Classical Transient Receptor Potential Research Articles

Podocyte Purinergic P2X4 Channels Are Mechanotransducers That Mediate Cytoskeletal Disorganization.

Podocytes are specialized, highly differentiated epithelial cells in the kidney glomerulus that are exposed to glomerular capillary pressure and possible increases in mechanical load. The proteins sensing mechanical forces in podocytes are unconfirmed, but the classic transient receptor potential channel 6 (TRPC6) interacting with the MEC-2 homolog podocin may form a mechanosensitive ion channel complex in podocytes. Here, we observed that podocytes respond to mechanical stimulation with increased intracellular calcium concentrations and increased inward cation currents. However, TRPC6-deficient podocytes responded in a manner similar to that of control podocytes, and mechanically induced currents were unaffected by genetic inactivation of TRPC1/3/6 or administration of the broad-range TRPC blocker SKF-96365. Instead, mechanically induced currents were significantly decreased by the specific P2X purinoceptor 4 (P2X4) blocker 5-BDBD. Moreover, mechanical P2X4 channel activation depended on cholesterol and podocin and was inhibited by stabilization of the actin cytoskeleton. Because P2X4 channels are not intrinsically mechanosensitive, we investigated whether podocytes release ATP upon mechanical stimulation using a fluorometric approach. Indeed, mechanically induced ATP release from podocytes was observed. Furthermore, 5-BDBD attenuated mechanically induced reorganization of the actin cytoskeleton. Altogether, our findings reveal a TRPC channel-independent role of P2X4 channels as mechanotransducers in podocytes.

Classical Transient Receptor Potential 1 (TRPC1): Channel or Channel Regulator?

In contrast to other Classical Transient Receptor Potential TRPC channels the function of TRPC1 as an ion channel is a matter of debate, because it is often difficult to obtain substantial functional signals over background in response to over-expression of TRPC1 alone. Along these lines, heterologously expressed TRPC1 is poorly translocated to the plasma membrane as a homotetramer and may not function on its own physiologically, but may rather be an important linker and regulator protein in heteromeric TRPC channel tetramers. However, due to the lack of specific TRPC1 antibodies able to detect native TRPC1 channels in primary cells, identification of functional TRPC1 containing heteromeric TRPC channel complexes in the plasma membrane is still challenging. Moreover, an extended TRPC1 cDNA, which was recently discovered, may seriously question results obtained in heterologous expression systems transfected with shortened cDNA versions. Therefore, this review will focus on the current status of research on TRPC1 function obtained in primary cells and a TRPC1-deficient mouse model.

Extracellular disulfide bridges stabilize TRPC5 dimerization, trafficking, and activity.

Crucial cysteine residues can be involved in the modulation of protein activity via the modification of thiol (-SH) groups. Among these reactions, disulfide bonds (S-S) play a key role in the folding, stability, and activity of membrane proteins. However, the regulation of extracellular cysteines in classical transient receptor potential (TRPC) channels remains controversial. Here, we examine the functional importance of the extracellular disulfide bond in TRPC5 in modulating channel gating and trafficking. Specifically, we investigated TRPC5 activity in transiently transfected HEK293 cells with wild-type (WT) or cysteine (C553 and C558) mutants in the pore loop. Using reducing agents, we determined that a disulfide linkage mediates the tetrameric formation of the TRPC5 channel. By measuring the TRPC5 current, we observed that C553S or C558S mutants completely lose channel activity induced by lanthanides or receptor stimulation. Co-expression of TRPC5 (WT) with mutants demonstrated a dominant-negative function in mutants, which inhibited the activity of TRPC5 (WT). We generated TRPC5-TRPC5 dimers and observed reduced activity of WT-mutant (C553S or C558S) dimers compared to WT-WT dimers. When pretreated with reducing agents for 12 h, the TRPC5 current decreased due to a reduction in membrane TRPC5 distribution. In addition, we identified a reduced expression of C553S mutant in plasma membrane. We analyzed a dimeric interaction of wild-type and mutant TRPC5 using co-immunoprecipitation and FRET method, indicating a weak interaction between dimeric partners. These results indicated that the disulfide bond between conserved extracellular cysteines, especially C553, is essential for functional TRPC5 activity by channel multimerization and trafficking.

Classical Transient Receptor Potential 1 and 6 Contribute to Hypoxic Pulmonary Hypertension Through Differential Regulation of Pulmonary Vascular Functions

Hypoxic pulmonary hypertension is characterized by increased vascular tone, altered vasoreactivity, and vascular remodeling, which are associated with alterations in Ca 2+ homeostasis in pulmonary arterial smooth muscle cells. We have previously shown that classical transient receptor potential 1 and 6 (TRPC1 and TRPC6) are upregulated in pulmonary arteries (PAs) of chronic hypoxic rats, but it is unclear whether these channels are essential for the development of pulmonary hypertension. Here we found that pulmonary hypertension was suppressed in TRPC1 and TRPC6 knockout ( Trpc1 −/− and Trpc6 −/− ) mice compared with wild-type after exposure to 10% O 2 for 1 and 3 weeks. Muscularization of pulmonary microvessels was inhibited, but rarefaction was unaltered in hypoxic Trpc1 −/− and Trpc6 −/− mice. Small PAs of normoxic wild-type mice exhibited vasomotor tone, which was significantly enhanced by chronic hypoxia. Similar vasomotor tone was found in normoxic Trpc1 −/− PAs, but the hypoxia-induced enhancement was blunted. In contrast, there was minimal vascular tone in normoxic Trpc6 −/− PAs, but the hypoxia-enhanced tone was preserved. Chronic hypoxia caused significant increase in serotonin-induced vasoconstriction; the augmented vasoreactivity was attenuated in Trpc1 −/− and eliminated in Trpc6 −/− PAs. Moreover, the effects of 3-week hypoxia on pulmonary arterial pressure, right ventricular hypertrophy, and muscularization of microvessels were further suppressed in TRPC1-TRPC6 double-knockout mice. Our results, therefore, provide clear evidence that TRPC1 and TRPC6 participate differentially in various pathophysiological processes, and that the presence of TRPC1 and TRPC6 is essential for the full development of hypoxic pulmonary hypertension in the mouse model.

Classical Transient Receptor Potential Channel 1 in Hypoxia-induced Pulmonary Hypertension

Pulmonary hypertension (PH) is a life-threatening disease, characterized by pulmonary vascular remodeling. Abnormal smooth muscle cell proliferation is a primary hallmark of chronic hypoxia-induced PH. Essential for cell growth are alterations in the intracellular Ca(2+) homeostasis. Classical transient receptor potential (TRPC) proteins have been suggested to contribute to PH development, as TRPC1 and TRPC6 are predominantly expressed in precapillary pulmonary arterial smooth muscle cells (PASMC). Studies in a TRPC6-deficient mouse model revealed an essential function of TRPC6 in acute but not in chronic hypoxia. We aimed to identify the importance of TRPC1 in the pathogenesis of chronic hypoxia-induced PH in mice. TRPC1 expression analysis was performed using real-time polymerase chain reaction. TRPC1 function was assessed by in vivo experiments in TRPC1(-/-) animals as well as in isolated precapillary murine PASMC after TRPC1 knockdown by TRPC1-specific small interfering RNAs. Only TRPC1 mRNA was up-regulated under hypoxia in isolated murine PASMC (1% O2 for 72 h). Hypoxia-induced proliferation of murine PASMC was attenuated in cells treated with small interfering RNA against TRPC1 and in cells isolated from TRPC1(-/-) animals compared with untreated and wild-type cells. TRPC1(-/-) mice did not develop PH in response to chronic hypoxia (FI(O2) 0.10 for 21 d) and had less vascular muscularization but a similar degree of right ventricular hypertrophy compared with wild-type mice. Our results indicate an important role of TRPC1 in pulmonary vascular remodeling underlying the development of hypoxia-induced PH.

TRPC6 Regulates CXCR2-Mediated Chemotaxis of Murine Neutrophils

Unraveling the mechanisms involved in chemotactic navigation of immune cells is of particular interest for the development of new immunoregulatory therapies. It is generally agreed upon that members of the classical transient receptor potential channel family (TRPC) are involved in chemotaxis. However, the regulatory role of TRPC channels in chemoattractant receptor-mediated signaling has not yet been clarified in detail. In this study, we demonstrate that the TRPC6 channels play a pronounced role in CXCR2-mediated intermediary chemotaxis, whereas N-formyl-methionine-leucine-phenylalanine receptor-mediated end-target chemotaxis is TRPC6 independent. The knockout of TRPC6 channels in murine neutrophils led to a strongly impaired intermediary chemotaxis after CXCR2 activation which is not further reinforced by CXCR2, PI3K, or p38 MAPK inhibition. Furthermore, CXCR2-mediated Ca(2+) influx but not Ca(2+) store release was attenuated in TRPC6(-/-) neutrophils. We demonstrate that the TRPC6 deficiency affected phosphorylation of AKT and MAPK downstream of CXCR2 receptor activation and led to altered remodeling of actin. The relevance of this TRPC6-depending defect in neutrophil chemotaxis is underscored by our in vivo findings. A nonseptic peritoneal inflammation revealed an attenuated recruitment of neutrophils in the peritoneal cavity of TRPC6(-/-) mice. In summary, this paper defines a specific role of TRPC6 channels in CXCR2-induced intermediary chemotaxis. In particular, TRPC6-mediated supply of calcium appears to be critical for activation of downstream signaling components.

Stretch-Induced Activation of NFAT Signaling in Hl-1 Cardiomyocytes

Mechanical forces are efficiently converted into cellular signals to initiate structural and functional remodeling of cardiac myocytes. Molecular mechanisms underlying cardiac mechanotransduction are still barely understood. Nuclear Factor of Activated T-cells (NFAT) is an important calcium-responsive transcription factor and a key player in cardiac remodeling processes. In this study, we investigated the involvement of Ca2+ signaling molecules as a link between mechanical stretch and NFAT translocation in HL-1 atrial myocytes. We specifically focused on the potential role of receptor-operated and/or store-operated Ca2+ permeable channels of the TRPCs (classical transient receptor potential) and the Orai family. Populations of HL-1 cells overexpressing GFP-NFAT were exposed to a single-stretch by 20% for 20 minutes on commercially available silicon membranes and NFAT localization was then analyzed by fluorescence microscopy. Results revealed that stretched HL-1 cells significantly increased NFAT nuclear translocation when compared to unstretched cells. Similar NFAT activation was obtained when HL-1 cells were stimulated by either endothelin-1 (100 nM) or thapsigargin (1μM) in the absence of a stretch stimulus. These results demonstrate that similar levels of NFAT activation are induced in HL-1 cells by either mechanical stress, cell activation via Gq-coupled receptors or depletion of internal Ca2+ stores. HL-1 cells were found to express several TRPC isoforms that might contribute to mechanotransduction. Our data suggest a linkage between mechanical stretch and cardiac remodeling involving Ca2+ entry and NFAT activation.This work is funded by FWF (Austrian research fund) projects P21925-B19, P22565-B18 and the DK Metabolic and Cardiovascular Disease grant W1226-B18.

Transient Receptor Potential Channels in Human Platelets: Expression and Functional Role

Recent studies have demonstrated that mammalian homologues of Drosophila transient receptor potential (TRP) channels are widely expressed in human platelets. Occupation of G protein-coupled receptors by agonists results in activation of these channels, which results in Na+ and Ca2+ entry. Canonical or classic TRP (TRPC) family members have been reported to associate with different Ca2+-handling proteins, including the type II inositol 1,4,5-trisphosphate receptor, the endoplasmic reticulum Ca2+ sensor STIM1 (STromal Interaction Molecule-1) or the Ca2+ permeable channel Orai1. The dynamic interaction of TRPC channels with the above mentioned proteins has been found to be important for both store-operated and capacitative Ca2+ entry, as well as for non-capacitative Ca2+ influx. The former is a major mechanism for Ca2+ entry in human platelets. This mechanism, activated by a reduction in the concentration of free Ca2+ in the intracellular stores, results in the formation of signaling complexes involving STIM proteins, Orai1, Orai2, TRPC1 and TRPC6. There is a growing body of evidence supporting that Ca2+ signaling dysfunction plays an important role in the pathogenesis of several platelet-linked disorders, including those associated to type 2 diabetes mellitus. Abnormal Ca2+ signals in response to physiological agonists have been associated to platelet hyperactivity. The expression of several TRPCs, STIM1 and Orai1, as well as their interaction, has been reported to be altered in platelets from type 2 diabetic patients, which results in attenuated capacitative Ca2+ entry but enhanced non-capacitative Ca2+ influx; thus suggesting a role for Ca2+ handling proteins, including TRPs, in the pathomechanism of diabetic complications.

Transient Receptor Potential Channel 1 (TRPC1) Reduces Calcium Permeability in Heteromeric Channel Complexes

Specific biological roles of the classical transient receptor potential channel 1 (TRPC1) are still largely elusive. To investigate the function of TRPC1 proteins in cell physiology, we studied heterologously expressed TRPC1 channels and found that recombinant TRPC1 subunits do not form functional homomeric channels. Instead, by electrophysiological analysis TRPC1 was shown to form functional heteromeric, receptor-operated channel complexes with TRPC3, -4, -5, -6, and -7 indicating that TRPC1 proteins can co-assemble with all members of the TRPC subfamily. In all TRPC1-containing heteromers, TRPC1 subunits significantly decreased calcium permeation. The exchange of select amino acids in the putative pore-forming region of TRPC1 further reduced calcium permeability, suggesting that TRPC1 subunits contribute to the channel pore. In immortalized immature gonadotropin-releasing hormone neurons endogenously expressing TRPC1, -2, -5, and -6, down-regulation of TRPC1 resulted in increased calcium permeability and elevated basal cytosolic calcium concentrations. We did not observe any involvement of TRPC1 in store-operated cation influx. Notably, TRPC1 suppressed the migration of gonadotropin-releasing hormone neurons without affecting cell proliferation. Conversely, in TRPC1 knockdown neurons, specific migratory properties like distance covered, locomotion speed, and directionality were increased. These findings suggest a novel regulatory mechanism relying on the expression of TRPC1 and the subsequent formation of heteromeric TRPC channel complexes with reduced calcium permeability, thereby fine-tuning neuronal migration.

Activation of TRPC6 channels is essential for lung ischaemia–reperfusion induced oedema in mice

Lung ischaemia–reperfusion-induced oedema (LIRE) is a life-threatening condition that causes pulmonary oedema induced by endothelial dysfunction. Here we show that lungs from mice lacking nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox2y/−) or the classical transient receptor potential channel 6 (TRPC6−/−) are protected from LIR-induced oedema (LIRE). Generation of chimeric mice by bone marrow cell transplantation and endothelial-specific Nox2 deletion showed that endothelial Nox2, but not leukocytic Nox2 or TRPC6, are responsible for LIRE. Lung endothelial cells from Nox2- or TRPC6-deficient mice showed attenuated ischaemia-induced Ca2+ influx, cellular shape changes and impaired barrier function. Production of reactive oxygen species was completely abolished in Nox2y/− cells. A novel mechanistic model comprising endothelial Nox2-derived production of superoxide, activation of phospholipase C-γ, inhibition of diacylglycerol (DAG) kinase, DAG-mediated activation of TRPC6 and ensuing LIRE is supported by pharmacological and molecular evidence. This mechanism highlights novel pharmacological targets for the treatment of LIRE.

Diacylglycerol regulates acute hypoxic pulmonary vasoconstriction via TRPC6

BackgroundHypoxic pulmonary vasoconstriction (HPV) is an essential mechanism of the lung that matches blood perfusion to alveolar ventilation to optimize gas exchange. Recently we have demonstrated that acute but not sustained HPV is critically dependent on the classical transient receptor potential 6 (TRPC6) channel. However, the mechanism of TRPC6 activation during acute HPV remains elusive. We hypothesize that a diacylglycerol (DAG)-dependent activation of TRPC6 regulates acute HPV.MethodsWe investigated the effect of the DAG analog 1-oleoyl-2-acetyl-sn-glycerol (OAG) on normoxic vascular tone in isolated perfused and ventilated mouse lungs from TRPC6-deficient and wild-type mice. Moreover, the effects of OAG, the DAG kinase inhibitor R59949 and the phospholipase C inhibitor U73122 on the strength of HPV were investigated compared to those on non-hypoxia-induced vasoconstriction elicited by the thromboxane mimeticum U46619.ResultsOAG increased normoxic vascular tone in lungs from wild-type mice, but not in lungs from TRPC6-deficient mice. Under conditions of repetitive hypoxic ventilation, OAG as well as R59949 dose-dependently attenuated the strength of acute HPV whereas U46619-induced vasoconstrictions were not reduced. Like OAG, R59949 mimicked HPV, since it induced a dose-dependent vasoconstriction during normoxic ventilation. In contrast, U73122, a blocker of DAG synthesis, inhibited acute HPV whereas U73343, the inactive form of U73122, had no effect on HPV.ConclusionThese findings support the conclusion that the TRPC6-dependency of acute HPV is induced via DAG.

Novel pyrazole inhibitors for discrimination between receptor-operated and store-operated Ca2+ entry

Background Calcium governs a wide range of cellular processes. Specifically, control of gene transcription involves Ca entry channels that are activated by either voltage, second messengers or depletion of intracellular stores. The family of classical transient receptor potential channels (TRPC) has been implicated in both the receptor/second messenger as well as in store-operated Ca entry pathway, and represents an attractive target for therapeutic intervention.

Kisspeptin-Evoked Calcium Signals in Isolated Primary Rat Gonadotropin- Releasing Hormone Neurones

Background: Kisspeptin and its cognate receptor GPR54 are the central driving forces in the hypothalamus-pituitary-gonadal axis essential for sexual maturation and reproduction. Kisspeptin/GPR54 signalling stimulates gonadotropin-releasing hormone (GnRH) neurones and induces pulsatile GnRH release. The molecular signalling pathway by which kisspeptin stimulates GnRH neurones is currently under investigation. Methods: Primary GnRH neurones were isolated from young adult rats and loaded with the calcium indicator Fura Red. Cytosolic calcium was measured while the cells were stimulated with kisspeptin. Results: GnRH neurones show a maintained increase of cytosolic calcium upon stimulation with 100 nM kisspeptin-10. The calcium elevation was inhibited 30% by 1 µM tetrodotoxin, a voltage-gated sodium channel blocker, and 76% by 30 µM SKF96365, an inhibitor of receptor-mediated calcium entry. Furthermore, removal of extracellular calcium completely abolished the kisspeptin-induced calcium elevation. Conclusion: Our results suggest that the major part of the kisspeptin-evoked calcium signal is generated by an action potential-independent calcium influx, possibly through channels of the classical transient receptor potential type, with an additional influx through voltage-gated calcium channels activated by sodium action potentials.

Evidence for a supportive role of classical transient receptor potential 6 (TRPC6) in the exploration behavior of mice

Non-selective classical transient receptor potential (TRPC) cation channels share important roles in processes of neuronal development and function. To test the influence of TRPC6 activity on behavior, we developed a TRPC6-deficient (TRPC6(-/-)) mouse model in a BALB/c genetic background. Both, TRPC6(-/-) and wild-type (WT) mice were analyzed first for their general health and reflex status (modified SHIRPA protocol) and then in three different behavioral tests (marble-burying test, square open field and elevated star maze). No abnormalities were detected in the SHIRPA protocol. Most interestingly, TRPC6(-/-) mice showed no significant differences in anxiety in a marble-burying test, but demonstrated reduced exploration in the square open field and the elevated star maze. Therefore, TRPC6 channel activity may play a yet unknown role for exploration behavior.

Attenuation of store‐operated Ca2+ entry and enhanced expression of TRPC channels in caudal artery smooth muscle from Type 2 diabetic Goto‐Kakizaki rats

1. Previously, we found that Ca(2+) entry from the extracellular space via alpha(1)-adrenoceptor-activated, Ca(2+)-permeable channels, but not voltage-gated Ca(2+) channels, is impaired in endothelium-denuded caudal artery smooth muscle from Type 2 diabetic Goto-Kakizaki (GK) rats. In the present study, we investigated the impairment of Ca(2+) entry mechanisms via Ca(2+)-permeable channels from the extracellular space in response to alpha(1)-adrenoceptor stimulation (cirazoline) in endothelium-denuded caudal artery strips isolated from GK rats. 2. The contraction of caudal artery strips from GK rats in response to the sarcoplasmic reticulum Ca(2+)-ATPase inhibitor cyclopiazonic acid (10 micromol/L), which causes depletion of Ca(2+) stores and subsequent store-operated Ca(2+) (SOC) entry, was significantly depressed compared with that of Wistar rats (maximal force 0.023 +/- 0.004 vs 0.058 +/- 0.005 mN/mg tissue wet weight, respectively). These results suggest that receptor-activated Ca(2+) entry through SOC channels is impaired in caudal artery smooth muscle in GK rats. 3. The classic transient receptor potential (TRPC) channels, which constitute SOC and receptor-operated cation channels, play an important role in Ca(2+) regulation. Therefore, we investigated the mRNA and protein expression of TRPC channels in caudal artery smooth muscle from Wistar and GK rats using reverse transcription-polymerase chain reaction and immunoblotting. 4. Expression of TRPC1, TRPC3 and TRPC6 mRNA and protein was found in Wistar rats. However, in GK rats, in addition to the expression of these TRPC channels, mRNA and protein expression of TRPC4 was found. The expression of TRPC1 and TRPC6, but not TRPC3, was increased approximately twofold in GK rats compared with Wistar rats. 5. These results suggest that changes in TRPC channel expression may be responsible, in part, for the dysfunction of receptor-mediated Ca(2+) entry in caudal artery smooth muscle of GK rats.

Classical transient receptor potential channel 6 (TRPC6) is essential for ischemia‐reperfusion injury of the lung

Lung endothelial damage is a characteristic feature of ischemia‐reperfusion (I/R) injury. However the molecular steps involved in the loss of endothelial integrity and subsequent invasion of immune cells are still poorly understood. Most notably, in isolated lungs from TRPC6‐deficient mice we observed a resistance to I/R injury, while lungs from wild‐type mice displayed pulmonary edema formation and organ dysfunction. To differentiate between the role of TRPC6 in circulating and/or resident leukocytes versus endothelial cells, TRPC6‐deficient bone marrow cells were transplanted into lethally irradiated wild‐type mice and vice versa. Based on these experiments in chimeric mice it is evident that endothelial but not leukocytic TRPC6 deficency protects from I/R injury of the lung. Therefore, pulmonary endothelial cells from wild‐type and TRPC6‐deficient mice were isolated and further analyzed. TRPC1, TRPC4 and TRPC6 were the predominantly expressed TRPC isoforms in these cells. Primary isolated endothelial cells from TRPC6‐deficient lungs show reduced ischemia‐induced Ca2+ influx and subsequent actin stress‐fiber formation as well as a decreased cellular shape change when compared to wild‐type cells. Thus, TRPC6 represents a promising novel pharmacological target to control critical early stages of I/R injury in the lung. Supported by the von‐Behring‐Röntgen Stiftung.

Structure–functional intimacies of transient receptor potential channels

Although a unifying characteristic common to all transient receptor potential (TRP) channel functions remains elusive, they could be described as tetramers formed by subunits with six transmembrane domains and containing cation-selective pores, which in several cases show high calcium permeability. TRP channels constitute a large superfamily of ion channels, and can be grouped into seven subfamilies based on their amino acid sequence homology: the canonical or classic TRPs, the vanilloid receptor TRPs, the melastatin or long TRPs, ankyrin (whose only member is the transmembrane protein 1 [TRPA1]), TRPN after the nonmechanoreceptor potential C (nonpC), and the more distant cousins, the polycystins and mucolipins. Because of their role as cellular sensors, polymodal activation and gating properties, many TRP channels are activated by a variety of different stimuli and function as signal integrators. Thus, how TRP channels function and how function relates to given structural determinants contained in the channel-forming protein has attracted the attention of biophysicists as well as molecular and cell biologists. The main purpose of this review is to summarize our present knowledge on the structure of channels of the TRP ion channel family. In the absence of crystal structure information for a complete TRP channel, we will describe important protein domains present in TRP channels, structure-function mutagenesis studies, the few crystal structures available for some TRP channel modules, and the recent determination of some TRP channel structures using electron microscopy.

Dependence of STIM1/Orai1-mediated Calcium Entry on Plasma Membrane Phosphoinositides

Recent studies identified two main components of store-operated calcium entry (SOCE): the endoplasmic reticulum-localized Ca2+ sensor protein, STIM1, and the plasma membrane (PM)-localized Ca2+ channel, Orai1/CRACM1. In the present study, we investigated the phosphoinositide dependence of Orai1 channel activation in the PM and of STIM1 movements from the tubular to PM-adjacent endoplasmic reticulum regions during Ca2+ store depletion. Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) levels were changed either with agonist stimulation or by chemically induced recruitment of a phosphoinositide 5-phosphatase domain to the PM, whereas PtdIns4P levels were decreased by inhibition or down-regulation of phosphatidylinositol 4-kinases (PI4Ks). Agonist-induced phospholipase C activation and PI4K inhibition, but not isolated PtdIns(4,5)P(2) depletion, substantially reduced endogenous or STIM1/Orai1-mediated SOCE without preventing STIM1 movements toward the PM upon Ca2+ store depletion. Patch clamp analysis of cells overexpressing STIM1 and Orai1 proteins confirmed that phospholipase C activation or PI4K inhibition greatly reduced I(CRAC) currents. These results suggest an inositide requirement of Orai1 activation but not STIM1 movements and indicate that PtdIns4P rather than PtdIns(4,5)P2 is a likely determinant of Orai1 channel activity.

Deletion of TRPC4 and TRPC6 in Mice Impairs Smooth Muscle Contraction and Intestinal Motility In Vivo

Downstream effects of muscarinic receptor stimulation in intestinal smooth muscle include contraction and intestinal transit. We thought to determine whether classic transient receptor potential (TRPC) channels integrate the intracellular signaling cascades evoked by the stimulated receptors and thereby contribute to the control of the membrane potential, Ca-influx, and cell responses. We created trpc4-, trpc6-, and trpc4/trpc6-gene-deficient mice and analyzed them for intestinal smooth muscle function in vitro and in vivo. In intestinal smooth muscle cells TRPC4 forms a 55 pS cation channel and underlies more than 80% of the muscarinic receptor-induced cation current (mI(CAT)). The residual mI(CAT) depends on the expression of TRPC6, indicating that TRPC6 and TRPC4 determine mI(CAT) channel activity independent of other channel subunits. In TRPC4-deficient ileal myocytes the carbachol-induced membrane depolarizations are diminished greatly and the atropine-sensitive contraction elicited by acetylcholine release from excitatory motor neurons is reduced greatly. Additional deletion of TRPC6 aggravates these effects. Intestinal transit is slowed down in mice lacking TRPC4 and TRPC6. In intestinal smooth muscle cells TRPC4 and TRPC6 channels are gated by muscarinic receptors and are responsible for mI(CAT). They couple muscarinic receptors to depolarization of intestinal smooth muscle cells and voltage-activated Ca(2+)-influx and contraction, and thereby accelerate small intestinal motility in vivo.

Functional TRPC5‐containing channels in human saphenous vein endothelial cells

Vascular endothelium plays pivotal roles in regulation of vascular tone, blood coagulation, wound healing and cell‐cell adhesion via the expression of vasoactive mediators. Ca2+ plays a central role in the regulation of endothelial cell function. Classical transient receptor potential channels (TRPC1‐7) are widely expressed and some are suggested to play critical roles in regulating endothelial functions. Here, we demonstrate the expression and functionality of TRPC5‐containing channels in human saphenous vein endothelial cells (HSVEC). RT‐PCR analysis suggested that TRPC1, 4, and 5 are expressed in HSVEC. Cells were then studied using fura‐2 real‐time 96‐well plate assays. Challenging of HSVEC with 10 µM histamine or lysophosphatidylcholine (LPC), 1 mM H2O2 or 100 ng/mL vascular endothelial growth factor (VEGF) induced rises in [Ca2+ ]i, which was inhibited by a TRPC5‐specific polyclonal antibody. The inhibition varied from ~80% in the case of LPC, to ~40, 50 and 70% for the histamine, H2O2 and VEGF responses. ATP (100 µM) mediated Ca2+ rise was not affected. Our data support earlier murine studies, and suggest the involvement of TRPC5‐containing channels in mediating Ca2+ signals in human endothelial cells. The channel may be important in inflammatory, oxidative or angiogenic conditions, and is a potential target for therapeutic development.Work supported by the Wellcome Trust and The Egyptian Ministry of Higher Education.Grant Funding SourceWellcome Trust and Egyptian Ministry of Higher Education