Yasunori Takayama

Functional Role for Piezo1 in Stretch-evoked Ca2+ Influx and ATP Release in Urothelial Cell Cultures

Background: The Piezo1 channel was recently identified as a genuine mechanosensor in mammalian cells. Results: Urothelial cells exhibited a Piezo1-dependent increase in cytosolic Ca2+ concentrations in response to mechanical stretch stimuli, leading to ATP release. Conclusion: Piezo1 senses extension of the bladder urothelium, which is converted into an ATP signal. Significance: Inhibition of Piezo1 might provide a new treatment for bladder dysfunction. The urothelium is a sensory structure that contributes to mechanosensation in the urinary bladder. Here, we provide evidence for a critical role for the Piezo1 channel, a newly identified mechanosensory molecule, in the mouse bladder urothelium. We performed a systematic analysis of the molecular and functional expression of Piezo1 channels in the urothelium. Immunofluorescence examination demonstrated abundant expression of Piezo1 in the mouse and human urothelium. Urothelial cells isolated from mice exhibited a Piezo1-dependent increase in cytosolic Ca2+ concentrations in response to mechanical stretch stimuli, leading to potent ATP release; this response was suppressed in Piezo1-knockdown cells. In addition, Piezo1 and TRPV4 distinguished different intensities of mechanical stimulus. Moreover, GsMTx4, an inhibitor of stretch-activated channels, attenuated the Ca2+ influx into urothelial cells and decreased ATP release from them upon stretch stimulation. These results suggest that Piezo1 senses extension of the bladder urothelium, leading to production of an ATP signal. Thus, inhibition of Piezo1 might provide a promising means of treating bladder dysfunction.

Pain-enhancing mechanism through interaction between TRPV1 and anoctamin 1 in sensory neurons

Significance The capsaicin receptor transient receptor potential cation channel vanilloid 1 (TRPV1) and anoctamin 1 (ANO1) work as receptors activated by noxious stimuli in sensory nerve endings. It is believed that activation of the two channels causes cation influx and anion efflux, respectively, both of which lead to depolarization. We show that ANO1 is activated by calcium ions entering neurons through TRPV1 activation based on their physical binding on the cell membrane. Indeed, both capsaicin-activated inward currents in sensory neurons and capsaicin-induced pain-related behaviors in mice were inhibited significantly by ANO1 blockade. To our knowledge, this is the first evidence for a mechanism by which interaction between TRPV1 and ANO1 functions as a pain-enhancing mechanism. The capsaicin receptor transient receptor potential cation channel vanilloid 1 (TRPV1) is activated by various noxious stimuli, and the stimuli are converted into electrical signals in primary sensory neurons. It is believed that cation influx through TRPV1 causes depolarization, leading to the activation of voltage-gated sodium channels, followed by the generation of action potential. Here we report that the capsaicin-evoked action potential could be induced by two components: a cation influx-mediated depolarization caused by TRPV1 activation and a subsequent anion efflux-mediated depolarization via activation of anoctamin 1 (ANO1), a calcium-activated chloride channel, resulting from the entry of calcium through TRPV1. The interaction between TRPV1 and ANO1 is based on their physical binding. Capsaicin activated the chloride currents in an extracellular calcium-dependent manner in HEK293T cells expressing TRPV1 and ANO1. Similarly, in mouse dorsal root ganglion neurons, capsaicin-activated inward currents were inhibited significantly by a specific ANO1 antagonist, T16Ainh-A01 (A01), in the presence of a high concentration of EGTA but not in the presence of BAPTA [1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid]. The generation of a capsaicin-evoked action potential also was inhibited by A01. Furthermore, pain-related behaviors in mice treated with capsaicin, but not with αβ-methylene ATP, were reduced significantly by the concomitant administration of A01. These results indicate that TRPV1–ANO1 interaction is a significant pain-enhancing mechanism in the peripheral nervous system.

Modulation of water efflux through functional interaction between TRPV4 and TMEM16A/anoctamin 1

Transient receptor potential vanilloid 4 (TRPV4), a calcium‐permeable channel, is highly expressed in the apical membrane of choroid plexus epithelial cells (CPECs) in the brain. The function of TRPV4 is unknown. Here, we show physical and functional interaction between TRPV4 and anoctamin 1 (ANO1) in HEK293T cells and CPECs. Chloride currents induced by a TRPV4 activator (GSK1016790A) were markedly increased in an extracellular calcium‐dependent manner in HEK293T cells expressing TRPV4 with ANO1, but not with ANO4, ANO6, or ANO10, the mRNAs of which were expressed in the choroid plexus. We also found physical interaction between TRPV4 and ANO1 in both HEK293T cells and choroid plexus. We observed that ANO1 was activated at a warm temperature (37°C) in HEK293T cells and that the heat‐evoked chloride currents were markedly enhanced after GSK1016790A application in CPECs. Simultaneous stimulation by warmth and hyposmosis induced chloride current activation in wild‐type, but not in TRPV4‐deficient, CPECs. Cell volume changes were induced by ANO1‐mediated chloride currents in parallel with membrane potential changes, and the cell volume was significantly decreased at negative membrane potentials by TRPV4‐induced ANO1 activation. Thus, physical and functional interactions between TRPV4 and ANO1 can modulate water transport in the choroid plexus.—Takayama, Y., Shibasaki, K., Suzuki, Y., Yamanaka, A., Tominaga, M. Modulation of water efflux through functional interaction between TRPV4 and TMEM16A/anoctamin 1. FASEB J. 28, 2238–2248 (2014). www.fasebj.org

Functional roles of TRPV1 and TRPV4 in control of lower urinary tract activity: dual analysis of behavior and reflex during the micturition cycle

The present study used a dual analysis of voiding behavior and reflex micturition to examine lower urinary tract function in transient receptor potential vanilloid (TRPV)1 knockout (KO) mice and TRPV4 KO mice. In metabolic cage experiments conducted under conscious conditions (i.e., voluntary voiding behavior), TRPV4 KO mice showed a markedly higher voiding frequency (VF; 19.3 ± 1.2 times/day) and a smaller urine volume/voiding (UVV; 114 ± 9 μl) compared with wild-type (WT) littermates (VF: 5.2 ± 0.5 times/day and UVV: 380 ± 34 μl). Meanwhile, TRPV1 KO mice showed a similar VF to WT littermates (6.8 ± 0.5 times/day) with a significantly smaller UVV (276 ± 20 μl). Water intake among these genotypes was the same, but TRPV4 KO mice had a larger urine output than the other two groups. In cystometrogram experiments conducted in decerebrate unanesthetized mice (i.e., reflex micturition response), no differences between the three groups were found in any cystometrogram variables, including voided volume, volume threshold for inducing micturition contraction, maximal voiding pressure, and bladder compliance. However, both TRPV1 KO and TRPV4 KO mice showed a significant number of nonvoiding bladder contractions (NVCs; 3.5 ± 0.9 and 2.8 ± 0.7 contractions, respectively) before each voiding, whereas WT mice showed virtually no NVCs. These results suggest that in the reflex micturition circuit, a lack of either channel is involved in NVCs during bladder filling, whereas in the forebrain, it is involved in the early timing of urine release, possibly in the conscious response to the bladder instability.

Lack of TRPV2 impairs thermogenesis in mouse brown adipose tissue

Brown adipose tissue (BAT), a major site for mammalian non‐shivering thermogenesis, could be a target for prevention and treatment of human obesity. Transient receptor potential vanilloid 2 (TRPV2), a Ca2+‐permeable non‐selective cation channel, plays vital roles in the regulation of various cellular functions. Here, we show that TRPV2 is expressed in brown adipocytes and that mRNA levels of thermogenic genes are reduced in both cultured brown adipocytes and BAT from TRPV2 knockout (TRPV2KO) mice. The induction of thermogenic genes in response to β‐adrenergic receptor stimulation is also decreased in TRPV2KO brown adipocytes and suppressed by reduced intracellular Ca2+ concentrations in wild‐type brown adipocytes. In addition, TRPV2KO mice have more white adipose tissue and larger brown adipocytes and show cold intolerance, and lower BAT temperature increases in response to β‐adrenergic receptor stimulation. Furthermore, TRPV2KO mice have increased body weight and fat upon high‐fat‐diet treatment. Based on these findings, we conclude that TRPV2 has a role in BAT thermogenesis and could be a target for human obesity therapy.

TRPV1 is crucial for proinflammatory STAT3 signaling and thermoregulation-associated pathways in the brain during inflammation.

Transient receptor potential vanilloid receptor 1 (TRPV1) is a non-selective cation channel that is stimulated by heat (>43 °C), mechanical/osmotic stimuli, and low pH. The importance of TRPV1 in inflammatory responses has been demonstrated, whereas its participation in brains remains unclear. In the present study, the intracerebroventricular (icv) administration of the TRPV1 agonist resiniferatoxin (RTX) induced the activation of signal transducer and activator of transcription 3 (STAT3) in circumventricular organs (CVOs) and thermoregulation-associated brain regions with a similar patttern to the peripheral and icv administration of lipopolysaccharide (LPS). With the peripheral and icv LPS stimuli, STAT3 activation was significantly lower in Trpv1−/− mice than in Trpv1+/+ mice. The icv administration of RTX induced transient hypothermia, whereas that of the TRPV1 antagonist capsazepine enhanced the magnitude and period of LPS-induced hyperthermia. These results indicate that TRPV1 is important for activating proinflammatory STAT3 signaling and thermoregulation-associated brain pathways in the brain.

Infantile Pain Episodes Associated with Novel Nav1.9 Mutations in Familial Episodic Pain Syndrome in Japanese Families.

Painful peripheral neuropathy has been correlated with various voltage-gated sodium channel mutations in sensory neurons. Recently Nav1.9, a voltage-gated sodium channel subtype, has been established as a genetic influence for certain peripheral pain syndromes. In this study, we performed a genetic study in six unrelated multigenerational Japanese families with episodic pain syndrome. Affected participants (n = 23) were characterized by infantile recurrent pain episodes with spontaneous mitigation around adolescence. This unique phenotype was inherited in an autosomal-dominant mode. Linkage analysis was performed for two families with 12 affected and nine unaffected members, and a single locus was identified on 3p22 (LOD score 4.32). Exome analysis (n = 14) was performed for affected and unaffected members in these two families and an additional family. Two missense variants were identified: R222H and R222S in SCN11A. Next, we generated a knock-in mouse model harboring one of the mutations (R222S). Behavioral tests (Hargreaves test and cold plate test) using R222S and wild-type C57BL/6 (WT) mice, young (8–9 weeks old; n = 10–12 for each group) and mature (36–38 weeks old; n = 5–6 for each group), showed that R222S mice were significantly (p < 0.05) more hypersensitive to hot and cold stimuli than WT mice. Electrophysiological studies using dorsal root ganglion neurons from 8–9-week-old mice showed no significant difference in resting membrane potential, but input impedance and firing frequency of evoked action potentials were significantly increased in R222S mice compared with WT mice. However, there was no significant difference among Nav1.9 (WT, R222S, and R222H)-overexpressing ND7/23 cell lines. These results suggest that our novel mutation is a gain-of-function mutation that causes infantile familial episodic pain. The mouse model developed here will be useful for drug screening for familial episodic pain syndrome associated with SCN11A mutations.

Interaction between TRP and Ca2+-activated chloride channels

The transient receptor potential (TRP) channel superfamily includes a large number of proteins that constitute 7 subfamilies (TRPC, TRPV, TRPM, TRPML, TRPN, TRPA and TRPP). These channels have been strongly conserved evolutionarily. The family was first cloned from a mutant Drosophila strain in which receptor potentials in the eye were transient upon light stimulation. There are 27 known channels in the 6 subfamilies, excluding TRPN in humans. Some of the TRP channels respond to a wide variety of sensory stimuli, including chemicals and temperature changes. Thus, they are termed “thermosensitive” TRP channels. Three in particular (TRPV1, a capsaicin receptor, TRPA1, a receptor for mustard oil, and TRPM8, a menthol receptor) are involved in temperature sensation in sensory neurons. The structure of TRPV1 was recently solved at a 3.4 Å level using a single particle analysis with cryo-electron microscopy. One of the key characteristics of thermosensitive TRP channels is their high Ca permeability that is more than 5 times larger than that of sodium. Therefore, thermosensitive TRP channels might well play important roles involving Ca. There are many Ca binding proteins in the cytosol that contribute to tight regulation of intracellular Ca concentrations. Nonetheless, upon entering the cells through TRP channels, Ca is certainly involved in mediating various intracellular events. Indeed, TRPV4, a thermosensitive TRP channel (reportedly an osmoor mechano-sensor, and anoctamin 1 (TMEM16A; a Caactivated chloride channel), functionally interact in choroid plexus epithelial cells in the brain. Upon entering choroid plexus epithelial cells, Ca activates anoctamin 1, leading to chloride efflux because the chloride equilibrium potential in choroid plexus epithelial cells is smaller than the resting membrane potentials due to relatively high intracellular chloride concentrations. The chloride efflux could drive water movement from the choroid plexus epithelial cells to ventricles, a process that could be viewed as a mechanism for release of cerebrospinal fluid from the choroid plexus. Aquaporin (AQP) 1 and AQP4 water channels are well-expressed in choroid plexus epithelial cells, and TRPV4 directly interacts with AQP4. Activation of anoctamin 1 was also observed downstream of Gq-coupled receptor activation that can increase intracellular Ca concentrations though Ca release from intracellular Ca stores by Ins(1,4,5) P 3 receptor activation. However, activation of anoctamin 1 was not frequently observed following a simple increase in intracellular Ca concentration compared with that seen after TRP channel activation. This result indicated that Ca entering the cells through Ca-permeable channels activated anoctamin 1 more effectively. In fact, co-immunoprecipitation studies showed that TRPV4 physically interacted with anoctamin 1, suggesting that co-localization of the 2 proteins is important. The findings presented in the work illustrate the significance of TRPV4 in choroid plexus epithelial cells and emphasize the importance of the interaction between Ca-permeable channels and Ca-activated proteins Interaction between TRP and Ca-activated chloride channels

Adipose hypothermia in obesity and its association with period homolog 1, insulin sensitivity, and inflammation in fat.

Visceral fat adiposity plays an important role in the development of metabolic syndrome. We reported previously the impact of human visceral fat adiposity on gene expression profile of peripheral blood cells. Genes related to circadian rhythm were highly associated with visceral fat area and period homolog 1 (PER1) showed the most significant negative correlation with visceral fat area. However, regulation of adipose Per1 remains poorly understood. The present study was designed to understand the regulation of Per1 in adipose tissues. Adipose Per1 mRNA levels of ob/ob mice were markedly low at 25 and 35 weeks of age. The levels of other core clock genes of white adipose tissues were also low in ob/ob mice at 25 and 35 weeks of age. Per1 mRNA was mainly expressed in the mature adipocyte fraction (MAF) and it was significantly low in MAF of ob/ob mice. To examine the possible mechanisms, 3T3-L1 adipocytes were treated with H2O2, tumor necrosis factor-α (TNF-α), S100A8, and lipopolysaccharide (LPS). However, no significant changes in Per1 mRNA level were observed by these agents. Exposure of cultured 3T3-L1 adipocytes to low temperature (33°C) decreased Per1 and catalase, and increased monocyte chemoattractant protein-1 (Mcp-1) mRNA levels. Hypothermia also worsened insulin-mediated Akt phosphorylation in 3T3-L1 adipocytes. Finally, telemetric analysis showed low temperature of adipose tissues in ob/ob mice. In obesity, adipose hypothermia seems to accelerate adipocyte dysfunction.

Cell surface flip-flop of phosphatidylserine is critical for PIEZO1-mediated myotube formation

Myotube formation by fusion of myoblasts and subsequent elongation of the syncytia is essential for skeletal muscle formation. However, molecules that regulate myotube formation remain elusive. Here we identify PIEZO1, a mechanosensitive Ca2+ channel, as a key regulator of myotube formation. During myotube formation, phosphatidylserine, a phospholipid that resides in the inner leaflet of the plasma membrane, is transiently exposed to cell surface and promotes myoblast fusion. We show that cell surface phosphatidylserine inhibits PIEZO1 and that the inward translocation of phosphatidylserine, which is driven by the phospholipid flippase complex of ATP11A and CDC50A, is required for PIEZO1 activation. PIEZO1-mediated Ca2+ influx promotes RhoA/ROCK-mediated actomyosin assemblies at the lateral cortex of myotubes, thus preventing uncontrolled fusion of myotubes and leading to polarized elongation during myotube formation. These results suggest that cell surface flip-flop of phosphatidylserine acts as a molecular switch for PIEZO1 activation that governs proper morphogenesis during myotube formation.Myotube formation by fusion of myoblasts is essential for skeletal muscle formation, but which molecules regulate this process remains elusive. Here authors identify the mechanosensitive PIEZO1 channel as a key element, whose activity is regulated by phosphatidylserine during myotube formation.