TRPC Channels Research Articles

Transient receptor potential canonical 5 channel is involved in the cardiac damage related to obstructive sleep apnea-hypopnea syndrome in rats.

Obstructive sleep apnea-hypopnea syndrome (OSAHS) is recognized as an independent risk factor of cardiovascular disease. The release of Ca2+ mediated by transient receptor potential canonical (TRPC) channels participates in the hypoxia-induced pathophysiological changes in the cardiovascular systems in case of OSAHS. This study aimed to investigate which subtypes of TRPCs were involved in OSAHS in a rat model of intermittent hypoxia. OSAHS was induced by exposure of rats to intermittent hypoxia. The expression of TRPCrelated genes and proteins in the cardiomyocytes by qRT-PCR and Western Blotting, respectively. The mRNA expression of TRPC3/TRPC4/TRPC5 increased significantly in OSAHS group compared with the control group (P<0.05). The TRPC5 protein expression was significantly higher in the OSAHS control than the control group (P<0.05). The TRPC5 channel is likely to be involved in the OSAHS induced pathophysiological changes in the myocardium and may become a target to prevent OSAHS related cardiac damage.

RNA-seq analysis reveals TRPC genes to impact an unexpected number of metabolic and regulatory pathways

The seven-member transient receptor potential canonical genes (TRPC1-7) encode cation channels linked to several human diseases. There is little understanding of the participation of each TRPC in each pathology, considering functional redundancy. Also, most of the inhibitors available are not specific. Thus, we developed mice that lack all of the TRPCs and performed a transcriptome analysis in eight tissues. The aim of this research was to address the impact of the absence of all TRPC channels on gene expression. We obtained a total of 4305 differentially expressed genes (DEGs) in at least one tissue where spleen showed the highest number of DEGs (1371). Just 21 genes were modified in all the tissues. Performing a pathway enrichment analysis, we found that many important signaling pathways were modified in more than one tissue, including PI3K (phosphatidylinositol 3-kinase/protein kinase-B) signaling pathway, cytokine-cytokine receptor interaction, extracellular matrix (ECM)-receptor interaction and circadian rhythms. We describe for the first time the changes at the transcriptome level due to the lack of all TRPC proteins in a mouse model and provide a starting point to understand the function of TRPC channels and their possible roles in pathologies.

Critical Components for Spontaneous Activity and Rhythm Generation in Spinal Cord Circuits in Culture.

Neuronal excitability contributes to rhythm generation in central pattern generating networks (CPGs). In spinal cord CPGs, such intrinsic excitability partly relies on persistent sodium currents (INaP), whereas respiratory CPGs additionally depend on calcium-activated cation currents (ICAN). Here, we investigated the contributions of INaP and ICAN to spontaneous rhythm generation in neuronal networks of the spinal cord and whether they mainly involve Hb9 neurons. We used cultures of ventral and transverse slices from the E13–14 embryonic rodent lumbar spinal cord on multielectrode arrays (MEAs). All cultures showed spontaneous bursts of network activity. Blocking synaptic excitation with the AMPA receptor antagonist CNQX suppressed spontaneous network bursts and left asynchronous intrinsic activity at about 30% of the electrodes. Such intrinsic activity was completely blocked at all electrodes by both the INaP blocker riluzole as well as by the ICAN blocker flufenamic acid (FFA) and the more specific TRPM4 channel antagonist 9-phenanthrol. All three antagonists also suppressed spontaneous bursting completely and strongly reduced stimulus-evoked bursts. Also, FFA reduced repetitive spiking that was induced in single neurons by injection of depolarizing current pulses to few spikes. Other antagonists of unspecific cation currents or calcium currents had no suppressing effects on either intrinsic activity (gadolinium chloride) or spontaneous bursting (the TRPC channel antagonists clemizole and ML204 and the T channel antagonist TTA-P2). Combined patch-clamp and MEA recordings showed that Hb9 interneurons were activated by network bursts but could not initiate them. Together these findings suggest that both INaP through Na+-channels and ICAN through putative TRPM4 channels contribute to spontaneous intrinsic and repetitive spiking in spinal cord neurons and thereby to the generation of network bursts.

TRPC6-Mediated Ca2+ Entry Essential for the Regulation of Nano-ZnO Induced Autophagy in SH-SY5Y Cells.

Recently, possible applications of zinc oxide nanoparticles (nano-ZnO) have been extensively studied owing to their ease of synthesis. However, the effect of nano-ZnO on the nervous system remains unclear. This study investigates the action of nano-ZnO on SH-SY5Y neuroblastoma cells. We found that nano-ZnO (0-50 µg/mL) induced a significant decrease in cell survival rate in a dose-dependent manner, and increased LC3 puncta formation. However, the apoptosis was not affected by nano-ZnO, because the protein levels of cytochrome c, caspase-3, Bcl-xL, and BAX were not varied by the nano-ZnO treatment. Nano-ZnO increased Ca2+ entry and the expression of TRPC6.The results suggested that nano-ZnO increased [Ca2+] through the TRPC-dependent Ca2+ influx, since Ca2+ influx can be prevented by the TRPC inhibitor. Furthermore, cells on nano-ZnO-treatment groups displayed loss of F-actin in a dose dependent manner, which also could be prevented by TRPC inhibitor. Herein, we demonstrated that the nano-ZnO activated TRPC6 channel, thereby increasing the Ca2+ flux and resulting in increased autophagy. Nano-ZnO could have possible anticancer effects in neuroblastoma by inhibiting the proliferation of tumor cells. However, we should also pay attention toward the biosecurity of nano materials.

Analysis of Mrgprb2 Receptor-Evoked Ca 2+ Signaling in Bone Marrow Derived (BMMC) and Peritoneal (PMC) Mast Cells of TRPC-Deficient Mice.

Mast cells are a heterogeneous group of immune cells. The simplest and commonly accepted classification divides them in two groups according to their protease content. We have compared the action of diverse secretagogues on bone marrow derived (BMMC) and peritoneal (PMC) mast cells which represent classical models of mucosal and connective tissue type mast cells in mice. Whereas, antigen stimulation of the FcεRI receptors was similarly effective in triggering elevations of free intracellular Ca2+ concentration ([Ca2+]i) in both BMMC and PMC, robust [Ca2+]i rise following Endothelin-1 stimulation was observed only in a fraction of BMMC. Leukotriene C4 activating cysteinyl leukotriene type I receptors failed to evoke [Ca2+]i rise in either mast cell model. Stimulation of the recently identified target of many small-molecule drugs associated with systemic pseudo-allergic reactions, Mrgprb2, with compound 48/80, a mast cell activator with unknown receptor studied for many years, triggered Ca2+ oscillations in BMMC and robust [Ca2+]i rise in PMCs similarly to that evoked by FcεRI stimulation. [Ca2+]i rise in PMC could also be evoked by other Mrgprb2 agonists such as Tubocurarine, LL-37, and Substance P. The extent of [Ca2+]i rise correlated with mast cell degranulation. Expression analysis of TRPC channels as potential candidates mediating agonist evoked Ca2+ entry revealed the presence of transcripts of all members of the TRPC subfamily of TRP channels in PMCs. The amplitude and AUC of compound 48/80-evoked [Ca2+]i rise was reduced by ~20% in PMC from Trpc1/4/6−/− mice compared to Trpc1/4−/− littermatched control mice, whereas FcεRI-evoked [Ca2+]i rise was unaltered. Whole-cell patch clamp recordings showed that the reduction in compound 48/80-evoked [Ca2+]i rise in Trpc1/4/6−/− PMC was accompanied by a reduced amplitude of Compound 48/80-induced cation currents which exhibited typical features of TRPC currents. Together, this study demonstrates that PMC are an appropriate mast cell model to study mechanisms of Mrgprb2 receptor-mediated mast cell activation, and it reveals that TRPC channels contribute at least partially to Mrgprb2-mediated mast cellactivation but not following FcεRI stimulation. However, the channels conducting most of the Ca2+ entry in mast cells triggered by Mrgprb2 receptor stimulation remains to be identified.

Contribution of TRPC channels in evoking the exercise pressor reflex and mechanoreflex in healthy and type 1 diabetic rats

The exercise pressor reflex and the mechanoreflex are exaggerated in type 1 diabetes mellitus (T1DM) rats. Studies using GsMTx‐4, which has high potency for Piezo channels, have suggested that Piezo channels play a significant role in evoking this exaggerated response. However, studies have also reported that GsMTx‐4 may block TRPC1 and TRPC6 channels at similar concentrations. Therefore, to account for the possible effects of GsMTx‐4 on TRPC1 and TRPC6 channels, the purpose of this study was to determine whether TRPC channels contribute to the exercise pressor reflex and the mechanoreflex in both healthy and T1DM rats.MethodsStudies were conducted in adult unanesthetized, decerebrated T1DM (STZ; Streptozotocin 50 mg/kg i.p) and healthy control (CTL) Sprague Dawley rats. The exercise pressor reflex was evoked by intermittently contracting the hindlimb for 30 s and the mechanoreflex was evoked by stretching the Achilles tendon for 30 s. Changes in mean arterial pressure (MAP), heart rate (HR) and renal sympathetic nerve activity (RSNA) were continuously measured and compared before and after injecting a TRPC channel antagonist, namely SKF 96365 (0.1ml, 10μM or 100μM).ResultsSKF 96365 (100μM) had no effect on the peak pressor response to muscle contraction in either STZ rats (ΔMAP before: 37 ± 9 mmHg, ΔMAP after: 32 ± 12 mmHg, n=2, p&gt;0.05) or CTL rats (before: 16 ± 3 mmHg, after: 5 ± 2 mmHg, n=3, p&gt;0.05). The cardioaccelerator response to muscle contraction was unaffected by SKF 96365 in STZ rats (before: 25 ± 1 bpm, after: 21 ± 6 bpm, n=2, p&gt;0.05), whereas it was attenuated in CTL rats (before: 13 ± 4 bpm, after: 4 ± 4 bpm, n=3, p&lt;0.05). The sympathetic response to muscle contraction was unaffected by SKF 96365 in CTL rats (%ΔRSNA before: 138 ± 108 %, %ΔRSNA after: 181 ± 41 %, n=2, p&gt;0.05). The peak pressor response to tendon stretch was unaffected by SKF 96365 in STZ (before: 42 ± 9 mmHg, after: 34 ± 10 mmHg, n=3, p&gt;0.05) and CTL rats (before: 16 ± 3 mmHg, after: 16 ± 2 mmHg, n=4, p&gt;0.05). Likewise, the cardioaccelerator response to tendon stretch was unaffected by SKF 96365 in STZ (before: 25 ± 4 bpm, after: 36 ± 12 bpm, n=3, p&gt;0.05) and CTL rats (before: 13 ± 2 bpm, after: 14 ± 2 bpm, n=4, p&gt;0.05). The sympathetic response to tendon stretch was unaffected by SKF 96365 in CTL rats (%ΔRSNA before: 98 ± 94 %, %ΔRSNA after: 94 ± 104 %, n=2, p&gt;0.05). Furthermore, a lower concentration of SKF 96365 (10μM) had no effect on the peak pressor (before: 33 ± 20 mmHg, after: 31 ± 18 mmHg, n=2, p&gt;0.05) or cardioaccelerator (before: 23 ± 9 bpm, after: 22 ± 6 bpm, p&gt;0.05) responses to muscle contraction in STZ rats. Similarly, the peak pressor (before: 29 ± 15 mmHg, after: 16 ± 3 mmHg, n=2, p&gt;0.05) and cardioaccelerator (before: 29 ± 15 bpm, after: 16 ± 3 bpm, n=2, p&gt;0.05) responses to tendon stretch were unaffected by SKF 96365 (10μM) in STZ rats.ConclusionThese preliminary findings suggest that TRPC channels do not play a role in evoking the exercise pressor reflex or the mechanoreflex in T1DM and healthy rats. Thus, it is unlikely that the attenuating effect of GsMTx‐4 on the exercise pressor reflex is due to its effect on TRPC channels.

Structure‐Function Relationship for Spermine‐mediated Inward Rectification of Transient Receptor Potential Canonical (TRPC) 4 Channels.

Transient Receptor Potential Canonical (TRPC) channels constitute calcium‐permeable, non‐selective cation channels in mammalian cells. The expression of TRPC4 channels can be found in gastrointestinal (GI) tract, endometrium, ventricular myocytes, vascular smooth muscle cells, endothelium and many others. In such tissues, it is well‐known that TRPC4 channels exert membrane excitatory effect due to inwardly‐rectifying nature of current‐voltage (I‐V) relationship of the channels. Based on the electrophysiological similarity between inwardly‐rectifying potassium channel and TRPC4 channel, we investigated the molecular mechanism of inward‐rectification of TRPC4 channels, especially by intracellular magnesium and polyamine, which is two major cytoplasmic blocking agent of IRKs.Based on the pre‐established theory for inward‐rectification of IRKs, we have constructed a number of mutant TRPC4 channels whose acidic amino acid (glutamate or aspartate) at cytoplasmic side (C‐terminus) was changed to alanine. Such mutant channels were overexpressed in HEK293 cells and the activity and efficacy of cytoplasmic blocking action of spermine was measured using whole‐cell patch‐clamp technique. Structural analysis was based on published Cryo‐EM structure of mouse TRPC4 channels (PDB Accession Number: 5z96). The modeled 3D structures of TRPC4 mutants were visualized and analyzed using the PyMOL software. For the calculation of electrical distance, two‐barrier model with one reaction‐intermediate potential was conveyed.As a result, the electrical distance of spermine demonstrated that spermine barely penetrates into the pore but rather exerts its action on the entrance of putative inner vestibule of the channel. The voltage‐dependency of Kd value was not significant compared to other well‐known blocking agents such as proton in Na channels and polyamine in IRKs. Among various mutant channels, D629A, E648A, E728A and E729A channels showed significant recovery from spermine block, demonstrating that such residues are crucial for spermine‐mediated inward rectification. Identification of such residues in Cryo‐EM structure of TRPC4 channel showed rather consistent structural implication for residues D629, E728 and E729; they all lined inner vestibule of the channel. E648 is located at the connecting‐helix of the channel and faces the into cytoplasmic face. Hence we postulated that the residue may contribute to the inward‐rectification by reducing access resistance of spermine from channel periphery into inner vestibule. The location of residues fitted well with electrical distance.Support or Funding Information*This work was supported by the National Research Foundation of Korea, which is funded by the Ministry of Science, ICT (Information &amp; Communication Technology), and Future Planning (MSIP) of the Korean Government (2018R1A41023822 to I. So) and by the Education and Research Encouragement Fund of Seoul National University Hospital (I. So). Christine Haewon Park was supported by the BK plus program from the MSIP.

Modulation of TRPC store‐operated channels by Orai and STIM proteins

Store‐operated calcium entry is significant pathway of calcium concentration rise in cytoplasm. The main players in this process are Orai store‐operated channels, which are activated directly by STIM calcium sensors. Data on participation of TRPC channels in store‐operated calcium entry is controversial.Study was focused on interaction between STIM and Orai proteins with endogenous TRPC and its role in pathology. We investigated activity of TRPC channels in a model object — HEK293 cells and in the podocytes of the rat model of diabetic nephropathy, using single channel recording patch camp technique.To eliminate Orai channels activity in HEK293 cells we used several experimental approaches: overexpression of dominant‐negative Orai mutant, Orai genes knockout and knockdown via CRISPR‐Cas 9 system and shRNA. Endogenous TRPC1 channels remain active in cells without functional Orai proteins and become insensitive to store depletion.In experiments with different extracellular calcium concentration we confirmed that calcium entry is needed for TRPC1 activation by store depletion.In experiments with calcium, barium or sodium as a charge carrier we observed similarities in dependence of open channel probability versus membrane potential between TRPC1 and Orai channels.The role of STIM calcium sensors in TRPC1 channels activation was investigated in cells with different expression level of STIM1 and STIM2 proteins.The findings allow us to propose that STIM proteins don’t activate the endogenous TRPC channels directly. TRPC channels are activated downstream to calcium entry through the Orai channels.Support or Funding InformationThe study was supported by grant of Russian science foundation № 19‐14‐00114 (Glushankova L., Skopin A., Kaznacheyeva E.), and by grant of Russian foundation for basic research № 19‐315‐90065 (Shalygin A., Kolesnikov D.).

Endothelins contribute to early renal insufficiency engendered by acute urinary tract obstruction in neonatal pigs

Urinary tract obstruction reduces kidney functions, which, if left uncorrected, may lead to irreversible renal loss, especially in infants. Despite the removal of obstruction, a decline in steady‐state renal blood flow (RBF) persists, vascular mechanisms of which are poorly understood. Urinary tract obstruction‐induced increase in the levels of vasoactive endothelin (ET)‐1 has been reported in humans and rodents. However, whether obstructive renal vasculopathy is associated with increased renal production of all ET isoforms (ET1‐3) remains unresolved. Also, the mechanisms that underlie ET‐induced renal vasoregulation in neonates are unclear. Here, we show that ET receptor (ETR)‐operated calcium entry via TRPC3 channels increases renal vascular resistance and reduces RBF in neonatal pigs. Acute unilateral urinary tract obstruction (UUO) in neonatal pigs increased peptidase endothelin converting enzyme 1 (ECE1)‐dependent renal production of ET1‐3 and diacylglycerol (DAG). Despite the removal of obstruction, acute UUO reduced kidney perfusion and caused an increase in renal vascular resistance (RVR) and the urinary levels of NGAL, the early predictive biomarker of acute kidney injury. The protein expression levels of ETRs (ETA and ETB) and TRPC3 channels in renal microvessels were unaltered in neonatal pigs subjected to acute UUO. However, pharmacological inhibition of ECE1, ETR, and TRPC3 mitigated acute UUO‐induced increase in RVR and urine NGAL in the pigs. These data suggest that increased ECE1 production and proteolytic processing of all big ETs to their vasoactive isoforms contribute to early neonatal renal insufficiency engendered by UUO. ET‐derived DAG activates renal vascular smooth muscle cell TRPC3 channels, leading to extracellular calcium entry, vasoconstriction, increased RVR and sustained hypoperfusionSupport or Funding InformationNIH/NIDDK: R01DK101668 and R56DK120595

Regulation of transient receptor potential canonical 4 activity by phospholipase C δ1

Transient receptor potential canonical (TRPC)4 and TRPC5 channels are non‐selective calciumpermeable cation channels that maintained by phosphatidylinositol 4,5‐bisphosphate (PI(4,5)P2) and inactivated due to its depletion. Phospholipase C (PLC) is an enzyme that cleaves phospholipids and the PLCδ is the most calcium‐sensitive form among the isozymes that stimulated by physiological concentration of Ca2+. Here, we investigated the regulation mechanism of TRPC4 by the Ca2+‐PLCδ‐PI(4,5)P2 signaling cascade. In order to identify the interaction between ion channel and PLCδ protein, we implied co‐immunoprecipitation and fluorescence imaging including Föster Resonance Energy Transfer. We evaluated the activity of channels with electrophysiological recording in HEK293 cells expressing TRPC channels. Here, we demonstrate that TRPC4 directly interacts with PLCδ1. We also found that PLCδ isozymes are activated by the calcium through opened channels but not by the cytosolic calcium increase. Our study established the PLCδ1 inhibits overall TRPC4’s currents activity, but mainly regulates the basal TRPC4 currents to have a characteristic low basal activity. These regulation of PLCδ1 on TRPC4 activity occurs depend on PI(4,5)P2 hydrolysis. Resultantly, PLCδ1 is considered to be a negative regulator of TRPC4.Support or Funding InformationThis research was supported by the National Research Foundation of Korea, which is funded by the Ministry of Science, ICT (Information &amp; Communication Technology), and Future Planning (MSIP) of the Korean government (2018R1A4A1023822 to I.S.S.). H.Kang, J.Ko., J.Myeong., and M.Kwak. were supported by the BK plus program from the MSIP.

The Role of Opioid Receptors in Podocyte Injury and Kidney Damage During the Development of Salt‐Induced Hypertension

The global increase in opioid use requires to better understand the side effects of opioids in order to avoid additional complications. Opioids are common analgesics for patients with hypertension and CKD despite its association with increased albuminuria and reduced glomerular filtration rate. Opioids work by binding to kappa‐, mu‐ and delta‐opioid receptors (ORs). We hypothesize that stimulation of ORs modulates calcium flux in podocytes ultimately leading to cell apoptosis, development of albuminuria, glomeruli damage and progression of hypertension. To test this hypothesis, we applied specific OR agonists to: 1) in vitro ‐ immortalized cultured human podocytes (hPod), 2) ex vivo ‐ freshly isolated human and rat glomeruli; and 3) in vivo ‐ Dahl salt‐sensitive (SS) rats. Calcium response in the podocytes was analyzed via ratiometric confocal fluorescent microscopy using Fluo4/FuraRed and Fluo8 fluorescent dyes. For chronic studies Dahl SS rats were on a 0.4% (LS) or 8% (HS) NaCl diets for 14 days with or without a daily i.v. bolus infusion of BRL52537, a potent and selective kappa‐OR agonist. The hPods displayed a fast increase in fluorescence following an addition of BRL52537 (EC50 = 162 ± 2 μM) in a 2 mM calcium solution. A pre‐application of kappa‐OR antagonist (NorBNI) disrupted the effect of BRL52537 on calcium flux. The effect of BRL52537 on calcium in podocytes was completely abolished when we used the 0 mM calcium solution or a pre‐application of SAR7334 (a TRPC6 channel inhibitor), which revealed that calcium flux is mediated by activation of TRPC6 channels rather than other plasma membrane channels or depletion of intracellular calcium stores. Immunofluorescent analysis of human kidney validated kappa‐OR protein expression in podocytes. Podocytes of the freshly isolated human glomeruli also revealed robust increases in fluorescent intensity following a single application of BRL52537, which was abolished by a pre‐application of SAR7334. Effect of kappa‐OR agonist was specific since application of Damgo (mu‐OR agonist) or DPDPE (delta‐OR agonist) had either no or minimal effect in normotensive rats. Chronic studies in Dahl SS rats showed that in BRL‐treated group animals had elevation of microalbuminuria (13 ± 3 vs. 3 ± 1 Albumin/Creatinine at day 7 of HS), higher mean arterial pressures (MAP, 179 ± 15 mmHg vs. 151 ± 11 mmHg), and nephrinuria (50,232 ± 22,254 vs. 15,230 ± 4,647 a.u./Creatinine) compared to control rats. Podocytes isolated from the glomeruli from the hypertensive opioid treated animals had a significantly higher level of basal calcium (245 ± 23 vs. 139 ± 9 nM) compared to non‐treated group. Additionally, acute application of BRL52537 to podocytes of hypertensive animals resulted in a sustained increase in total [Ca2+]i influx. Our data established that stimulation of kappa‐ORs were specifically involved in increasing of intracellular calcium in podocytes through the TRPC6 pathway, which caused calcium overload and cellular damage. The opioid‐induced increase in the calcium flux in podocytes is expected to contribute to podocytopathy, proteinuria, kidney injury and progression of salt‐induced hypertension.

What Is the Mechanism Underlying Pressure‐induced Chronotropy in Collecting Lymphatic Vessels?

The rhythmic contractions of lymphatic smooth muscle (LSM) cells enable lymph propulsion against adverse pressure gradients. These contractions are initiated by LSM action potentials (APs), generated by an ionic pacemaker, whose rate is sensitive to the transmural pressure difference. We recently showed that SM‐specific deletion of the Ca2+‐activated Cl− channel, anoctamin1 (Ano1), reduced the basal AP firing rate of mouse inguinal‐axillary lymphatic (IALs) vessels to ~25% of normal and abrogated pressure‐induced chronotopy. However, IALs are efferent vessels that show only a modest (2‐fold) increase in contraction frequency over the presumed physiological pressure range 0.5 – 8 cmH2O. In contrast, popliteal afferent lymphatics (PLs) exhibit an 8‐fold increase in chronotropy from 0.5 – 5 cmH2O and SM‐specific deletion of Ano1 attenuates but does not abolish their pressure‐induced chronotropy. Here we asked: 1) what other ionic mechanisms participate in pressure‐induced chronotropy of PLs? 2) what accounts for the residual, pressure‐independent pacemaking in IALs and PLs after Ano1 deletion?To address these questions we measured the frequency‐pressure relationship (F‐P) of cannulated, pressurized PLs from control mice and mice deficient in key mechanosensitive and second‐messenger‐gated ion channels implicated in arterial pressure sensing. SM‐specific deletion of the mechanosensitive cation channel Piezo1 had no significant effect on F‐P. Global deletion of the osmosensitive cation channel TRPV4, or the DAG‐gated cation channels TRPC3 or TRPC6, or both, had no significant effect on F‐P. TRPM4−/− mice were not available, but the selective TRPM4 inhibitors, CBA and NBA, had minimal effects on F‐P. SM‐specific deletion of the cation channel TRPP1 (Pkd2) had no significant effect on F‐P. Deletion of T‐type Ca2+ channels Cav3.1 and/or Cav3.2 (previously implicated in lymphatic pacemaking), had no significant effect on F‐P. However, SM‐specific deletion of IP3R1, the only IP3R isoform expressed in mouse LSM, produced a phenotype similar to Ano1 deletion: a reduction in basal frequency and blunting of the F‐P relationship, suggesting a pressure‐induced IP3 generation downstream from mechanosensitive G‐protein(s). We then tested the prominent arterial SM mechanosensitive GPCR, AT1R. The inverse agonist losartan had no effect on F‐P nor did global knock out of AT1bR, the only isoform expressed in mouse LSM. In conclusion, our results point to a GPCR mechanosensing mechanism, independent of AT1R, that generates IP3 in a pressure‐sensitive manner to activate Ano1 and regulate the cycling rate of the ionic pacemaker in lymphatic smooth muscle.Support or Funding InformationNIH‐HL‐122578

Cellular mechanisms underlying the rapid depolarization caused by oxygen and glucose deprivation in layer III pyramidal cells of the somatosensory cortex

Cortical pyramidal neurons show rapid and irreversible membrane depolarization in response to oxygen-glucose depolarization (OGD). In this study, we investigated cellular mechanisms responsible for rapid depolarization caused by OGD in layer III pyramidal neurons of the mouse somatosensory cortex. When OGD solution was perfused in the presence of Ca2+ chelator and inhibitors of ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors (IP3Rs) in the pipette solution or in the presence of inhibitors of NMDA receptors (NMDARs), voltage-gated Ca2+ channels (VGCCs), and canonical transient receptor potential (TRPC) channels in the perfusion solution, the latency of the rapid depolarization was significantly prolonged compared to the control. In addition, when OGD solution was perfused in the presence of scavengers of nitric oxide and reactive oxygen species in the perfusion solution or in the presence of calcineurin inhibitors in the pipette solution, the latency of the rapid depolarization was significantly prolonged compared to the control. These data indicate that OGD-induced intracellular Ca2+ increases mediated by Ca2+ influx through NMDARs, VGCCs and TRPC channels as well as by Ca2+ release from RyRs and IP3Rs lead to mitochondrial impairment, which may facilitate the generation of the rapid depolarization via dysfunction of Na+-K+-ATPase due to decreased ATP production.

Canonical Transient Receptor Potential (TRPC) Channels in Nociception and Pathological Pain.

Chronic pathological pain is one of the most intractable clinical problems faced by clinicians and can be devastating for patients. Despite much progress we have made in understanding chronic pain in the last decades, its underlying mechanisms remain elusive. It is assumed that abnormal increase of calcium levels in the cells is a key determinant in the transition from acute to chronic pain. Exploring molecular players mediating Ca2+ entry into cells and molecular mechanisms underlying activity-dependent changes in Ca2+ signaling in the somatosensory pain pathway is therefore helpful towards understanding the development of chronic, pathological pain. Canonical transient receptor potential (TRPC) channels form a subfamily of nonselective cation channels, which permit the permeability of Ca2+ and Na+ into the cells. Initiation of Ca2+ entry pathways by these channels triggers the development of many physiological and pathological functions. In this review, we will focus on the functional implication of TRPC channels in nociception with the elucidation of their role in the detection of external stimuli and nociceptive hypersensitivity.

Novel Targets for Stroke Therapy: Special Focus on TRPC Channels and TRPC6.

Stroke remains a leading cause of death, disability, and medical care burden worldwide. However, transformation from laboratory findings toward effective pharmacological interventions for clinical stroke has been unsatisfactory. Novel evidence has been gained on the underlying mechanisms and therapeutic potential related to the transient receptor potential (TRP) channels in several disorders. The TRP superfamily consists of a diverse group of Ca2+ permeable non-selective cation channels. In particular, the members of TRP subfamilies, TRP canonical (TRPC) channels and TRPC6, have been found in different cell types in the whole body and have high levels of expression in the central nervous system (CNS). Notably, the TRPCs and TRPC6 channel have been implicated in neurite outgrowth and neuronal survival during normal development and in a range of CNS pathological conditions. Recent studies have shown that suppression of TRPC6 channel degradation prevents ischemic neuronal cell death in experimental stroke. Accumulating evidence supports the important functions of TRPC6 in brain ischemia. We have highlighted some crucial advancement that points toward an important involvement of TRPCs and TRPC6 in ischemic stroke. This review will make an overview of the TRP and TRPC channels due to their roles as targets for clinical trials and CNS disorders. Besides, the primary goal is to discuss and update the critical role of TRPC6 channels in stroke and provide a promising target for stroke prevention and therapy.

High-intensity interval training increases myocardial levels of Klotho and protects the heart against ischaemia-reperfusion injury.

What is the central question of this study? Can short-term high-intensity interval training (HIIT) contribute to the reduction of ischaemia-reperfusion (IR) injury by enhancing the levels of Klotho and its related axes, including myocardial TRPC6 expression, and antioxidant defence as novel possible mechanisms of exercise-induced cadioprotection (EICP) against IR injury? What is the main finding and its importance? The increase of plasma and myocardial levels of Klotho as a result of preconditioning with HIIT and prevention of a significant reduction of Klotho during IR injury can promote cardioprotection and reduce damage by attenuating myocardial TRPC6 expression and increasing antioxidant defence. The present findings may provide a new mechanism in EICP and IR injury, and provide the knowledge to develop preventive and therapeutic approaches. Cardiovascular disease, especially coronary artery disease, remains a major cause of morbidity and mortality in the world, and ischaemia-reperfusion (IR) insult is the main pathological cause leading to death. Exercise training is associated with a reduced risk of cardiovascular disease and the development of cardioprotection against IR injury. Therefore, the purpose of this study was to investigate the effect of preconditioning with high-intensity interval training (HIIT) on myocardial and plasma levels of Klotho and its related axes as novel mechanisms of exercise-induced cardioprotection against IR injury. Seventy male Wistar rats were randomly divided into five groups of control, HIIT, sham, IR and HIIT group that underwent IR injury (H-IR). The training group performed five sessions of HIIT on the treadmill. The cardiac IR injury was induced by ligation of the left anterior descending coronary artery for 30min followed by 24h reperfusion. Infarct size and histopathological assessment of cardiac tissues were determined through Evans Blue-triphenyltetrazolium chloride and haematoxylin-eosin staining, respectively. We investigated lipid peroxidation and markers of cardiac injury, antioxidant enzymes and the plasma levels of Klotho using enzyme-linked immunosorbent assays. Also, myocardial levels of Klotho and TRPC6 expression were determined by western blot assays. The results demonstrated a significant increase in myocardial and plasma levels of Klotho following HIIT and a significant decrease during IR injury. Myocardial TRPC6 channel expression increased following IR. HIIT also prevented a significant reduction of Klotho during IR and consequently reduced the expression of the TRPC6 channel in the H-IR group compared with the IR group. Furthermore, HIIT decreased the infarct size, cardiac injury, lipid peroxidation, lactate dehydrogenase, creatine kinase myocardial band and cardiac troponin-I, and improved total antioxidant capacity and catalase, superoxide dismutase and glutathione peroxidase activities following IR injury. The findings of the present study suggest that HIIT improves cardioprotection against IR injury and reduces cardiac damages through an increase in myocardial and plasma levels of Klotho and its related axes (TRPC6 and antioxidant defence). These findings can help to develop preventive and therapeutic approaches.

Engineering an enhanced voltage-sensing phosphatase.

Voltage-sensing phosphatases (VSP) consist of a membrane-spanning voltage sensor domain and a cytoplasmic region that has enzymatic activity toward phosphoinositides (PIs). VSP enzyme activity is regulated by membrane potential, and its activation leads to rapid and reversible alteration of cellular PIP levels. These properties enable VSPs to be used as a tool for studying the effects of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) binding to ion channels and transporters. For example, by applying simple changes in the membrane potential, Danio rerio VSP (Dr-VSP) has been used effectively to manipulate PI(4,5)P2 in mammalian cells with few, if any, side effects. In the present study, we report an enhanced version of Dr-VSP as an improved molecular tool for depleting PI(4,5)P2 from cultured mammalian cells. We modified Dr-VSP in two ways. Its voltage-dependent phosphatase activity was enhanced by introducing an aromatic residue at the position of Leu-223 within a membrane-interacting region of the phosphatase domain called the hydrophobic spine. In addition, selective plasma membrane targeting of Dr-VSP was facilitated by fusion with the N-terminal region of Ciona intestinalis VSP. This modified Dr-VSP (CiDr-VSPmChe L223F, or what we call eVSP) induced more drastic voltage-evoked changes in PI(4,5)P2 levels, using the activities of Kir2.1, KCNQ2/3, and TRPC6 channels as functional readouts. eVSP is thus an improved molecular tool for evaluating the PI(4,5)P2 sensitivity of ion channels in living cells.

Shape Similarity by Fractal Dimensionality: An Application in the de novo Design of (-)-Englerin A Mimetics.

Molecular shape and pharmacological function are interconnected. To capture shape, the fractal dimensionality concept was employed, providing a natural similarity measure for the virtual screening of de novo generated small molecules mimicking the structurally complex natural product (-)-englerin A. Two of the top-ranking designs were synthesized and tested for their ability to modulate transient receptor potential (TRP) cation channels which are cellular targets of (-)-englerin A. Intracellular calcium assays and electrophysiological whole-cell measurements of TRPC4 and TRPM8 channels revealed potent inhibitory effects of one of the computer-generated compounds. Four derivatives of this identified hit compound had comparable effects on TRPC4 and TRPM8. The results of this study corroborate the use of fractal dimensionality as an innovative shape-based molecular representation for molecular scaffold-hopping.

Molecular Regulations and Functions of the Transient Receptor Potential Channels of the Islets of Langerhans and Insulinoma Cells.

Insulin secretion from the β-cells of the islets of Langerhans is triggered mainly by nutrients such as glucose, and incretin hormones such as glucagon-like peptide-1 (GLP-1). The mechanisms of the stimulus-secretion coupling involve the participation of the key enzymes that metabolize the nutrients, and numerous ion channels that mediate the electrical activity. Several members of the transient receptor potential (TRP) channels participate in the processes that mediate the electrical activities and Ca2+ oscillations in these cells. Human β-cells express TRPC1, TRPM2, TRPM3, TRPM4, TRPM7, TRPP1, TRPML1, and TRPML3 channels. Some of these channels have been reported to mediate background depolarizing currents, store-operated Ca2+ entry (SOCE), electrical activity, Ca2+ oscillations, gene transcription, cell-death, and insulin secretion in response to stimulation by glucose and GLP1. Different channels of the TRP family are regulated by one or more of the following mechanisms: activation of G protein-coupled receptors, the filling state of the endoplasmic reticulum Ca2+ store, heat, oxidative stress, or some second messengers. This review briefly compiles our current knowledge about the molecular mechanisms of regulations, and functions of the TRP channels in the β-cells, the α-cells, and some insulinoma cell lines.

Structural basis for pharmacological modulation of the TRPC6 channel.

Transient receptor potential canonical (TRPC) proteins form nonselective cation channels that play physiological roles in a wide variety of cells. Despite growing evidence supporting the therapeutic potential of TRPC6 inhibition in treating pathological cardiac and renal conditions, mechanistic understanding of TRPC6 function and modulation remains obscure. Here we report cryo-EM structures of TRPC6 in both antagonist-bound and agonist-bound states. The structures reveal two novel recognition sites for the small-molecule modulators corroborated by mutagenesis data. The antagonist binds to a cytoplasm-facing pocket formed by S1-S4 and the TRP helix, whereas the agonist wedges at the subunit interface between S6 and the pore helix. Conformational changes upon ligand binding illuminate a mechanistic rationale for understanding TRPC6 modulation. Furthermore, structural and mutagenesis analyses suggest several disease-related mutations enhance channel activity by disrupting interfacial interactions. Our results provide principles of drug action that may facilitate future design of small molecules to ameliorate TRPC6-mediated diseases.