanide, significantly attenuated TBI-induced brain edema through decreasing the phosphorylation of MEK, ERK, and Akt proteins. Bumetanide injection inhibited TRPV4 expression, which suggests NKCC1 activation is critical to TRPV4 activation. Our results showed that hippocampal NKCC1 activation increased TRPV4 expression after TBI and then induced severe brain edema and neuronal damage through activation of the MAPK cascade and Akt-related signaling pathway.

Background We have recently shown that the transient receptor potential vanilloid type 1 (TRPV1), a ligand-gated cation channel expressed in sensory nerves innervating the heart, plays an important role in protecting the heart from ischemia/reperfusion injury. However, the role of TRPV1 in pressure overload-induced hypertrophy of the heart is unknown. Objective - This study tests the hypothesis that TRPV1 protects the heart from pressure overload-induced hypertrophy and functional deterioration using gene-targeted TRPV1-null mutant (TRPV1 −/− ) or wild-type (WT) mice. Methods and Results Four weeks after transverse aortic constriction ( TAC ) in TRPV1 −/− and WT mice, echo cardiography demonstrated that cardiac fractional shortening (FS) and ejection fraction (EF) were suppressed in a greater extent in TRPV1 −/− than WT mice (FS: 14 ± 3% in TRPV1 −/− vs. 18 ± 2% in WT mice, P &lt;0.05; EF: 34 ± 6% in TRPV1 −/− vs. 43 ± 3% mice, P &lt;0.05), while there was no difference in FS and EF in TRPV1 −/− and WT sham-operated mice. The ratio of heart weight to body weight (mg/g) was greater in TRPV1 −/− than in WT mice (7.39±1.34 in TRPV1 −/− vs. 6.26±1.07 in WT mice, P &lt;0.05), and the increase in heart weigh was accompanied by an increase in cardiomyocyte cross-sectional areas in TRPV1 −/− compared to WT mice (248±8.2 in TRPV1 −/− vs 224±8.7 in WT μm 2 , P &lt;0.05). Immunohistochemistry study showed enhanced cellular infiltration and fibrosis in TRPV1 −/− compared to WT mice. Consistently, hydroxyproline assay showed that the collagen level was higher in TRPV1 −/− than WT mice (1.7±0.3 in TRPV1 −/− vs 1.3±0.4 μg/mg dry tissue in WT, P &lt;0.05), and ELISA assay showed that interleukin-6 levels were higher in TRPV1 −/− than in WT mice (14.2±4.6 in TRPV1 −/− vs. 10.6±2.6 pg/mg protein in WT, P &lt;0.05). Conclusions These data show that ablation of TRPV1 results in exaggerated cardiac hypertrophy, fibrosis, and dysfunction induced by pressure overload, which may result from enhanced inflammatory responses in the absence of TRPV1.

As aortic valve stenosis develops, valve tissue becomes stiffer. In response to this change in environmental mechanical stiffness, valvular interstitial cells (VICs) activate into myofibroblasts. We aimed to investigate the role of mechanosensitive calcium channel Transient Receptor Potential Vanilloid type 4 (TRPV4) in stiffness induced myofibroblast activation. We verified TRPV4 functionality in VICs using live calcium imaging during application of small molecule modulators of TRPV4 activity. We designed hydrogel biomaterials that mimic mechanical features of healthy or diseased valve tissue microenvironments, respectively, to investigate the role of TRPV4 in myofibroblast activation and proliferation. Our results show that TRPV4 regulates VIC proliferation in a microenvironment stiffness-independent manner. While there was a trend toward inhibiting myofibroblast activation on soft microenvironments during TRPV4 inhibition, we observed near complete deactivation of myofibroblasts on stiff microenvironments. We further identified Yes-activated protein (YAP) as a downstream target for TRPV4 activity on stiff microenvironments. Mechanosensitive TRPV4 channels regulate VIC myofibroblast activation, whereas proliferation regulation is independent of the microenvironmental stiffness. Collectively, the data suggests differential regulation of stiffness-induced proliferation and myofibroblast activation. Our data further suggest a regulatory role for TRPV4 regarding YAP nuclear localization. TRPV4 is an important regulator for VIC myofibroblast activation, which is linked to the initiation of valve fibrosis. Although more validation studies are necessary, we suggest TRPV4 as a promising pharmaceutical target to slow aortic valve stenosis progression.

Obesity is associated with an increase in renal sympathetic nerve activity (RSNA) and impairment in baroreflex (BR) that raises cardiovascular morbidity and mortality. This study tests the hypothesis that ablation of the transient receptor potential vanilloid type 1 (TRPV1) channels exacerbates impairment of BR in mice fed a western diet (WD) and leads to distinct diurnal and nocturnal blood pressure patterns. TRPV1-null mutant (TRPV1 -/- ) or wild type (WT) mice were given a WD (42% kcal from fat) or normal diet (CON) for 4 months. Perfusion of capsaicin (CAP, 10 -6 M ), a selective TRPV1 agonist, into the left renal pelvis increased ipsilateral afferent renal nerve activity (ARNA) in WT but not TRPV1 -/- mice, with a smaller magnitude in WT-WD compared to WT-CON (P&lt;0.05). The sensitivity of RSNA and heart rate (HR) responses to BR was assessed during changes in mean arterial pressure (MAP) induced by IV infusion of sodium nitroprusside (SNP)- or phenylephrine (PE). The sensitivity of RSNA and HR responses to BR was reduced to the same degree in TRPV1-/-CON and WT-WD and further decreased in TRPV1-/-WD (SNP infusion-RSNA: WT-CON183 ±37.7; TRPV1-/-CON 114.5 ± 15.3; WT-WD 92.1 ± 33.3; TRPV1-/-WD 53.9 ± 26.6%; P&lt;0.05; SNP infusion-HR: WT-CON 47.4 ± 7.6; TRPV1-/-CON 38.6 ± 5.3; WT-WD 36.6 ± 3.0; TRPV1-/-WD 28.2 ± 3.9 beats/min; P&lt;0.05; PE infusion-RSNA: WT-CON -93.9 ±6.4; TRPV1-/-CON -74.3 ± 9.2; WT-WD -66.1 ± 9.0; TRPV1-/-WD -40.6 ± 10.2%; P&lt;0.05; PE infusion-HR: WT-CON -35.5 ± 6.5; TRPV1-/-CON -29.0 ± 3.2.; WT-WD -27.6± 2.9; TRPV1-/-WD -20.2± 3.3 beats/min; P&lt;0.05). Urinary norepinephrine levels at day and night times were increased in WT-WD and TRPV1-/-WD, with further elevated levels at night time in TRPV1-/-WD ( P &lt;0.05). Urinary albumin and plasma creatinine levels were increased in WT-WD and TRPV1-/-WD, with further elevated urinary albumin levels in TRPV1-/-WD ( P &lt;0.05). Telemetry MAP was not different at the day time between all groups, but increased at the night time in TRPV1-/-WD compared to WT-CON, TRPV1-/-CON, and WT-WD (P&lt;0.05). Thus, TRPV1 ablation exacerbates impairment of BR and BR control of RSNA in the face of WD intake, and leads to elevated nocturnal but not diurnal blood pressure possibly attributed to further enhanced sympathetic drives at night time.

Transient receptor potential vanilloid type 4 (TRPV4) in urinary bladder structure and function.

The Ca2+-permeable transient receptor potential vanilloid type 4 (TRPV4) channel serves as the sensor of tubular flow, thus being well suited to govern mechanosensitive K+ transport in the distal renal tubule. Here, we directly tested whether the TRPV4 function is significant in affecting K+ balance. We used balance metabolic cage experiments and systemic measurements with different K+ feeding regimens [high (5% K+), regular (0.9% K+), and low (<0.01% K+)] in newly created transgenic mice with selective TRPV4 deletion in the renal tubule (TRPV4fl/fl-Pax8Cre) and their littermate controls (TRPV4fl/fl). Deletion was verified by the absence of TRPV4 protein expression and lack of TRPV4-dependent Ca2+ influx. There were no differences in plasma electrolytes, urinary volume, and K+ levels at baseline. In contrast, plasma K+ levels were significantly elevated in TRPV4fl/fl-Pax8Cre mice on high K+ intake. K+-loaded knockout mice exhibited lower urinary K+ levels than TRPV4fl/fl mice, which was accompanied by higher aldosterone levels by day 7. Moreover, TRPV4fl/fl-Pax8Cre mice had more efficient renal K+ conservation and higher plasma K+ levels in the state of dietary K+ deficiency. H+-K+-ATPase levels were significantly increased in TRPV4fl/fl-Pax8Cre mice on a regular diet and especially on a low-K+ diet, pointing to augmented K+ reabsorption in the collecting duct. Consistently, we found a significantly faster intracellular pH recovery after intracellular acidification, as an index of H+-K+-ATPase activity, in split-opened collecting ducts from TRPV4fl/fl-Pax8Cre mice. In summary, our results demonstrate an indispensable prokaliuretic role of TRPV4 in the renal tubule in controlling K+ balance and urinary K+ excretion during variations in dietary K+ intake. NEW & NOTEWORTHY The mechanoactivated transient receptor potential vanilloid type 4 (TRPV4) channel is expressed in distal tubule segments, where it controls flow-dependent K+ transport. Global TRPV4 deficiency causes impaired adaptation to variations in dietary K+ intake. Here, we demonstrate that renal tubule-specific TRPV4 deletion is sufficient to recapitulate the phenotype by causing antikaliuresis and higher plasma K+ levels in both states of K+ load and deficiency.

Objectives:Mechanical ventilation can cause ventilator-induced brain injury via afferent vagal signaling and hippocampal neurotransmitter imbalances. The triggering mechanisms for vagal signaling during mechanical ventilation are unknown. The objective of this study was to assess whether pulmonary transient receptor potential vanilloid type-4 (TRPV4) mechanoreceptors and vagal afferent purinergic receptors (P2X) act as triggers of ventilator-induced brain injury.Design:Controlled, human in vitro and ex vivo studies, as well as murine in vivo laboratory studies.Setting:Research laboratory.Subjects:Wild-type, TRPV4-deficient C57BL/6J mice, 8–10 weeks old. Human postmortem lung tissue and human lung epithelial cell line BEAS-2B.Intervention:Mice subjected to mechanical ventilation were studied using functional MRI to assess hippocampal activity. The effects of lidocaine (a nonselective ion-channel inhibitor), P2X-purinoceptor antagonist (iso-PPADS), or genetic TRPV4 deficiency on hippocampal dopamine-dependent pro-apoptotic signaling were studied in mechanically ventilated mice. Human lung epithelial cells (BEAS-2B) were used to study the effects of mechanical stretch on TRPV4 and P2X expression and activation. TRPV4 levels were measured in postmortem lung tissue from ventilated and nonventilated patients.Measurements and Main Results:Hippocampus functional MRI analysis revealed considerable changes in response to the increase in tidal volume during mechanical ventilation. Intratracheal lidocaine, iso-PPADS, and TRPV4 genetic deficiency protected mice against ventilationinduced hippocampal pro-apoptotic signaling. Mechanical stretch in both, BEAS-2B cells and ventilated wild-type mice, resulted in TRPV4 activation and reduced Trpv4 and P2x expression. Intratracheal replenishment of adenosine triphosphate in Trpv4–/– mice abrogated the protective effect of TRPV4 deficiency. Autopsy lung tissue from ventilated patients showed decreased lung TRPV4 levels compared with nonventilatedConclusions:TRPV4 mechanosensors and purinergic receptors are involved in the mechanisms of ventilator-induced brain injury. Inhibition of this neural signaling, either using nonspecific or specific inhibitors targeting the TRPV4/adenosine triphosphate/P2X signaling axis, may represent a novel strategy to prevent or treat ventilator-induced brain injury.

Transient receptor potential vanilloid type 1 (TRPV1) is a plasma membrane Ca(2+) channel involved in transduction of painful stimuli. Dorsal root ganglion (DRG) neurons express ectopic but functional TRPV1 channels in the endoplasmic reticulum (ER) (TRPV1(ER)). We have studied the properties of TRPV1(ER) in DRG neurons and HEK293T cells expressing TRPV1. Activation of TRPV1(ER) with capsaicin or other vanilloids produced an increase of cytosolic Ca(2+) due to Ca(2+) release from the ER. The decrease of [Ca(2+)](ER) was directly revealed by an ER-targeted aequorin Ca(2+) probe, expressed in DRG neurons using a herpes amplicon virus. The sensitivity of TRPV1(ER) to capsaicin was smaller than the sensitivity of the plasma membrane TRPV1 channels. The low affinity of TRPV1(ER) was not related to protein kinase A- or C-mediated phosphorylations, but it was due to inactivation by cytosolic Ca(2+) because the sensitivity to capsaicin was increased by loading the cells with the Ca(2+) chelator BAPTA. Decreasing [Ca(2+)](ER) did not affect the sensitivity of TRPV1(ER) to capsaicin. Disruption of the TRPV1 calmodulin-binding domains at either the C terminus (Delta35AA) or the N terminus (K155A) increased 10-fold the affinity of TRPV1(ER) for capsaicin, suggesting that calmodulin is involved in the inactivation. The lack of TRPV1 sensitizers, such as phosphatidylinositol 4,5-bisphosphate, in the ER could contribute to decrease the affinity for capsaicin. The low sensitivity of TRPV1(ER) to agonists may be critical for neuron health, because otherwise Ca(2+) depletion of ER could lead to ER stress, unfolding protein response, and cell death.