Increased vascular smooth muscle contractility in TRPC6-/- mice

Among the TRPC subfamily of TRP (classical transient receptor potential) channels, TRPC3, -6, and -7 are gated by signal transduction pathways that activate C-type phospholipases as well as by direct exposure to diacylglycerols. Since TRPC6 is highly expressed in pulmonary and vascular smooth muscle cells, it represents a likely molecular candidate for receptor-operated cation entry. To define the physiological role of TRPC6, we have developed a TRPC6-deficient mouse model. These mice showed an elevated blood pressure and enhanced agonist-induced contractility of isolated aortic rings as well as cerebral arteries. Smooth muscle cells of TRPC6-deficient mice have higher basal cation entry, increased TRPC-carried cation currents, and more depolarized membrane potentials. This higher basal cation entry, however, was completely abolished by the expression of a TRPC3-specific small interference RNA in primary TRPC6(-)(/)(-) smooth muscle cells. Along these lines, the expression of TRPC3 in wild-type cells resulted in increased basal activity, while TRPC6 expression in TRPC6(-/-) smooth muscle cells reduced basal cation influx. These findings imply that constitutively active TRPC3-type channels, which are up-regulated in TRPC6-deficient smooth muscle cells, are not able to functionally replace TRPC6. Thus, TRPC6 has distinct nonredundant roles in the control of vascular smooth muscle tone.     

The TRPC3/6/7 subfamily of cation channels

The mammalian transient receptor potential (TRP) proteins consist of a superfamily of Ca2+-permeant non-selective cation channels with structural similarities to Drosophila TRP. The TRP superfamily can be divided into three major families, among them the "canonical TRP" family (TRPC). The seven protein products of the mammalian TRPC family of genes (designated TRPC1-7) share in common the activation through PLC-coupled receptors and have been proposed to encode components of native store-operated channels in different cell types. In addition, the three members of the TRPC3/6/7 subfamily of TRPC channels can be activated by diacylglycerol analogs, providing a possible mechanism of activation of these channels by PLC-coupled receptors. This review summarizes the current knowledge about the mechanism of activation of the TRPC3/6/7 subfamily, as well as the potential role of these proteins as components of native Ca2+-permeant channels.     

Molecular Linkage of TRP Proteins to Smooth Muscle Receptor-Operated Ca2+-Permeable Cationic Channels

The molecular mechanisms underlying Ca²⁺ entry evoked by cell surface receptors in smooth muscle have long been enigmatic, but an important breakthrough has been made by recent investigations on mammalian homologues of Drosophila transient receptor potential (TRP) protein. There is now growing evidence that TRPC6 plays an integrative role in vascular tone regulation, Ca²⁺ entry channels activated by the sympathetic nerve stimulation, vasoactive peptides, and mechanosensitive mechanisms. Other TRPC isoforms, such as TRPC1 and TRPC4 (and perhaps TRPC5), are also expressed abundantly in smooth muscle and may contribute to muscle contraction, cell proliferation, and cholinergic control of the gut motility. This paper briefly overviews the current knowledge about these TRP proteins in smooth muscle physiology.     

TRP expression pattern and the functional importance of TRPC3 in primary human T-cells

TRP proteins form ion channels which are activated following receptor stimulation. In T-cell lines, expression data of TRP proteins have been published. However, almost no data about TRP expression is available in primary human T-cells. Using RT-PCR and quantitative RT-PCR, we compare the expression of TRP mRNA in 1) human peripheral blood lymphocytes, which are a mix of mostly mono-nuclear blood lymphocytes but contain other leucocytes, 2) a pure human CD4⁺ T-helper cell population in the resting (= naïve) and activated (= effector) state, and 3) two commonly used CD4⁺ Jurkat T-cell lines, E6-1 and parental. To mimic physiological cell stimulation, we analyzed TRP expression in primary human cells in a quantitative way over several days following formation of an immunological synapse through stimulation with antibody-coated beads. The TRP expression profile of primary human T-cells was significantly different from Jurkat T-cells. Among the TRP mRNAs of the TRPC, TRPM, and TRPV family, we found consistent expression of TRPC1, TRPC3, TRPV1, TRPM2, and TRPM7 in primary human CD4⁺ T-cells of all analyzed blood donors. Among these, TRPC3 and TRPM2 were strongly up-regulated following stimulation, but with different kinetics. We found that TRPC3 modulates Ca²⁺-dependent proliferation of primary CD4⁺ T-cells indicating that TRPC3 may be involved in Ca²⁺ homeostasis in T-cells besides the well-established STIM and ORAI proteins which are responsible for store-operated Ca²⁺ entry.     

Enkurin is a novel calmodulin and TRPC channel binding protein in sperm

The TRPC cation channel family has been implicated in receptor- or phospholipase C (PLC)-mediated Ca2+ entry into animal cells. These channels are present in mammalian sperm and are assigned a role in ZP3-evoked Ca2+ influx that drives acrosome reactions. However, the mechanisms controlling channel activity and coupling Ca2+ entry through these channels to cellular responses are not well understood. A yeast two-hybrid screen was carried out to identify TRPC-interacting proteins that would be candidate regulators or effectors. We identified a novel protein, enkurin, that is expressed at high levels in the testis and vomeronasal organ and at lower levels in selected other tissues. Enkurin interacts with several TRPC proteins (TRPC1, TRPC2, TRPC5, but not TRPC3) and colocalizes with these channels in sperm. Three protein-protein interaction domains were identified in enkurin: a C-terminal region is essential for channel interaction; an IQ motif binds the Ca2+ sensor, calmodulin, in a Ca2+-dependent manner; and a proline-rich N-terminal region contains predicted ligand sequences for SH3 domain proteins, including the SH3 domain of the p85 regulatory subunit of 1-phosphatidylinositol-3-kinase. We suggest that enkurin is an adaptor that functions to localize a Ca2+ sensitive signal transduction machinery in sperm to a Ca2+-permeable ion channel.     

In vivo TRPC functions in the cardiopulmonary vasculature

Cardiovascular diseases are the leading cause of death in the industrialized countries. The cardiovascular system includes the systemic blood circulation, the heart and the pulmonary circulation providing sufficient blood flow and oxygen to peripheral tissues and organs according to their metabolic demand. This review focuses on three major cell types of the cardiovascular system: myocytes of the heart as well as smooth muscle cells and endothelial cells from the systemic and pulmonary circulation. Ion channels initiate and regulate contraction in all three cell types, and the identification of their genes has significantly improved our knowledge of signal transduction pathways in these cells. Among the ion channels expressed in smooth muscle cells, cation channels of the TRPC family allow for the entry of Na(+) and Ca(2+). Physiological functions of TRPC1, TRPC3, TRPC4, TRPC5, TRPC6 and TRPC7 in the cardiovascular system, dissected by down-regulating channel activity in isolated tissues or by the analysis of gene-deficient mouse models, are reviewed. Possible functional roles and physiological regulation of TRPCs as homomeric or heteromeric channels in these cell types are discussed. Moreover, TRP channels may also be responsible for pathophysiological processes of the cardiovascular system like hypertension as well as cardiac hypertrophy and increased endothelial permeability.     

Inhibition of TRPC6 by protein kinase C isoforms in cultured human podocytes

Transient receptor potential canonical‐6 (TRPC6) ion channels, expressed at high levels in podocytes of the filtration barrier, are recently implicated in the pathogenesis of various forms of proteinuric kidney diseases. Indeed, inherited or acquired up‐regulation of TRPC6 activities are suggested to play a role in podocytopathies. Yet, we possess limited information about the regulation of TRPC6 in human podocytes. Therefore, in this study, we aimed at defining how the protein kinase C (PKC) system, one of the key intracellular signalling pathways, regulates TRPC6 function and expression. On human differentiated podocytes, we identified the molecular expressions of both TRPC6 and several PKC isoforms. We also showed that TRPC6 channels are functional since the TRPC6 activator 1‐oleoyl‐2‐acetyl‐sn‐glycerol (OAG) induced Ca²⁺‐influx to the cells. By assessing the regulatory roles of the PKCs, we found that inhibitors of the endogenous activities of classical and novel PKC isoforms markedly augmented TRPC6 activities. In contrast, activation of the PKC system by phorbol 12‐myristate 13‐acetate (PMA) exerted inhibitory actions on TRPC6 and suppressed its expression. Importantly, PMA treatment markedly down‐regulated the expression levels of PKCα, PKCβ, and PKCη reflecting their activation. Taken together, these results indicate that the PKC system exhibits a ‘tonic’ inhibition on TRPC6 activity in human podocytes suggesting that pathological conditions altering the expression and/or activation patterns of podocyte‐expressed PKCs may influence TRPC6 activity and hence podocyte functions. Therefore, it is proposed that targeted manipulation of certain PKC isoforms might be beneficial in certain proteinuric kidney diseases with altered TRPC6 functions.     

Trafficking mechanisms and regulation of TRPC channels

TRPC channels are Ca²⁺-permeable cation channels which are regulated downstream from receptor-coupled PIP₂ hydrolysis. These channels contribute to a wide variety of cellular functions. Loss or gain of channel function has been associated with dysfunction and aberrant physiology. TRPC channel functions are influenced by their physical and functional interactions with numerous proteins that determine their regulation, scaffolding, trafficking, as well as their effects on the downstream cellular processes. Such interactions also compartmentalize the Ca²⁺ signals arising from TRPC channels. A large number of studies demonstrate that trafficking is a critical mode by which plasma membrane localization and surface expression of TRPC channels are regulated. This review will provide an overview of intracellular trafficking pathways as well as discuss the current state of knowledge regarding the mechanisms and components involved in trafficking of the seven members of the TRPC family (TRPC1–TRPC7).     

Expression of the Argonaute protein PiwiL2 and piRNAs in adult mouse mesenchymal stem cells

Piwi (P-element-induced wimpy testis) first discovered in Drosophila is a member of the Argonaute family of micro-RNA binding proteins with essential roles in germ-cell development. The murine homologue of PiwiL2, also known as Mili is selectively expressed in the testes, and mice bearing targeted mutations of the PiwiL2 gene are male-sterile. PiwiL2 proteins are thought to protect the germ line genome by suppressing retrotransposons, stabilizing heterochromatin structure, and regulating target genes during meiosis and mitosis. Here, we report that PiwiL2 and associated piRNAs (piRs) may play similar roles in adult mouse mesenchymal stem cells. We found that PiwiL2 is expressed in the cytoplasm of metaphase mesenchymal stem cells from the bone marrow of adult and aged mice. Knockdown of PiwiL2 with a specific siRNA enhanced cell proliferation, significantly increased the number of cells in G1/S and G2/M cell cycle phases and was associated with increased expression of cell cycle genes CCND1, CDK8, microtubule regulation genes, and decreased expression of tumor suppressors Cables 1, LATS, and Cxxc4. The results suggest broader roles for Piwi in genome surveillance beyond the germ line and a possible role in regulating the cell cycle of mesenchymal stem cells.     

Selective Gαi subunits as novel direct activators of transient receptor potential canonical (TRPC)4 and TRPC5 channels

The ubiquitous transient receptor potential canonical (TRPC) channels function as non-selective, Ca(2+)-permeable channels and mediate numerous cellular functions. It is commonly assumed that TRPC channels are activated by stimulation of Gα(q)-PLC-coupled receptors. However, whether the Gα(q)-PLC pathway is the main regulator of TRPC4/5 channels and how other Gα proteins may regulate these channels are poorly understood. We previously reported that TRPC4/TRPC5 can be activated by Gα(i). In the current work, we found that Gα(i) subunits, rather than Gα(q), are the primary and direct activators of TRPC4 and TRPC5. We report a novel molecular mechanism in which TRPC4 is activated by several Gα(i) subunits, most prominently by Gα(i2), and TRPC5 is activated primarily by Gα(i3). Activation of Gα(i) by the muscarinic M2 receptors or expression of the constitutively active Gα(i) mutants equally and fully activates the channels. Moreover, both TRPC4 and TRPC5 are activated by direct interaction of their conserved C-terminal SESTD (SEC14-like and spectrin-type domains) with the Gα(i) subunits. Two amino acids (lysine 715 and arginine 716) of the TRPC4 C terminus were identified by structural modeling as mediating the interaction with Gα(i2). These findings indicate an essential role of Gα(i) proteins as novel activators for TRPC4/5 and reveal the molecular mechanism by which G-proteins activate the channels.     

Activation of TRPC4β by Gαi subunit increases Ca selectivity and controls neurite morphogenesis in cultured hippocampal neuron

The ubiquitous transient receptor potential canonical (TRPC) channels function as non-selective, Ca²⁺-permeable channels. TRPC channels are activated by stimulation of Gα_q-PLC-coupled receptors. Here, we report that TRPC4/TRPC5 can be activated by Gα_i. We studied the essential role of Gα_i subunits in TRPC4 activation and investigated changes in ion selectivity and pore dilation of the TRPC4 channel elicited by the Gα_i2 subunit. Activation of TRPC4 by Gα_i2 increased Ca²⁺ permeability and Ca²⁺ influx through TRPC4 channels. Co-expression of the muscarinic receptor (M2) and TRPC4 in HEK293 cells induced TRPC4-mediated Ca²⁺ influx. Moreover, both TRPC4β and the TRPC4β-Gα_i2 signaling complex induced inhibition of neurite growth and arborization in cultured hippocampal neurons. Cells treated with KN-93, a CaMKII inhibitor, prevented TRPC4- and TRPC4-Gα_i2^Q205L-mediated inhibition of neurite branching and growth. These findings indicate an essential role of Gα_i proteins in TRPC4 activation and extend our knowledge of the functional role of TRPC4 in hippocampal neurons.     

Identification of ML204, a novel potent antagonist that selectively modulates native TRPC4/C5 ion channels

Transient receptor potential canonical (TRPC) channels are Ca(2+)-permeable nonselective cation channels implicated in diverse physiological functions, including smooth muscle contractility and synaptic transmission. However, lack of potent selective pharmacological inhibitors for TRPC channels has limited delineation of the roles of these channels in physiological systems. Here we report the identification and characterization of ML204 as a novel, potent, and selective TRPC4 channel inhibitor. A high throughput fluorescent screen of 305,000 compounds of the Molecular Libraries Small Molecule Repository was performed for inhibitors that blocked intracellular Ca(2+) rise in response to stimulation of mouse TRPC4β by μ-opioid receptors. ML204 inhibited TRPC4β-mediated intracellular Ca(2+) rise with an IC(50) value of 0.96 μm and exhibited 19-fold selectivity against muscarinic receptor-coupled TRPC6 channel activation. In whole-cell patch clamp recordings, ML204 blocked TRPC4β currents activated through either μ-opioid receptor stimulation or intracellular dialysis of guanosine 5'-3-O-(thio)triphosphate (GTPγS), suggesting a direct interaction of ML204 with TRPC4 channels rather than any interference with the signal transduction pathways. Selectivity studies showed no appreciable block by 10-20 μm ML204 of TRPV1, TRPV3, TRPA1, and TRPM8, as well as KCNQ2 and native voltage-gated sodium, potassium, and calcium channels in mouse dorsal root ganglion neurons. In isolated guinea pig ileal myocytes, ML204 blocked muscarinic cation currents activated by bath application of carbachol or intracellular infusion of GTPγS, demonstrating its effectiveness on native TRPC4 currents. Therefore, ML204 represents an excellent novel tool for investigation of TRPC4 channel function and may facilitate the development of therapeutics targeted to TRPC4.     

Expression of TRPC6 in renal cortex and hippocampus of mouse during postnatal development

TRPC6, a member of the TRPC family, attracts much attention from the public because of its relationship with the disease. In both the brain and kidney, TRPC6 serves a variety of functions. The aim of the present study was to observe the expression and effects of TRPC6 in renal cortex and hippocampus during early?postnatal development of?the mouse. In the present study, immunohistochemistry and Western blotting were used to detect the expression of TRPC6 in the mouse kidney and hippocampus of postnatal day 1, 3, 5, 7, 14, 21, 28 and 49 (P1, P3, P5, P7, P14, P21, P28 and P49). Results showed that the expression of TRPC6 was increased in the mouse hippocampus, and there was a significant increase between P7 and P14 during the postnatal development. Meanwhile, the expression of TRPC6 was also detected in glomerulus and tubules, and a decreased expression was found during postnatal maturation of mouse renal cortex. From these in vivo experiments, we concluded that the expression of TRPC6 was active in the developing mouse kidney cortex, and followed a loss of expression with the development of kidney. Meanwhile, an increased expression was found in the hippocampus with the development. Together, these data suggested that the developmental changes in TRPC6 expression might be required for proper postnatal kidney cortex development, and played a critical role in the hippocampus during development, which formed the basis for understanding the nephrogenesis and neurogenesis in mice and provided a practically useful knowledge to the clinical and related research.     

Plasticity of TRPC expression in arterial smooth muscle: correlation with store-operated Ca2+ entry

Loss of the smooth muscle contractile phenotype is critical in atherosclerosis and in restenosis after angioplasty, but its early signals are incompletely understood. In this study, we have explored the role of transient receptor potential canonical (TRPC) proteins, which have been suggested to mediate store-operated Ca²⁺ entry (SOCE). Contractility of rat cerebral arteries in organ culture is preserved for several days, whereas SOCE is increased. In correlation with this increase is that nifedipine-insensitive whole cell current, activated by depletion of intracellular Ca²⁺ stores, was increased by 50% in cells isolated from arteries cultured for 3 days. TRPC1 and TRPC6 mRNA were more than fivefold increased in cells isolated after organ culture, whereas TRPC3 was decreased. Immunofluorescent staining and/or Western blotting of arteries and isolated cells showed upregulation of TRPC1 and TRPC6 proteins during organ culture. In intact arteries, TRPC4 expression correlated with the amount of endothelium present. Ca²⁺ addition after store depletion caused a contraction in cultured, but not in freshly dissected, arteries. A polyclonal TRPC1 antibody directed against an extracellular epitope inhibited this contraction by 50%. To investigate the basis of the TRPC upregulation and assess its possible clinical significance, segments of human internal mammary artery were organ cultured for 24 h and then exposed to balloon dilatation in vitro, followed by further culturing for up to 48 h. After dilatation, TRPC1 and TRPC6 mRNA were progressively increased compared with undilated control segments. The results of this study indicate that vascular injury enhances plasticity in TRPC expression, that TRPC expression correlates with cellular Ca²⁺ handling, and that TRPC1 is a subunit of upregulated store-operated Ca²⁺ channels. differentiation; ion channels; angioplasty; organ culture     

TRPC6 and TRPC4 Heteromultimerization Mediates Store Depletion-Activated NCX1 Reversal in Proliferative Vascular Smooth Muscle Cells

Store depletion has been shown to induce Ca²⁺ entry by Na+/Ca+ exchange (NCX) 1 reversal in proliferative vascular smooth muscle cells (VSMCs). The study objective was to investigate the role of transient receptor potential canonical (TRPC) channels in store depletion and NCX1 reversal in proliferative VSMCs. In cultured VSMCs, expressing TRPC1, TRPC4, and TRPC6, the removal of extracellular Na⁺ was followed by a significant increase of cytosolic Ca²⁺ concentration that was inhibited by KBR, a selective NCX1 inhibitor. TRPC1 knockdown significantly suppressed store-operated, channel-mediated Ca²⁺ entry, but TRPC4 knockdown and TRPC6 knockdown had no effect. Separate knockdown of TRPC1, TRPC4, or TRPC6 did not have a significant effect on thapsigargin-initiated Na⁺ increase in the peripheral regions with KBR treatment, but knockdown of both TRPC4 and TRPC6 did. Stromal interaction molecule (STIM)1 knockdown significantly reduced TRPC4 and TRPC6 binding. The results demonstrated that TRPC4–TRPC6 heteromultimerization linked Ca²⁺ store depletion and STIM1 accumulation with NCX reversal in proliferative VSMCs.     

Role of phosphoinositol 4,5-bisphosphate and diacylglycerol in regulating native TRPC channel proteins in vascular smooth muscle

Stimulation of receptor-operated (ROCs) and store-operated (SOCs) Ca²⁺-permeable cation channels by vasoconstrictors has many important physiological functions in vascular smooth muscle. The present review indicates that ROCs and SOCs with diverse properties in different blood vessels are likely to be explained by composition of different subunits from the canonical transient receptor potential (TRPC) family of cation channel proteins. In addition we illustrate that activation of native TRPC ROCs and SOCs involves different phospholipase-mediated transduction pathways linked to generation of diacylglycerol (DAG). Moreover we describe recent novel data showing that the endogenous phospholipid phosphoinositol 4,5-bisphosphate (PIP₂) has profound and contrasting actions on TRPC ROCs and SOCs. Optimal activation of a native TRPC6 ROC by angiotensin II (Ang II) requires both depletion of PIP₂ and generation of DAG which leads to stimulation of TRPC6 via a PKC-independent mechanism. The data also indicate that PIP₂ has a marked constitutive inhibitory action of TRPC6 and DAG and PIP₂ are physiological antagonists on TRPC6 ROCs. In contrast PIP₂ stimulates TRPC1 SOCs and has an obligatory role in activation of these channels by store-depletion which requires PKC-dependent phosphorylation of TRPC1 proteins. Finally, we conclude that interactions between PIP₂ bound to TRPC proteins at rest, generation of DAG and PKC-dependent phosphorylation of TRPC proteins have a fundamental role in activation mechanisms of ROCs and SOCs in vascular smooth muscle.     

TRPC channels: Regulation,dysregulation and contributions to chronic kidney disease

Mutations in the gene encoding canonical transient receptor potential-6 (TRPC6) channels result in severe nephrotic syndromes that typically lead to end-stage renal disease. Many but not all of these mutations result in a gain in the function of the resulting channel protein. Since those observations were first made, substantial work has supported the hypothesis that TRPC6 channels can also contribute to progression of acquired (non-genetic) glomerular diseases, including primary and secondary FSGS, glomerulosclerosis during autoimmune glomerulonephritis, and possibly in type-1 diabetes. Their regulation has been extensively studied, especially in podocytes, but also in mesangial cells and other cell types present in the kidney. More recent evidence has implicated TRPC6 in renal fibrosis and tubulointerstitial disease caused by urinary obstruction. Consequently TRPC6 is being extensively investigated as a target for drug discovery. Other TRPC family members are present in kidney. TRPC6 can form a functional heteromultimer with TRPC3, and it has been suggested that TRPC5 may also play a role in glomerular disease progression, although the evidence on this is contradictory. Here we review literature on the expression and regulation of TRPC6, TRPC3 and TRPC5 in various cell types of the vertebrate kidney, the evidence that these channels are dysregulated in disease models, and research showing that knock-out or pharmacological inhibition of these channels can reduce the severity of kidney disease. We also summarize several areas that remain controversial, and some of the large gaps of knowledge concerning the fundamental role of these proteins in regulation of renal function.