Determining the functional role of TRPC channels in primary cells

Although the TRPC members of the mammalian transient receptor potential TRP cation channel family were the first to be described in 1995, the depth of knowledge of TRPC channels has fallen behind that of their counterparts in the TRPV and TRPM subfamilies in the intervening years. The complexities and controversies of TRPC channel composition and regulation have hindered their progress as therapeutic targets in the drug discovery environment to date, however embracing these challenges as opportunities may bring TRPC channels to the forefront of the discovery of novel therapies for many diseases. These challenges and opportunities of exploring TRPC channels as therapeutic targets are highlighted and discussed in this review with respect to respiratory diseases.     

Functional role of TRPC channels in the regulation of endothelial permeability

The endothelial cells (ECs) form a semipermeable barrier between the blood and the tissue. An important function of the endothelium is to maintain the integrity of the barrier function of the vessel wall. Ca²⁺ signaling in ECs plays a key role in maintaining the barrier integrity. Transient receptor potential canonical (TRPC) channels are mammalian homologs of Drosophila TRP Ca²⁺-permeable channels expressed in EC. TRPC channels are thought to function as a Ca²⁺ entry channel operated by store-depletion as well as receptor-activated channels in a variety of cell types, including ECs. Inflammatory mediators such as thrombin, histamine, bradykinin, and others increase endothelial permeability by actin polymerization-dependent EC rounding and formation of inter-endothelial gaps, a process critically dependent on the increase in EC cytosolic [Ca²⁺] ([Ca²⁺]_i). Increase in endothelial permeability depends on both intracellular Ca²⁺ release and extracellular Ca²⁺ entry through TRPC channels. This review summarizes recent findings on the role of TRPC channels in the mechanism of Ca²⁺ entry in ECs, and, in particular, the role of TRPC channels in regulating endothelial barrier function.     

The puzzling role of TRPC3 channels in motor coordination

Transient receptor potential canonical 3 (TRPC3) proteins are nonselective cation channels activated downstream of phospholipase-C-coupled receptors. TRPC3 channels have emerged as major players in the function of the central nervous system. They have been described as important contributors to brain-derived neurotrophic factor mediated survival and growth-cone guidance of cerebellar granule neurons. TRPC3 were also identified as postsynaptic cation channels essential for metabotropic glutamate receptor1-dependent synaptic transmission in cerebellar Purkinje neurons. A recent report described motor coordination defects in TRPC3 knockout mice while a subsequent study reported a similar phenotype in so-called moonwalker mice, harboring a TRPC3 gain-of-function mutation. How can opposing aspects of TRPC3 channel activation lead to the same phenotype? Here we discuss the salient features of TRPC3 knockout mice and moonwalker mice and attempt to reconcile the apparently conflicting findings from these two animal models.     

The possible role of TRPC6 in atopic dermatitis

Skin barrier dysfunction is involved in the pathogenesis of atopic dermatitis (AD). The impaired barrier function is related to disturbed keratinocyte differentiation through improper Ca²⁺-regulated pathway in AD. Transient receptor potential canonical (subtype) 6 (TRPC6) is a Ca²⁺-permeable nonselective cation channel expressed in keratinocytes. TRPC6 contributes to the process of keratinocyte differentiation by regulating Ca²⁺ influx. Furthermore, TRPC6 alone is considered to be sufficient for nearly full physiological response of keratinocyte differentiation. Until now, the correlation between TRPC6 and AD has not been investigated. We hypothesize that dysfunction of TRPC6 may trigger the process of AD, and that restoring or enhancing the activity of TRPC6 may serve as a new modality in treating AD.     

Sub-miniature junction potentials in excitatory neuromuscular synapses of the locust

By recording miniature excitatory junction potentials (mejps) intracellularly at two points from a multiterminally innervated muscle fibre it is possible to select mejps whose amplitudes are not substantially affected by electrotonic decay. Many amplitude histograms of such selected mejps from untreated locust jumping muscle show a bimodal distribution with a high proportion of small-amplitude mejps (sub-mejps). Most amplitudes of excitatory junction potentials (ejps) resulting from the release of a single transmitter quantum correspond to the large-mode mejps. Tetanic nerve stimulation, in high [Mg2+]o without Ca2+, greatly reduces the proportion of sub-mejps. It is concluded that there are two modes of spontaneous transmitter release from the motor nerve endings.     

Developmental alterations in the functional properties of excitatory neocortical synapses

In the neocortex, most excitatory, glutamatergic synapses are established during the first 4–5 weeks after birth. During this period profound changes in the properties of synaptic transmission occur. Excitatory postsynaptic potentials (EPSPs) at immature synaptic connections are profoundly and progressively reduced in response to moderate to high frequency (5–100 Hz) stimulation. With maturation, this frequency-dependent depression becomes progressively weaker and may eventually transform into a weak to moderate EPSP facilitation. In parallel to changes in the short-term plasticity, a reduction in the synaptic reliability occurs at most glutamatergic neocortical synapses: immature synapses show a high probability of neurotransmitter release as indicated by their low failure rate and small EPSP amplitude variation. This high reliability is reduced in mature synapses, which show considerably higher failure rates and more variable EPSP amplitudes. During early neocortical development synaptic vesicle pools are not yet fully differentiated and their replenishment may be slow, thus resulting in EPSP amplitude depression. The decrease in the probability of neurotransmitter release may be the result of an altered Ca²⁺ control in the presynaptic terminal with a reduced Ca²⁺ influx and/or a higher Ca²⁺ buffering capacity. This may lead to a lower synaptic reliability and a weaker short-term synaptic depression with maturation.     

Intramembranous organization of lobster excitatory neuromuscular synapses

Summary The fine structure of identified neuromuscular synapses of the single excitatory axon to the distal accessory flexor muscle in lobster limbs was examined with freeze-fracture and serial thin-section electron microscopy. The latter technique reveals presynaptic dense bars with synaptic vesicles aligned on either side of these bars and often fused to the membrane, suggesting exocytosis and confirming our previous contention that these bars are active zones of transmitter release. The intramembranous organization of these active zones, as revealed in freeze-etched tissue, is a ridge-like elevation of the P-face of the axolemma with a matching trough on the complementary E-face. The ridge on the P-face has rows of large scattered intramembranous particles along the apex and is often bordered by a series of small, circular depressions which are presumed to represent exocytotic vesicles attached to the presynaptic membrane. Complementing these depressions are a few volcano-like protuberances seen occasionally on the E-face membrane. Because such evidence for transmitter release occurred in both stimulated and non-stimulated preparations, it demonstrates that chemical fixatives employing aldehydes induce transmitter release. The postsynaptic receptor sites of these excitatory synapses are characterized by oval-shaped patches of densely packed particles on the E-face, arranged in a random pattern on the sarcolemma. The complementary P-face view exhibits a regular square array of particle imprints or pits.     

Functional roles of TRPC channels in the developing brain

Transient receptor potential canonical (TRPC) channels are Ca²⁺-permeable, nonselective cation channels formed by homomeric or heteromeric complexes of TRPC proteins that contain six transmembrane domains. These channels can be activated through a phospholipase-C-dependent mechanism, making them sensors for environmental cues. Their expression begins early in embryonic days and remains in adulthood. These channels have important roles in the processes of neuronal development, including neural stem cell proliferation, cerebellar granule cell survival, axon path finding, neuronal morphogenesis, and synaptogenesis. In this review, we will discuss functional implications of TRPC channels during brain development.     

Epileptiform activity in rat hippocampus strengthens excitatory synapses

Although epileptic seizures are characterized by excessive excitation, the role of excitatory synaptic transmission in the induction and expression of epilepsy remains unclear. Here, we show that epileptiform activity strengthens excitatory hippocampal synapses by increasing the number of functional (RS)-α-amino-3hydroxy-5methyl-4-isoxadepropionate (AMPA)-type glutamate receptors in CA3–CA1 synapses. This form of synaptic strengthening occludes long-term potentiation (LTP) and enhances long-term depression (LTD), processes involved in learning and memory. These changes in synaptic transmission and plasticity, which are fully blocked with N -methyl-D-aspartate (NMDA) receptor antagonists, may underlie epilepsy induction and seizure-associated memory deficits.     

Nitric oxide lacks direct effect on TRPC5 channels but suppresses endogenous TRPC5-containing channels in endothelial cells

TRPC5 is a member of the canonical transient receptor potential (TRPC) family of proteins that forms cationic channels either through homomultimeric assembly or heteromultimeric coordination with other TRPC proteins. It is expressed in a variety of cells including central neurones and endothelial cells and has susceptibility to stimulation by multiple factors. Here we investigated if TRPC5 is sensitive to nitric oxide. Mouse TRPC5 or human TRPC5 was over-expressed in HEK293 cells, and TRPC5 activity was determined by measuring the cytosolic Ca²⁺ concentration with an indicator dye or by recording membrane current under voltage clamp. TRPC5 activity could be evoked by carbachol acting at muscarinic receptors, lanthanum, or a reducing agent. However, S-nitroso-N-acetylpenicillamine (SNAP) and diethylamine NONOate (DEA-NONOate) failed to stimulate or inhibit TRPC5 at concentrations that generated nitric oxide, caused vasorelaxation, or suppressed activity of TRPC6 via protein kinase G. At high concentrations, SNAP (but not DEA-NONOate) occasionally stimulated TRPC5 but the effect was confounded by background TRPC5-independent Ca²⁺ signals. Endogenous Ca²⁺-entry in bovine aortic endothelial cells (BAECs) was suppressed by SNAP; TRPC5 blocking antibody or dominant-negative mutant TRPC5 suppressed this Ca²⁺ entry and occluded the effect of SNAP. The data suggest that nitric oxide is not a direct modulator of homomeric TRPC5 channels but may inhibit endogenous BAEC channels that contain TRPC5.     

Layer-specific generation and propagation of seizures in slices of developing neocortex: role of excitatory GABAergic synapses

The neonatal period is critical for seizure susceptibility, and neocortical networks are central in infantile epilepsies. We report that application of 4-aminopyridine (4-AP) to immature (P6–P9) neocortical slices generates layer-specific interictal seizures (IISs) that transform after recurrent seizures to ictal seizures (ISs). During IISs, cell-attached recordings show action potentials in interneurons and pyramidal cells in L5/6 and interneurons but not pyramidal neurons in L2/3. However, L2/3 pyramidal neurons also fire during ISs. Using single N-methyl-D-aspartate (NMDA) channel recordings for measuring the cell resting potential (E_m), we show that transition from IISs to ISs is associated with a gradual E_m depolarization of L2/3 and L5/6 pyramidal neurons that enhances their excitability. Bumetanide, a NKCC1 co-transporter antagonist, inhibits generation of IISs and prevents their transformation to ISs, indicating the role excitatory GABA in epilepsies. Therefore deep layer neurons are more susceptible to seizures than superficial ones. The initiating phase of seizures is characterized by IISs generated in L5/6 and supported by activation of both L5/6 interneurons and pyramidal cells. IISs propagate to L2/3 via activation of L2/3 interneurons but not pyramidal cells, which are mostly quiescent at this phase. In superficial layers, a persistent increase in excitability of pyramidal neurons caused by E_m depolarization is associated with a transition from largely confined GABAergic IIS to ictal events that entrain the entire neocortex.     

Obligatory role for phosphatidylinositol 4,5-bisphosphate in activation of native TRPC1 store-operated channels in vascular myocytes

In the present study the effect of phosphatidylinositol 4,5-bisphosphate (PIP₂) was studied on a native TRPC1 store-operated channel (SOC) in freshly dispersed rabbit portal vein myocytes. Application of diC8-PIP₂, a water soluble form of PIP₂, to quiescent inside-out patches evoked single channel currents with a unitary conductance of 1.9 pS. DiC8-PIP₂-evoked channel currents were inhibited by anti-TRPC1 antibodies and these characteristics are identical to SOCs evoked by cyclopiazonic acid (CPA) and BAPTA-AM. SOCs stimulated by CPA, BAPTA-AM and the phorbol ester phorbol 12,13-dibutyrate (PDBu) were reduced by anti-PIP₂ antibodies and by depletion of tissue PIP₂ levels by pre-treatment of preparations with wortmannin and LY294002. However, these reagents did not alter the ability of PIP₂ to activate SOCs in inside-out patches. Co-immunoprecipitation techniques demonstrated association between TRPC1 and PIP₂ at rest, which was greatly decreased by wortmannin and LY294002. Pre-treatment of cells with PDBu, which activates protein kinase C (PKC), augmented SOC activation by PIP₂ whereas the PKC inhibitor chelerythrine decreased SOC stimulation by PIP₂. Co-immunoprecipitation experiments provide evidence that PKC-dependent phosphorylation of TRPC1 occurs constitutively and was increased by CPA and PDBu but decreased by chelerythrine. These novel results show that PIP₂ can activate TRPC1 SOCs in native vascular myocytes and plays an important role in SOC activation by CPA, BAPTA-AM and PDBu. Moreover, the permissive role of PIP₂ in SOC activation requires PKC-dependent phosphorylation of TRPC1.     

TRPC1 channels regulate directionality of migrating cells

Cell migration depends on the generation of structural asymmetry and on different steps: protrusion and adhesion at the front and traction and detachment at the rear part of the cell. The activity of Ca²⁺ channels coordinate these steps by arranging intracellular Ca²⁺ signals along the axis of movement. Here, we investigated the role of the putative mechanosensitive canonical transient receptor potential channel 1 (TRPC1) in cell migration. We analyzed its function in transformed renal epithelial (Madin–Darby canine kidney-focus) cells with variation of TRPC1 expression. As shown by time lapse video microscopy, TRPC1 knockdown cells have partially lost their polarity and the ability to persistently migrate into a given direction. This failure is linked to the suppression of a local Ca²⁺ gradient at the front of migrating TRPC1 knockdown cells, whereas TRPC1 overexpression leads to steeper Ca²⁺ gradients. We propose that the Ca²⁺ signaling events regulated by TRPC1 within the lamellipodium determine polarity and directed cell migration. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Modulation of long-term synaptic plasticity at excitatory striatal synapses

Modulation of long-term plasticity by both the intrinsic activation of metabotropic glutamate receptors and dopamine released from the nigrostriatal pathway was investigated at excitatory striatal synapses. Intracellular recordings demonstrated that tetanic stimulation at an intensity equal to that used for synaptic sampling produced, on average, a slight long-term depression of excitatory postsynaptic potentials. The long-term response pattern was variable, however, with some cells showing potentiation and others no plasticity. Block of metabotropic glutamate receptors with 3-aminophosphonovaleric acid changed the pattern of responses, increasing the percentage of cells showing long-term potentiation. Similarly, 6-hydroxydopamine lesions to the substantia nigra changed the pattern of response to tetanic stimulation, increasing the expression of long-term potentiation. These data indicate that metabotropic glutamate receptor and dopamine receptor activation may function to regulate the expression of activity-dependent plasticity at corticostriatial synapses. Paired-pulse stimulation revealed that post-tetanic plasticity was negatively correlated with changes in paired-pulse plasticity in the control and 6-hydroxydopamine-lesioned groups, suggesting that the expression of long-term plasticity has a presynaptic component at corticostriatal synapses.     

The role of mechanical tension on lipid raft dependent PDGF-induced TRPC6 activation

Canonical transient receptor potential channel 6 (TRPC6) can play an important role in governing how cells perceive the surrounding material environment and regulate Ca²⁺ signaling. We have designed a TRPC6 reporter based on fluorescence resonance energy transfer (FRET) to visualize the TRPC6-mediated calcium entry and hence TRPC6 activity in live cells with high spatiotemporal resolutions. In mouse embryonic fibroblasts (MEFs), platelet-derived growth factor BB (PDGF) can activate the TRPC6 reporter, mediated by phospholipase C (PLC). This TRPC6 activation occurred mainly at lipid rafts regions of the plasma membrane because disruption of lipid raft/caveolae by methyl-β-cyclodextrin (MβCD) or the expression of dominant-negative caveolin-1 inhibited the TRPC6 activity. Culturing cells on soft materials or releasing the intracellular tension by ML-7 reduced this PDGF-induced activation of TRPC6 without affecting the PDGF-regulated Src or inositol 1,4,5-trisphosphate (IP₃) receptor function, suggesting a specific role of mechanical tension in regulating TRPC6. We further showed that the release of intracellular tension had similar effect on the diffusion coefficients of TRPC6 and a raft marker, confirming a strong coupling between TRPC6 and lipid rafts. Therefore, our results suggest that the TRPC6 activation mainly occurs at lipid rafts, which is regulated by the mechanical cues of surrounding materials.     

Synergism between inositol phosphates and diacylglycerol on native TRPC6-like channels in rabbit portal vein myocytes

In rabbit portal vein myocytes noradrenaline activates a non-selective cation current ( I _cat) which involves a transient receptor potential protein (TRPC6). Previously we have shown that the diaylglycerol (DAG) analogue 1-oleoyl-2-acetyl- sn -glycerol (OAG) stimulates I _cat via a protein kinase C (PKC)-independent mechanism, and in the present study we have investigated the interaction between inositol phosphates (InsPs) and OAG on I _cat. With whole-cell recording of I _cat from freshly isolated rabbit portal vein myocytes the amplitude and rate of activation of noradrenaline-evoked I _cat were much greater than those of OAG-induced I _cat. Inclusion of inositol 1,4,5-trisphosphate (Ins(1,4,5) P ₃) in the pipette solution did not evoke I _cat but greatly potentiated the amplitude and rate of activation of OAG-induced I _cat. With isolated outside-out patches Ins(1,4,5) P ₃ markedly increased the rate of activation and the open probability of OAG-evoked channel activity, with no change in unitary conductance, channel mean open times or burst durations. The effects of Ins(1,4,5) P ₃ were mimicked by Ins(2,4,5) P ₃, 3-F-Ins(1,4,5) P ₃ and Ins(1,4) P ₂ but not by Ins(1,3,4,5) P ₄ and the potentiating effects of InsPs were not inhibited by heparin. Therefore it is concluded that both DAG and InsPs are necessary for full activation of I _cat by noradrenaline and the effect of InsPs is via a heparin-insensitive mechanism and represents a novel action of InsPs.