Transient Receptor Potential Channels Channels Research Articles

Canonical transient receptor potential channels and hypothalamic control of homeostatic functions.

Recent molecular biological and electrophysiological studies have identified multiple transient receptor potential (TRP) channels in hypothalamic neurons as critical modulators of homeostatic functions. In particular, the canonical transient receptor potential channels (TRPCs) are expressed in hypothalamic neurons that are vital for the control of fertility and energy homeostasis. Classical neurotransmitters such as serotonin and glutamate and peptide neurotransmitters such as kisspeptin, neurokinin B and pituitary adenylyl cyclase-activating polypeptide signal through their cognate G protein-coupled receptors to activate TPRC 4, 5 channels, which are essentially ligand-gated calcium channels. In addition to neurotransmitters, circulating hormones like insulin and leptin signal through insulin receptor (InsR) and leptin receptor (LRb), respectively, to activate TRPC 5 channels in hypothalamic arcuate nucleus pro-opiomelanocortin (POMC) and kisspeptin (arcuate Kiss1 [Kiss1ARH]) neurons to have profound physiological (excitatory) effects. Besides its overt depolarizing effects, TRPC channels conduct calcium ions into the cytoplasm, which has a plethora of downstream effects. Moreover, not only the expression of Trpc5 mRNA but also the coupling of receptors to TRPC 5 channel opening are regulated in different physiological states. In particular, the mRNA expression of Trpc5 is highly regulated in kisspeptin neurons by circulating estrogens, which ultimately dictates the firing pattern of kisspeptin neurons. In obesity states, InsRs are "uncoupled" from opening TRPC 5 channels in POMC neurons, rendering them less excitable. Therefore, in this review, we will focus on the critical role of TRPC 5 channels in regulating the excitability of Kiss1ARH and POMC neurons in different physiological and pathological states.

Expression and Purification of the Human Lipid-sensitive Cation Channel TRPC3 for Structural Determination by Single-particle Cryo-electron Microscopy.

Transient receptor potential channels (TRPCs) of the canonical TRP subfamily are nonselective cation channels that play an essential role in calcium homeostasis, particularly store-operated calcium entry, which is critical to maintaining proper function of synaptic vesicle release and intracellular signaling pathways. Accordingly, TRPC channels have been implicated in a variety of human diseases including cardiovascular disorders such as cardiac hypertrophy, neurodegenerative disorders such as Parkinson's disease, and neurologic disorders such as spinocerebellar ataxia. Therefore, TRPC channels represent a potential pharmacologic target in human diseases. However, the molecular mechanisms of gating in these channels are still unclear. The difficulty in obtaining large quantities of stable, homogeneous, and purified protein has been a limiting factor in structure determination studies, particularly for mammalian membrane proteins such as the TRPC ion channels. Here, we present a protocol for the large-scale expression of mammalian ion channel membrane proteins using a modified baculovirus gene transfer system and the purification of these proteins by affinity and size-exclusion chromatography. We further present a protocol to collect single-particle cryo-electron microscopy images from purified protein and to use these images to determine the protein structure. Structure determination is a powerful method for understanding the mechanisms of gating and function in ion channels.

Characterization of TRPC Channels in a Heterologous System Using Calcium Imaging and the Patch-Clamp Technique.

The family of transient receptor potential (TRPs) channels contains 28 mammalian members, each a unique cellular sensor that responds to a wide variety of external and internal signals. TRP channels are expressed by most mammalian cells, where they are involved in many different physiological functions. Canonical TRP channels (TRPCs) form a family of nonselective cationic channels, although with greater selectivity for Ca2+. This family is made up of seven members (TRPC1-7), all of which contain a TRP box in the carboxyl terminal and 3-4 ankyrin repeats in the amino terminal. While these channels share some similar properties, they display diverse gating mechanisms and are involved in different signaling pathways (Gees M et al., Compr Physiol, 2012). The activation or inhibition of these channels has been studied using different approaches and techniques. Here, we characterize the activation of the TRPC5 channel expressed in a heterologous system, using calcium imaging and the patch-clamp technique in whole-cell configuration.

TRPCing around the hypothalamus.

All of the canonical transient receptor potential channels (TRPC) with the exception of TRPC 2 are expressed in hypothalamic neurons and are involved in multiple homeostatic functions. Although the metabotropic glutamate receptors have been shown to be coupled to TRPC channel activation in cortical and sub-cortical brain regions, in the hypothalamus multiple amine and peptidergic G protein-coupled receptors (GPCRs) and growth factor/cytokine receptors are linked to activation of TRPC channels that are vital for reproduction, temperature regulation, arousal and energy homeostasis. In addition to the neurotransmitters, circulating hormones like insulin and leptin through their cognate receptors activate TRPC channels in POMC neurons. Many of the post-synaptic effects of the neurotransmitters and hormones are regulated in different physiological states by expression of TRPC channels in the post-synaptic neurons. Therefore, TRPC channels are key targets not only for neurotransmitters but circulating hormones in their vital role to control multiple hypothalamic functions, which is the focus of this review.

The effects of transient receptor potential channel (TRPC) on airway smooth muscle cell isolated from asthma model mice.

This study aimed to validate whether transient receptor potential channel1 (TRPC1) and TRPC3 participate in the regulation the proliferation of airway smooth muscle cells (ASMCs) through modulating calcium ion (Ca2+ ) influx in vitro. Chronic model of murine asthma was induced and ASMCs isolated from asthmatic mice were used in this whole study. TRPC1 and TRPC3 were upregulated in asthmatic mouse ASMCs and selected for further investigation. Ca2+ concentration and the cell viability of asthmatic mouse ASMCs were significantly higher than that from non- asthma mice, however, TRPC channels blocker SKF96365 alleviated these effects. Furthermore, TRPC1 or TRPC3 overexpression markedly increased Ca2+ concentration and significantly induced the viability of ASMCs; whereas TRPC1 or TRPC3 knockdown exerted the completely conversed effects. Moreover, knockdown of TRPC1 and TRPC3 also exerted different effects on the protein expression of growth-related proteins p-p38, p-JNK, cleaved caspase-3 and Bcl-2, as well as on cell cycle. Finally, we found Ca2+ chelator EGTA or BAPTA-AM significantly diminished the effects of si-TRPC1 and si-TRPC3 on the cell viability, cell cycle, and the protein expression of p-p38, p-JNK, cleaved caspase-3, and Bcl-2 in asthmatic mouse ASMCs. Our findings demonstrated that the effects of TRPC1 and TRPC3 on the cell viability and cell cycle of ASMCs were, at least partially, through regulating Ca2+ influx.

Targeting Transient Receptor Potential Canonical Channels for Diseases of the Nervous System.

Ca2+ influx plays an essential role in the physiological and pathophysiologic processes of several nervous system diseases. The transient receptor-potential channels (TRPCs) form a family of voltage -sensitive calcium ion channels. In this review, we will discuss the importance of transient receptor potential canonical (TRPC) channels, which is a crucial family of calcium channels. This article reviews the role of TRPC channels in the pathogenesis of diseases such as brain hemorrhage, hemorrhagic transformation after cerebral infarction, subarachnoid hemorrhage, and brain injury. TRPC has especially high expression in the central nervous system (CNS), and was involved in several physiological functions. The TRPC family is associated with cerebral vasospasm after subarachnoid hemorrhage, neuronal damage after intracerebral hemorrhage, NMDA cytotoxicity in cerebral ischemia, nervous system tumors, neurodegenerative diseases, neural addiction and other diseases. The TRPC family has rich functionality and is widely distributed, with different functions in various nervous system diseases.

Major contribution of the 3/6/7 class of TRPC channels to myocardial ischemia/reperfusion and cellular hypoxia/reoxygenation injuries

The injury phase after myocardial infarcts occurs during reperfusion and is a consequence of calcium release from internal stores combined with calcium entry, leading to cell death by apoptopic and necrotic processes. The mechanism(s) by which calcium enters cells has(ve) not been identified. Here, we identify canonical transient receptor potential channels (TRPC) 3 and 6 as the cation channels through which most of the damaging calcium enters cells to trigger their death, and we describe mechanisms activated during the injury phase. Working in vitro with H9c2 cardiomyoblasts subjected to 9-h hypoxia followed by 6-h reoxygenation (H/R), and analyzing changes occurring in areas-at-risk (AARs) of murine hearts subjected to a 30-min ischemia followed by 24-h reperfusion (I/R) protocol, we found: (i) that blocking TRPC with SKF96365 significantly ameliorated damage induced by H/R, including development of the mitochondrial permeability transition and proapoptotic changes in Bcl2/BAX ratios; and (ii) that AAR tissues had increased TUNEL+ cells, augmented Bcl2/BAX ratios, and increased p(S240)NFATc3, p(S473)AKT, p(S9)GSK3β, and TRPC3 and -6 proteins, consistent with activation of a positive-feedback loop in which calcium entering through TRPCs activates calcineurin-mediated NFATc3-directed transcription of TRPC genes, leading to more Ca2+ entry. All these changes were markedly reduced in mice lacking TRPC3, -6, and -7. The changes caused by I/R in AAR tissues were matched by those seen after H/R in cardiomyoblasts in all aspects except for p-AKT and p-GSK3β, which were decreased after H/R in cardiomyoblasts instead of increased. TRPC should be promising targets for pharmacologic intervention after cardiac infarcts.

The TRPC family of TRP channels: roles inferred (mostly) from knockout mice and relationship to ORAI proteins.

Aside from entering into cells through voltage gated Ca channels and Na/Ca exchangers in those cells that express these proteins, for all cells be they excitable or non-excitable, Ca(2+) enters through channels that are activated downstream of phosphoinositide mobilization (activation of phospholipase C, PLC) and through channels that are activated secondary to depletion of internal stores. Depletion of internal stores activates plasma membrane channels known as ORAIs. Activation of PLCs activates the canonical class of transient receptor potential channels (TRPCs), and, because this activation also causes depletion of Ca(2+) stores, also ORAI based channels. Whereas the activation of ORAI is a well-accepted phenomenon, it appears that TRPC channels also participate in Ca(2+) entry triggered by store depletion with or without participation of ORAI molecules. Regardless of molecular makeup of TRPC containing channels, a plethora of studies have shown TRPCs to be important both in physiologic systems as well as in pathophysiologic phenomena. Particularly important in defining roles of TRPCs, have been studies with mice with targeted disruption of their genes, i.e., with TRPC KO mice. In this chapter we first focus on TRPCs as regulators of body functions in health and disease, and then focus on the possible make-up of the channels of which they participate. A hypothesis is set forth, whereby ORAI dimers are proposed to be regulatory subunits of tetrameric TRPC channels and serve as structural units that form ORAI channels either as dimers of dimers or trimers of dimers.

TRPC channels are involved in calcium‐sensing receptor mediated inhibition of renin release from Juxtaglomerular cells

Renin containing juxtaglomerular (JG) cells express the calcium‐sensing receptor (CaSR), whose activation increases intracellular calcium (Ca). Increases in intracellular Ca decrease renin release. Canonical transient receptor potential channels (TRPC), a seven‐member subfamily of the transient receptor potential (TRP) family of ion channels, can function either as receptor‐operated or as store‐operated Ca channels, whose activation increases intracellular Ca. However, it is not known if these channels are expressed in JG cells and are involved in CaSR‐mediated regulation of renin release. We hypothesized that TRPC channels are expressed in JG cells and participate in the Ca‐mediated inhibition of renin release in response to activation of the CaSR. RT‐ PCR revealed mRNA expression of TRPC isoforms ‐1, ‐2, ‐3, ‐4, ‐5 and ‐6 in freshly isolated mouse JG cells. Incubation of primary cultures of JG cells with the TRPC blocker (SKF 96365, 50μM) did not change control values (0.46±0.09 vs. 0.46±0.08 μg ANGI/ml/hr/mg prot.) CaSR agonist cinacalcet decreased renin release by 46% to 0.25±0.05 μg ANGI/ml/hr/mg prot.(p<0.04). Addition of SKF 96365 completely reversed the CaSR‐mediated inhibition by cinacalcet. Our data suggests that JG cells contain multiple isoforms of TRPC channels, and at least one of these is linked to down‐stream signaling of CaSR‐mediated renin inhibition. (NIH 5PO1HL090550–04)

TRPC Channels In Ca2+ Regulation And Endothelial Function During Cardioplegic Exposure

Elevation of [Ca2+]i is essential to endothelial function and depolarization of endothelial membrane results in a reduction of Ca2+ influx. We studied whether canonical transient receptor potential channels (TRPC), i.e., TRPC3, is involved in [Ca2+]i dysregulation and endothelial dysfunction caused by depolarizing cardioplegia. [Ca2+]i was measured in porcine coronary endothelial cells (PCECs) and relaxation was studied in porcine small coronary arteries. Ca2+ influx and Ca2+ influx via TRPC3 were decreased in PCECs exposed to hyperkalemic solutions (10, 20 or 120 mM K+) in a concentration‐dependent manner and similar reductions occurred in cardioplegic exposure (ST, HTK or UW solution). The reduction was prevented by TRPC3/6/7 activator OAG. Hyperkalemic or cardioplegic solutions decreased EDHF‐mediated relaxation that was restored by OAG‐addition (in 20 mM K+: 52.5±4.0% vs. 67.2±4.3%; in ST: 52.2±4.8% vs. 65.0±4.8%; p<0.01). We conclude that the decrease of Ca2+ influx via TRPC3 is involved in endothelial [Ca2+]i reduction caused by depolarizing cardioplegia. TRPC activation restores Ca2+ influx and prevents EDHF impairment. This study may provide new insight for improvement of cardioplegic solutions.Supported by GRF CUHK4789/09M; CUHK2041688; China Nat'l Ministry of Sci. & Tech. 2009DFB30560 & 2010CB529502(973); Tianjin Municipal Sci. & Tech. Commission 09ZCZDSF04200 & 10JCYBJC26400.

Heteromeric Canonical Transient Receptor Potential 1 and 4 Channels Play a Critical Role in Epileptiform Burst Firing and Seizure-Induced Neurodegeneration

Canonical transient receptor potential channels (TRPCs) are receptor-operated cation channels that are activated in response to phospholipase C signaling. Although TRPC1 is ubiquitously expressed in the brain, TRPC4 expression is the most restrictive, with the highest expression level limited to the lateral septum. The subunit composition of neuronal TRPC channels remains uncertain because of conflicting data from recombinant expression systems. Here we report that the large depolarizing plateau potential that underlies the epileptiform burst firing induced by metabotropic glutamate receptor agonists in lateral septal neurons was completely abolished in TRPC1/4 double-knockout mice, and was abolished in 74% of lateral septal neurons in TRPC1 knockout mice. Furthermore, neuronal cell death in the lateral septum and the cornu ammonis 1 region of hippocampus after pilocarpine-induced severe seizures was significantly ameliorated in TRPC1/4 double-knockout mice. Our data suggest that both TRPC1 and TRPC4 are essential for an intrinsic membrane conductance mediating the plateau potential in lateral septal neurons, possibly as heteromeric channels. Moreover, excitotoxic neuronal cell death, an underlying process for many neurological diseases, is not mediated merely by ionotropic glutamate receptors but also by heteromeric TRPC channels activated by metabotropic glutamate receptors. TRPC channels could be an unsuspected but critical molecular target for clinical intervention for excitotoxicity.

Ca2+ Waves Initiate Antigen-Stimulated Ca2+ Responses in Mast Cells

Ca(2+) mobilization is central to many cellular processes, including stimulated exocytosis and cytokine production in mast cells. Using single cell stimulation by IgE-specific Ag and high-speed imaging of conventional or genetically encoded Ca(2+) sensors in rat basophilic leukemia and bone marrow-derived rat mast cells, we observe Ca(2+) waves that originate most frequently from the tips of extended cell protrusions, as well as Ca(2+) oscillations throughout the cell that usually follow the initiating Ca(2+) wave. In contrast, Ag conjugated to the tip of a micropipette stimulates local, repetitive Ca(2+) puffs at the region of cell contact. Initiating Ca(2+) waves are observed in most rat basophilic leukemia cells stimulated with soluble Ag and are sensitive to inhibitors of Ca(2+) release from endoplasmic reticulum stores and to extracellular Ca(2+), but they do not depend on store-operated Ca(2+) entry. Knockdown of transient receptor potential channel (TRPC)1 and TRPC3 channel proteins by short hairpin RNA reduces the sensitivity of these cells to Ag and shifts the wave initiation site from protrusions to the cell body. Our results reveal spatially encoded Ca(2+) signaling in response to immunoreceptor activation that utilizes TRPC channels to specify the initiation site of the Ca(2+) response.

Calcium entry in skeletal muscle

Calcium entry in skeletal muscle

Canonical Transient Receptor Potential Channel (TRPC) 3 and TRPC6 Associate with Large-Conductance Ca2+-Activated K+(BKCa) Channels: Role in BKCaTrafficking to the Surface of Cultured Podocytes

Large-conductance (BK(Ca) type) Ca(2+)-activated K(+) channels encoded by the Slo1 gene and various canonical transient receptor potential channels (TRPCs) are coexpressed in many cell types, including podocytes (visceral epithelial cells) of the renal glomerulus. In this study, we show by coimmunoprecipitation and GST pull-down assays that BK(Ca) channels can associate with endogenous TRPC3 and TRPC6 channels in differentiated cells of a podocyte cell line. Both types of TRPC channels colocalize with Slo1 in podocytes and in human embryonic kidney (HEK) 293T cells transiently coexpressing the TRPC channels with Slo1. In HEK293T cells, coexpression of TRPC6 increased surface expression of a Slo1 subunit splice variant (Slo1(VEDEC)) that is typically retained in intracellular compartments, as assessed by cell-surface biotinylation assays and confocal microscopy. Corresponding currents through BK(Ca) channels were also increased with TRPC6 coexpression, as assessed by whole-cell and excised inside-out patch recordings. By contrast, coexpression of TRPC3 had no effect on the surface expression of BK(Ca) channels in HEK293T cells or on the amplitudes of currents in whole cells or excised patches. In podocytes, small interfering RNA knockdown of endogenous TRPC6 reduced steady-state surface expression of endogenous Slo1 channels, but knockdown of TRPC3 had no effect. TRPC6, but not TRPC3 knockdown also reduced voltage-evoked outward current through podocyte BK(Ca) channels. These data indicate that TRPC6 and TRPC3 channels can bind to Slo1, and this colocalization may allow them to serve as a source of Ca(2+) for the activation of BK(Ca) channels. TRPC6 channels also play a role in the regulation of surface expression of a subset of podocyte BK(Ca) channels.

Interaction with dopamine D2 receptor enhances expression of transient receptor potential channel 1 at the cell surface

Receptor signaling is mediated by direct protein interaction with various types of cytoskeletal, adapter, effector, and additional receptor molecules. In brain tissue and in cultured neurons, activation of dopamine D2 receptors (D2Rs) has been found to impact cellular calcium signaling. Using a yeast two-hybrid approach, we have uncovered a direct physical interaction between the D2R and the transient receptor potential channel (TRPC) subtypes 1, 4 and 5. The TRPC/D2R interaction was further validated by GST-pulldown assays and coimmunoprecipitation from mammalian brain. Ultrastructural analysis of TRPC1 and D2R expression indicates colocalization of the two proteins within the cell body and dendrites of cortical neurons. In cultured cells, expression of D2Rs was found to increase expression of TRPC1 at the cell surface by 50%. These findings shed new light on the constituents of the D2R signalplex, and support the involvement of D2Rs in cellular calcium signaling pathways via a novel link to TRPC channels.

Immunohistochemical Study on the Distribution of Canonical Transient Receptor Potential Channels in Rat Cerebellum

Channels formed by the transient receptor potential (TRP) family of proteins have a variety of physiological functions. In the present study, we examined the localization of canonical transient receptor potential channels (TRPCs) in rat cerebellum. Twelve adult (4~6 month old) Sprague-Dawley rats were examined in this study. We performed immunohistochemistry using specific antibodies against TRPCs to investigate the detailed and comparative distribution of six TRPCs in rat cerebellum. There was a high density of TRPC1, TRPC3, TRPC4, TRPC5 and TRPC7, with a much lower density of TRPC6 in the rat cerebellar cortex. The somatodendritic Purkinje cell areas were intensely stained with antiTRPC3, TRPC4, TRPC5 or TRPC7 antibodies, whereas the staining intensity of TRPC6 was relatively low in the Purkinje cell bodies. In the cerebellar nuclei, the cell bodies of cerebellar output neurons showed moderate TRPC1, TRPC3, TRPC5 and TRPC7 immunoreactivities, while TRPC6 immunoreactivity was observed in the surrounding neuropil. This study showing the differential localizations of TRPC channels in the cerebellum may provide useful data for the future investigations on the structural and functional properties of TRPCs.

E-selectin permits communication between PAF receptors and TRPC channels in human neutrophils

AbstractThe selectin family of molecules (L-, P-, and E-selectin) mediates adhesive interactions between leukocytes and endothelial cells required for recruitment of leukocytes to inflammatory sites. Soluble E-selectin levels are elevated in inflammatory diseases and act to promote neutrophil β2-integrin–mediated adhesion by prolonging Ca2+ mobilization. Although soluble E-selectin alone was unable to initiate Ca2+ signaling, it allowed a novel “permissive” store-operative calcium entry (SOCE) following the initial platelet-activating factor (PAF)–induced release of Ca2+ from inositol 1,4,5-trisphosphate (IP3)–sensitive stores. This induction of permissive SOCE in response to soluble E-selectin and PAF was shown to act through a G protein-coupled receptor (GPCR) coupled to pertussis toxin-insensitive Gq/11. Furthermore, we demonstrated that permissive SOCE was mediated by canonical transient receptor potential channel (TRPC) due to its sensitivity to specific inhibition by MRS1845 and Gd3+ and that TRPC6 was the principal TRPC family member expressed by human neutrophils. In terms of mechanism, we demonstrated that soluble E-selectin activated Src family tyrosine kinases, an effect that was upstream of phosphatidylinositol 3′-kinase in a signaling pathway that regulates permissive SOCE following exposure of neutrophils to PAF. In summary, this report provides the first evidence for communication between an inflammatory mediator and adhesion receptors at a molecular level, through selectin receptor ligation allowing permissive SOCE to occur following PAF stimulation of human neutrophils.

Involvement of calmodulin and myosin light chain kinase in activation of mTRPC5 expressed in HEK cells

The classic type of transient receptor potential channel (TRPC) is a molecular candidate for Ca(2+)-permeable cation channels in mammalian cells. Because TRPC channels have calmodulin (CaM) binding sites at their COOH termini, we investigated the effect of CaM on mTRPC5. TRPC5 was initially activated by muscarinic stimulation with 50 microM carbachol and then decayed rapidly even in the presence of carbachol. Intracellular CaM (150 microg/ml) increased the amplitude of mTRPC5 current activated by muscarinic stimulation. CaM antagonists (W-7 and calmidazolium) inhibited mTRPC5 currents when they were applied during the activation of mTRPC5. Pretreatment of W-7 and calmidazolium also inhibited the activation of mTRPC5 current. Inhibitors of myosin light chain kinase (MLCK) inhibited the activation of mTRPC5 currents, whereas inhibitors of CaM-dependent protein kinase II did not. Small interfering RNA against cardiac type MLCK also inhibited the activation of mTRPC5 currents. However, inhibitors of CaM or MLCK did not show any effect on GTPgammaS-induced currents. Application of both Rho kinase inhibitor and MLCK inhibitor inhibited GTPgammaS-induced currents. We conclude that CaM and MLCK modulates the activation process of mTRPC5.

Epidermal Growth Factor Induces Tyrosine Phosphorylation, Membrane Insertion, and Activation of Transient Receptor Potential Channel 4

Various members of the canonical family of transient receptor potential channels (TRPCs) exhibit increased cation influx following receptor stimulation or Ca(2+) store depletion. Tyrosine phosphorylation of TRP family members also results in increased channel activity; however, the link between the two events is unclear. We report that two tyrosine residues in the C terminus of human TRPC4 (hTRPC4), Tyr-959 and Tyr-972, are phosphorylated following epidermal growth factor (EGF) receptor stimulation of COS-7 cells. This phosphorylation was mediated by Src family tyrosine kinases (STKs), with Fyn appearing to be the dominant kinase. In addition, EGF receptor stimulation induced the exocytotic insertion of hTRPC4 into the plasma membrane dependent on the activity of STKs and was accompanied by a phosphorylation-dependent increase in the association of hTRPC4 with Na(+)/H(+) exchanger regulatory factor. Furthermore, this translocation and association was defective upon mutation of Tyr-959 and Tyr-972 to phenylalanine. Significantly, inhibition of STKs was concomitant with a reduction in Ca(2+) influx in both native COS-7 cells and hTRPC4-expressing HEK293 cells, with cells expressing the Y959F/Y972F mutant exhibiting a reduced EGF response. These findings represent the first demonstration of a mechanism for phosphorylation to modulate TRPC channel function.

The Role of Canonical Transient Receptor Potential 7 in B-cell Receptor-activated Channels

Phospholipase C signaling stimulates Ca2+ entry across the plasma membrane through multiple mechanisms. Ca2+ store depletion stimulates store-operated Ca2+-selective channels, or alternatively, other phospholipase C-dependent events activate Ca2+-permeable non-selective cation channels. Transient receptor potential 7 (TRPC7) is a non-selective cation channel that can be activated by both mechanisms when ectopically expressed, but the regulation of native TRPC7 channels is not known. We knocked out TRPC7 in DT40 B-cells, which expresses both forms of Ca2+ entry. No difference in the store-operated current I(crac) was detected between TRPC7-/- and wild-type cells. Wild-type cells demonstrated nonstore-operated cation entry and currents in response to activation of the B-cell receptor or protease-activated receptor 2, intracellular dialysis with GTPgammaS, or application of the synthetic diacylglycerol oleyl-acetyl-glycerol. These responses were absent in TRPC7-/- cells but could be restored by transfection with human TRPC7. In conclusion, in B-lymphocytes, TRPC7 appeared to participate in the formation of ion channels that could be activated by phospholipase C-linked receptors. This represents the first demonstration of a physiological function for endogenous TRPC7 channels.