TRPC Channels Research Articles

Endocannabinoids produced in photoreceptor cells in response to light activate Drosophila TRP channels.

Drosophila phototransduction is a model for signaling cascades that culminate in the activation of transient receptor potential (TRP) cation channels. TRP and TRPL are the canonical TRP (TRPC) channels that are regulated by light stimulation of rhodopsin and engagement of Gαq and phospholipase Cβ (PLC). Lipid metabolite(s) generated downstream of PLC are essential for the activation of the TRPC channels in photoreceptor cells. We sought to identify the key lipids produced subsequent to PLC stimulation that contribute to channel activation. Here, using genetics, lipid analysis, and Ca2+ imaging, we found that light increased the amount of an abundant endocannabinoid, 2-linoleoyl glycerol (2-LG), in vivo. The increase in 2-LG amounts depended on the PLC and diacylglycerol lipase encoded by norpA and inaE, respectively. This endocannabinoid facilitated TRPC-dependent Ca2+ influx in a heterologous expression system and in dissociated ommatidia from compound eyes. Moreover, 2-LG and mechanical stimulation cooperatively activated TRPC channels in ommatidia. We propose that 2-LG is a physiologically relevant endocannabinoid that activates TRPC channels in photoreceptor cells.

Inhibition of transient receptor potential cation channel 6 promotes capillary arterialization during post-ischaemic blood flow recovery.

Capillary arterialization, characterized by the coverage of pre-existing or nascent capillary vessels with vascular smooth muscle cells (VSMCs), is critical for the development of collateral arterioles to improve post-ischaemic blood flow. We previously demonstrated that the inhibition of transient receptor potential 6 subfamily C, member 6 (TRPC6) channels facilitate contractile differentiation of VSMCs under ischaemic stress. We here investigated whether TRPC6 inhibition promotes post-ischaemic blood flow recovery through capillary arterialization in vivo. Mice were subjected to hindlimb ischaemia by ligating left femoral artery. The recovery rate of peripheral blood flow was calculated by the ratio of ischaemic left leg to non-ischaemic right one. The number and diameter of blood vessels were analysed by immunohistochemistry. Expression and phosphorylation levels of TRPC6 proteins were determined by western blotting and immunohistochemistry. Although the post-ischaemic blood flow recovery is reportedly dependent on endothelium-dependent relaxing factors, systemic TRPC6 deletion significantly promoted blood flow recovery under the condition that nitric oxide or prostacyclin production were inhibited, accompanying capillary arterialization. Cilostazol, a clinically approved drug for peripheral arterial disease, facilitates blood flow recovery by inactivating TRPC6 via phosphorylation at Thr69 in VSMCs. Furthermore, inhibition of TRPC6 channel activity by pyrazole-2 (Pyr2; BTP2; YM-58483) promoted post-ischaemic blood flow recovery in Apolipoprotein E-knockout mice. Suppression of TRPC6 channel activity in VSMCs could be a new strategy for the improvement of post-ischaemic peripheral blood circulation.

TRPC and TRPM channels: New possible targets for cancer

Cancer is the second most common reason for death in the world. The cancer research over four decades has been reached to the prospective on dysregulation of ions like (Ca2+, Mg2+, Na +, K+, or Cl - ) recently. These ions are orchestrated through numerous proteins, ion channels, selectively or non-selectively. However, the dysregulation of these ions and their channel expression are being reported for various diseases but here we have reviewed precisely TRP channels (TRPC and TRPM) for their role in cancer. The transient receptor potential (TRP) channels were first discovered in Drosophila melanogaster in 1989 and since then the superfamily becomes a group of 30 members under six subsections. Interestingly, we found that the TRPC (Canonical) channels, with 6 members, were explored in nine different types of cancers in last two decades. Additionally, we included the TRPM (Melastatin) subfamily and reviewed their role in cancer. Conclusively, these studies support that TRP channel-based therapies must be taken forward for clinical studies. Some channels, such as TRPC6, TRPM7 and TRPM8 were explored extensively in many cancer types which may be a potential target for cancer treatment. However, TRPM8 in lung cancer was reported for reverse association with cell proliferation, which needs to be reverified in lung cancer and other cancers. Besides, some TRPC channels are associated with store-operated calcium entry (SOCE) such as TRPC1, TRPC4 and TRPC6. Interestingly, the TRPC6 role was reported in breast cancer for modulation of Ca2+ through translocation of Orai1 and Orai3.

Transient Receptor Potential (TRP) Channels in Airway Toxicity and Disease: An Update.

Our respiratory system is exposed to toxicants and pathogens from both sides: the airways and the vasculature. While tracheal, bronchial and alveolar epithelial cells form a natural barrier in the airways, endothelial cells protect the lung from perfused toxic compounds, particulate matter and invading microorganism in the vascular system. Damages induce inflammation by our immune response and wound healing by (myo)fibroblast proliferation. Members of the transient receptor potential (TRP) superfamily of ion channel are expressed in many cells of the respiratory tract and serve multiple functions in physiology and pathophysiology. TRP expression patterns in non-neuronal cells with a focus on TRPA1, TRPC6, TRPM2, TRPM5, TRPM7, TRPV2, TRPV4 and TRPV6 channels are presented, and their roles in barrier function, immune regulation and phagocytosis are summarized. Moreover, TRP channels as future pharmacological targets in chronic obstructive pulmonary disease (COPD), asthma, cystic and pulmonary fibrosis as well as lung edema are discussed.

Inhibition of Ca2+ entry by capsazepine analog CIDD-99 prevents oral squamous carcinoma cell proliferation.

Oral cancer patients have a poor prognosis, with approximately 66% of patients surviving 5-years after diagnosis. Treatments for oral cancer are limited and have many adverse side effects; thus, further studies are needed to develop drugs that are more efficacious. To achieve this objective, we developed CIDD-99, which produces cytotoxic effects in multiple oral squamous cell carcinoma (OSCC) cell lines. While we demonstrated that CIDD-99 induces ER stress and apoptosis in OSCC, the mechanism was unclear. Investigation of the Bcl-family of proteins showed that OSCC cells treated with CIDD-99 undergo downregulation of Bcl-XL and Bcl-2 anti-apoptotic proteins and upregulation of Bax (pro-apoptotic). Importantly, OSCC cells treated with CIDD-99 displayed decreased calcium signaling in a dose and time-dependent manner, suggesting that blockage of calcium signaling is the key mechanism that induces cell death in OSCC. Indeed, CIDD-99 anti-proliferative effects were reversed by the addition of exogenous calcium. Moreover, electrophysiological properties further established that calcium entry was via the non-selective TRPC1 channel and prolonged CIDD-99 incubation inhibited STIM1 expression. CIDD-99 inhibition of calcium signaling also led to ER stress and inhibited mitochondrial complexes II and V in vitro. Taken together, these findings suggest that inhibition of TRPC mediates induction of ER stress and mitochondrial dysfunction as a part of the cellular response to CIDD-99 in OSCC.

Ionic signalling mechanisms involved in neurokinin-3 receptor-mediated augmentation of fear-potentiated startle response in the basolateral amygdala.

The tachykinin peptides include substance P (SP), neurokinin A and neurokinin B, which interact with three G-protein-coupled neurokinin receptors, NK1Rs, NK2Rs and NK3Rs, respectively. Whereas high densities of NK3Rs have been detected in the basolateral amygdala (BLA), the functions of NK3Rs in this brain region have not been determined. We found that activation of NK3Rs by application of the selective agonist, senktide, persistently excited BLA principal neurons. NK3R-elicited excitation of BLA neurons was mediated by activation of a non-selective cation channel and depression of the inwardly rectifying K+ (Kir) channels. With selective channel blockers and knockout mice, we further showed that NK3R activation excited BLA neurons by depressing the G protein-activated inwardly rectifying K+ (GIRK) channels and activating TRPC4 and TRPC5 channels. The effects of NK3Rs required the functions of phospholipase Cβ (PLCβ), but were independent of intracellular Ca2+ release and protein kinase C. PLCβ-mediated depletion of phosphatidylinositol 4,5-bisphosphate was involved in NK3R-induced excitation of BLA neurons. Microinjection of senktide into the BLA of rats augmented fear-potentiated startle (FPS) and this effect was blocked by prior injection of the selective NK3R antagonist SB218795, suggesting that activation of NK3Rs in the BLA increased FPS. We further showed that TRPC4/5 and GIRK channels were involved in NK3R-elicited facilitation of FPS. Our results provide a cellular and molecular mechanism whereby NK3R activation excites BLA neurons and enhances FPS. KEY POINTS: Activation of NK3 receptors (NK3Rs) facilitates the excitability of principal neurons in rat basolateral amygdala (BLA). NK3R-induced excitation is mediated by inhibition of GIRK channels and activation of TRPC4/5 channels. Phospholipase Cβ and depletion of phosphatidylinositol 4,5-bisphosphate are necessary for NK3R-mediated excitation of BLA principal neurons. Activation of NK3Rs in the BLA facilitates fear-potentiated startle response. GIRK channels and TRPC4/5 channels are involved in NK3R-mediated augmentation of fear-potentiated startle.

Interactions between the Polysialylated Neural Cell Adhesion Molecule and the Transient Receptor Potential Canonical Channels 1, 4, and 5 Induce Entry of Ca2+ into Neurons.

The neural cell adhesion molecule (NCAM) plays important functional roles in the developing and mature nervous systems. Here, we show that the transient receptor potential canonical (TRPC) ion channels TRPC1, −4, and −5 not only interact with the intracellular domains of the transmembrane isoforms NCAM140 and NCAM180, but also with the glycan polysialic acid (PSA) covalently attached to the NCAM protein backbone. NCAM antibody treatment leads to the opening of TRPC1, −4, and −5 hetero- or homomers at the plasma membrane and to the influx of Ca2+ into cultured cortical neurons and CHO cells expressing NCAM, PSA, and TRPC1 and −4 or TRPC1 and −5. NCAM-stimulated Ca2+ entry was blocked by the TRPC inhibitor Pico145 or the bacterial PSA homolog colominic acid. NCAM-stimulated Ca2+ influx was detectable neither in NCAM-deficient cortical neurons nor in TRPC1/4- or TRPC1/5-expressing CHO cells that express NCAM, but not PSA. NCAM-induced neurite outgrowth was reduced by TRPC inhibitors and a function-blocking TRPC1 antibody. A characteristic signaling feature was that extracellular signal-regulated kinase 1/2 phosphorylation was also reduced by TRPC inhibitors. Our findings indicate that the interaction of NCAM with TRPC1, −4, and −5 contributes to the NCAM-stimulated and PSA-dependent Ca2+ entry into neurons thereby influencing essential neural functions.

NCX1 coupled with TRPC1 to promote gastric cancer via Ca2+/AKT/β-catenin pathway

Plasma membrane Na+/Ca2+ exchanger 1 (NCX1) is a bidirectional ion transporter to operate in Ca2+ entry or exit modes, and TRPC1 is Ca2+-permeable channel. Both NCX1 and TRPC1 play critical roles in maintaining cytosolic free Ca2+ ([Ca2+]cyt) homeostasis in mammalian cells. Although either TRPC1 channel or Ca2+ entry mode of NCX1 is implicated in some tumorigenesis, it has not been explored if a coordination of NCX1 and TRPC1 involves in the pathogenesis of H. pylori-associated human gastric cancer (GC). Here we found the protein expression of NCX1 was significantly enhanced in human GC specimens, which correlated with tumor progression and poor survival in GC patients. TRPC1 and NCX1 were parallelly enhanced, co-localized and bound in human GC cells. By a functional coupling, TRPC1 drives NCX1 to the Ca2+ entry mode, raising [Ca2+]cyt in GC cells. Moreover, CaCl2, H. pylori and their virulence factors all enhanced expressions and activities of NCX1 and TRPC1, and evoked aberrant Ca2+ entry to promote proliferation, migration, and invasion of GC cells through AKT/β-catenin pathway. Tumor growth and metastasis also depended on the enhanced expression of NCX1 in subcutaneously xenografted GC mouse model. Overall, our findings indicate that TRPC1/NCX1 coupling may promote H. pylori-associated GC through the Ca2+/AKT/β-catenin pathway. Since the Ca2+ exit mode and the Ca2+ entry mode of NCX1 play different roles under mostly physiological and pathological conditions respectively, targeting TRPC1/NCX1 coupling could be a novel strategy for selectively blocking Ca2+ entry mode to potentially treat digestive cancer with less side effect.

TRPC6 N338S is a gain-of-function mutant identified in patient with doxorubicin-induced cardiotoxicity

The canonical transient receptor potential 6 gene, TRPC6, has been implicated as a putative risk gene for chemotherapy-induced congestive heart failure, but knowledge of specific risk variants is lacking. Following our genome-wide association study and subsequent fine-mapping, a rare missense mutant of TRPC6 N338S, was identified in a breast cancer patient who received anthracycline-containing chemotherapy regiments and developed congestive heart failure. However, the function of N338S mutant has not been examined. Using intracellular Ca2+ imaging, patch clamp recording and molecular docking techniques, we assessed the function of N338S mutant heterologously expressed in HEK293 cells and HL-1 cardiac cells. We found that expression of TRPC6 N338S significantly increased intracellular Ca2+ levels ([Ca2+]i) and current densities in response to 50 μM 1-oleoyl 2-acetyl-sn-glycerol (OAG), an activator of TRPC6 channels, compared to those of TRPC6 WT. A 24-h pretreatment with 0.5 μM doxorubicin (DOX) further potentiated the OAG effects on TRPC6 N338S current densities and [Ca2+]i, and these effects were abolished by 1 μM BI-749327, a highly selective TRPC6 inhibitor. Moreover, DOX treatment significantly upregulated the mRNA and protein expressions of TRPC6 N338S, compared to those of TRPC6 WT. Molecular docking and dynamics simulation showed that OAG binds to the pocket constituted by the pore-helix, S5 and S6 domains of TRPC6. However, the N338S mutation strengthened the interaction with OAG, therefore stabilizing the OAG-TRPC6 N338S complex and enhancing OAG binding affinity. Our results indicate that TRPC6 N338S is a gain-of-function mutant that may contribute to DOX-induced cardiotoxicity by increasing Ca2+ influx and [Ca2+]i in cardiomyocytes.

The TRPC1 Channel Forms a PI3K/CaM Complex and Regulates Pancreatic Ductal Adenocarcinoma Cell Proliferation in a Ca2+-Independent Manner.

Dysregulation of the transient receptor canonical ion channel (TRPC1) has been found in several cancer types, yet the underlying molecular mechanisms through which TRPC1 impacts pancreatic ductal adenocarcinoma (PDAC) cell proliferation are incompletely understood. Here, we found that TRPC1 is upregulated in human PDAC tissue compared to adjacent pancreatic tissue and this higher expression correlates with low overall survival. TRPC1 is, as well, upregulated in the aggressive PDAC cell line PANC-1, compared to a duct-like cell line, and its knockdown (KD) reduced cell proliferation along with PANC-1 3D spheroid growth by arresting cells in the G1/S phase whilst decreasing cyclin A, CDK2, CDK6, and increasing p21CIP1 expression. In addition, the KD of TRPC1 neither affected Ca2+ influx nor store-operated Ca2+ entry (SOCE) and reduced cell proliferation independently of extracellular calcium. Interestingly, TRPC1 interacted with the PI3K-p85α subunit and calmodulin (CaM); both the CaM protein level and AKT phosphorylation were reduced upon TRPC1 KD. In conclusion, our results show that TRPC1 regulates PDAC cell proliferation and cell cycle progression by interacting with PI3K-p85α and CaM through a Ca2+-independent pathway.

TRPC1 channels regulate the activation of pancreatic stellate cells through ERK1/2 and SMAD2 pathways and perpetuate their pressure-mediated activation

Pancreatic stellate cell (PSC) activation is a major event occurring during pancreatic ductal adenocarcinoma (PDAC) development. Up to now mechanisms underlying their activation by mechanical cues such as the elevated tissue pressure in PDAC remain poorly understood. Here we investigate the role of one potential mechano-transducer, TRPC1 ion channel, in PSC activation. Using pre-activated human siTRPC1 and murine TRPC1-KO PSCs, we show that TRPC1 promotes αSMA (α-smooth muscle actin) expression, the main activation marker, in cooperation with the phosphorylated SMAD2, under normal and elevated pressure. Functional studies following TRPC1 silencing demonstrate the dual role of TRPC1 in the modulation of PSC proliferation and IL-6 secretion through the activation of ERK1/2 and SMAD2 pathways. Moreover, pressurization changes the mechanical behavior of PSCs by increasing their cellular stiffness and emitted traction forces in a TRPC1-dependent manner. In summary, these results point to a role of TRPC1 channels in sensing and transducing the characteristic mechanical properties of the PDAC microenvironment in PSCs.

Brief exposure to directionally-specific pulsed electromagnetic fields stimulates extracellular vesicle release and is antagonized by streptomycin: A potential regenerative medicine and food industry paradigm

Pulsing electromagnetic fields (PEMFs) have been shown to promote in vitro and in vivo myogeneses via mitohormetic survival adaptations of which secretome activation is a key component. A single 10-min exposure of donor myoblast cultures to 1.5 mT amplitude PEMFs produced a conditioned media (pCM) capable of enhancing the myogenesis of recipient cultures to a similar degree as direct magnetic exposure. Downwardly-directed magnetic fields produced greater secretome responses than upwardly-directed fields in adherent and fluid-suspended myoblasts. The suspension paradigm allowed for the rapid concentrating of secreted factors, particularly of extracellular vesicles. The brief conditioning of basal media from magnetically-stimulated myoblasts was capable of conferring myoblast survival to a greater degree than basal media supplemented with fetal bovine serum (5%). Downward-directed magnetic fields, applied directly to cells or in the form of pCM, upregulated the protein expression of TRPC channels, markers for cell cycle progression and myogenesis. Direct magnetic exposure produced mild oxidative stress, whereas pCM provision did not, providing a survival advantage on recipient cells. Streptomycin, a TRP channel antagonist, precluded the production of a myogenic pCM. We present a methodology employing a brief and non-invasive PEMF-exposure paradigm to effectively stimulate secretome production and release for commercial or clinical exploitation.

The light-activated TRP channel: the founding member of the TRP channel superfamily

The Drosophila light-activated Transient Receptor Potential (TRP) channel is the founding member of a large and diverse family of channel proteins. The Drosophila TRP (dTRP) channel, which generates the electrical response to light has been investigated in a great detail two decades before the first mammalian TRP channel was discovered. Thus, dTRP is unique among members of the TRP channel superfamily because its physiological role and the enzymatic cascade underlying its activation are established. In this article we outline the research leading to elucidation of dTRP as the light activated channel and focus on a major physiological property of the dTRP channel, which is indirect activation via a cascade of enzymatic reactions. These detailed pioneering studies, based on the genetic dissection approach, revealed that light activation of the Drosophila TRP channel is mediated by G-Protein-Coupled Receptor (GPCR)-dependent enzymatic cascade, in which phospholipase C β (PLC) is a crucial component. This physiological mechanism of Drosophila TRP channel activation was later found in mammalian TRPC channels. However, the initial studies on the mammalian TRPV1 channel indicated that it is activated directly by capsaicin, low pH and hot temperature (>42 °C). This mechanism of activation was apparently at odds with the activation mechanism of the TRPC channels in general and the Drosophila light activated TRP/TRPL channels in particular, which are target of a GPCR-activated PLC cascade. Subsequent studies have indicated that under physiological conditions TRPV1 is also target of a GPCR-activated PLC cascade in the generation of inflammatory pain. The Drosophila light-activated TRP channel is still a useful experimental paradigm because its physiological function as the light-activated channel is known, powerful genetic techniques can be applied to its further analysis, and signaling molecules involved in the activation of these channels are available.

Calcium sparks enhance the tissue fluidity within epithelial layers and promote apical extrusion of transformed cells.

In vertebrates, newly emerging transformed cells are often apically extruded from epithelial layers through cell competition with surrounding normal epithelial cells. However, the underlying molecular mechanism remains elusive. Here, using phospho-SILAC screening, we show that phosphorylation of AHNAK2 is elevated in normal cells neighboring RasV12 cells soon after the induction of RasV12 expression, which is mediated by calcium-dependent protein kinase C. In addition, transient upsurges of intracellular calcium, which we call calcium sparks, frequently occur in normal cells neighboring RasV12 cells, which are mediated by mechanosensitive calcium channel TRPC1 upon membrane stretching. Calcium sparks then enhance cell movements of both normal and RasV12 cells through phosphorylation of AHNAK2 and promote apical extrusion. Moreover, comparable calcium sparks positively regulate apical extrusion of RasV12-transformed cells in zebrafish larvae as well. Hence, calcium sparks play a crucial role in the elimination of transformed cells at the early phase of cell competition.

A TRPC3/6 Channel Inhibitor Promotes Arteriogenesis after Hind-Limb Ischemia.

Retarded revascularization after progressive occlusion of large conductance arteries is a major cause of bad prognosis for peripheral artery disease (PAD). However, pharmacological treatment for PAD is still limited. We previously reported that suppression of transient receptor potential canonical (TRPC) 6 channel activity in vascular smooth muscle cells (VSMCs) facilitates VSMC differentiation without affecting proliferation and migration. In this study, we found that 1-benzilpiperadine derivative (1-BP), a selective inhibitor for TRPC3 and TRPC6 channel activities, induced VSMC differentiation. 1-BP-treated mice showed increased capillary arterialization and improvement of peripheral circulation and skeletal muscle mass after hind-limb ischemia (HLI) in mice. 1-BP had no additive effect on the facilitation of blood flow recovery after HLI in TRPC6-deficient mice, suggesting that suppression of TRPC6 underlies facilitation of the blood flow recovery by 1-BP. 1-BP also improved vascular nitric oxide bioavailability and blood flow recovery after HLI in hypercholesterolemic mice with endothelial dysfunction, suggesting the retrograde interaction from VSMCs to endothelium. These results suggest that 1-BP becomes a potential seed for PAD treatments that target vascular TRPC6 channels.

Critical contributions of pre-S1 shoulder and distal TRP box in DAG-activated TRPC6 channel by PIP2 regulation

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2 or PIP2) regulates the activities of numerous membrane proteins, including diacylglycerol(DAG)-activated TRPC3/6/7 channels. Although PIP2 binding is known to support DAG-activated TRP channel activity, its binding site remains unknown. We screened for PIP2 binding sites within TRPC6 channels through extensive mutagenesis. Using voltage-sensitive phosphatase (DrVSP), we found that Arg437 and Lys442, located in the channel’s pre-S1 domain/shoulder, are crucial for interaction with PIP2. To gain structural insights, we conducted computer protein–ligand docking simulations with the pre-S1 domain/shoulder of TRPC6 channels. Further, the functional significance of PIP2 binding to the pre-S1 shoulder was assessed for receptor-operated channel functions, cross-reactivity to DAG activation, and the kinetic model simulation. These results revealed that basic residues in the pre-S1 domain/shoulder play a central role in the regulation of PIP2-dependent gating. In addition, neutralizing mutation of K771 in the distal TRP box reversed the effect of PIP2 depletion from inhibiting to potentiating channel activity. A similar effect was seen in TRPV1 channels, which suggests that TRPC6 possesses a common but robust polarity switch mediating the PIP2-dependent effect. Overall, these mutagenesis studies reveal functional and structural insights for how basic residues and channel segments in TRP channels are controlled through phosphoinositides recognition.

Trpc6 gain-of-function disease mutation enhances phosphatidylserine exposure in murine platelets.

Platelets enhance coagulation by exposing phosphatidylserine (PS) on their cell surface in response to strong agonist activation. Transient receptor potential channels, including TRPC6, have been implicated in the calcium influx central to this process. Here, we characterize the effect of a Trpc6 gain-of-function (GOF) disease-associated, and a dominant negative (DN), mutation on murine platelet activation. Platelets from mice harboring Trpc6E896K/E896K (GOF) and Trpc6DN/DN mutations were subject to in vitro analysis. Trpc6E896K/E896K and Trpc6DN/DN mutant platelets show enhanced and absent calcium influx, respectively, upon addition of the TRPC3/6 agonist GSK1702934A (GSK). GSK was sufficient to induce integrin αIIbβ3 activation, P-selection and PS exposure, talin cleavage, and MLC2 phosphorylation in Trpc6E896K/E896K, but not in wild-type, platelets. Thrombin-induced calcium influx and PS exposure were enhanced, and clot retraction delayed, by GOF TRPC6, while no differences were noted between wild-type and Trpc6DN/DN platelets. In contrast, Erk activation upon GSK treatment was absent in Trpc6DN/DN, and enhanced in Trpc6E896K/E896K, platelets, compared to wild-type. The positive allosteric modulator, TRPC6-PAM-C20, and fluoxetine maintained their ability to enhance and inhibit, respectively, GSK-mediated calcium influx in Trpc6E896K/E896K platelets. The data demonstrate that gain-of-function mutant TRPC6 channel can enhance platelet activation, including PS exposure, while confirming that TRPC6 is not necessary for this process. Furthermore, the results suggest that Trpc6 GOF disease mutants do not simply increase wild-type TRPC6 responses, but can affect pathways not usually modulated by TRPC6 channel activity, displaying a true gain-of-function phenotype.

TRPC6 Inactivation Reduces Albuminuria Induced by Protein Overload in Sprague Dawley Rats.

Canonical transient receptor potential-6 (TRPC6) channels have been implicated in familial and acquired forms of focal and segmental glomerulosclerosis (FSGS), and in renal fibrosis following ureteral obstruction in mice. TRPC6 channels also appear to play a role in driving glomerular disease in aging and in autoimmune glomerulonephritis. In the present study, we examine the role of TRPC6 in the proteinuric state caused by prolonged albumin overload (AO) in Sprague Dawley rats induced by daily injections of exogenous albumin. This was assessed in rats with a global and constitutive inactivation of TRPC6 channels (Trpc6del/del rats) and in wild-type littermates (Trpc6wt/wt rats). AO for 14 and 28 days caused increased urine albumin excretion that was significantly attenuated in Trpc6del/del rats compared to Trpc6wt/wt controls. AO overload did not induce significant glomerulosclerosis or azotemia in either genotype. AO induced mild tubulointerstitial disease characterized by fibrosis, hypercellularity and increased expression of markers of fibrosis and inflammation. Those changes were equally severe in Trpc6wt/wt and Trpc6del/del rats. Immunoblot analysis of renal cortex indicated that AO increased the abundances of TRPC3 and TRPC6, and caused a nearly complete loss of TRPC5 in Trpc6wt/wt rats. The increase in TRPC3 and the loss of TRPC5 occurred to the same extent in Trpc6del/del rats. These data also suggest that TRPC6 plays a role in the normal function of the glomerular filtration barrier. However, whether TRPC6 inactivation protects the tubulointerstitial compartments in Sprague Dawley rats depends on the disease model examined.

Characterization of DAG Binding to TRPC Channels by Target-Dependent cis-trans Isomerization of OptoDArG.

Azobenzene-based photochromic lipids are valuable probes for the analysis of ion channel–lipid interactions. Rapid photoisomerization of these molecules enables the analysis of lipid gating kinetics and provides information on lipid sensing. Thermal relaxation of the metastable cis conformation to the trans conformation of azobenzene photolipids is rather slow in the dark and may be modified by ligand–protein interactions. Cis photolipid-induced changes in pure lipid membranes as visualized from the morphological response of giant unilamellar vesicles indicated that thermal cis–trans isomerization of both PhoDAG-1 and OptoDArG is essentially slow in the lipid bilayer environment. While the currents activated by cis PhoDAG remained stable upon termination of UV light exposure (dark, UV-OFF), cis OptoDArG-induced TRPC3/6/7 activity displayed a striking isoform-dependent exponential decay. The deactivation kinetics of cis OptoDArG-induced currents in the dark was sensitive to mutations in the L2 lipid coordination site of TRPC channels. We conclude that the binding of cis OptoDArG to TRPC channels promotes transition of cis OptoDArG to the trans conformation. This process is suggested to provide valuable information on DAG–ion channel interactions and may enable highly selective photopharmacological interventions.

Synthesis of AC1903 analogs as potent transient receptor potential canonical channel 4/5 inhibitors and biological evaluation

Transient receptor potential canonical (TRPC) channels are a class of non-selective cation channels expressed in a variety of tissues and organ systems where they functionally regulate physiological and pathological processes. TRPC5 has been shown to be a promising target for focal segmental glomerulosclerosis treatment. In this study, we report the synthesis and biological evaluation of a novel series of benzimidazole-based TRPC5 inhibitors. One compound, 8b, is 100-fold more potent than the parent compound, AC1903, in the suppression of TRPC5 channel activity. Interestingly, both AC1903 and 8b also suppressed TRPC4 channel activity with similar potency. Compound 8b also significantly blunts protamine sulfate-induced reorganization of podocyte cytoskeleton, interleukin (IL)-17-induced cell proliferation, and the expression of proinflammatory mediators in human keratinocyte HaCaT cells.