Transient Receptor Potential Canonical Channel Activation Research Articles

Dual effect of nifedipine on pregnant human myometrium contractility: Implication of TRPC1.

Nifedipine, an L‐type voltage‐gated Ca2+ channel (L‐VGCC) blocker, is one of the most used tocolytics to treat preterm labor. In clinical practice, nifedipine efficiently decreases uterine contractions, but its efficacy is limited over time, and repeated or maintained nifedipine‐based tocolysis appears to be ineffective in preventing preterm birth. We aimed to understand why nifedipine has short‐lasting efficiency for the inhibition of uterine contractions. We used ex vivo term pregnant human myometrial strips treated with cumulative doses of nifedipine. We observed that nifedipine inhibited spontaneous myometrial contractions in tissues with high and regular spontaneous contractions. By contrast, nifedipine appeared to increase contractions in tissues with low and/or irregular spontaneous contractions. To investigate the molecular mechanisms activated by nifedipine in myometrial cells, we used the pregnant human myometrial cell line PHM1‐41 that does not express L‐VGCC. The in vitro measurement of intracellular Ca2+ showed that high doses of nifedipine induced an important intracellular Ca2+ entry in myometrial cells. The inhibition or downregulation of the genes encoding for store‐operated Ca2+ entry channels from the Orai and transient receptor potential‐canonical (TRPC) families in PHM1‐41 cells highlighted the implication of TRPC1 in nifedipine‐induced Ca2+ entry. In addition, the use of 2‐APB in combination with nifedipine on human myometrial strips tends to confirm that the pro‐contractile effect induced by nifedipine on myometrial tissues may involve the activation of TRPC channels.

Blockade of TRPC Channels Limits Cholinergic-Driven Hyperexcitability and Seizure Susceptibility After Traumatic Brain Injury.

We investigated the contribution of excitatory transient receptor potential canonical (TRPC) cation channels to posttraumatic hyperexcitability in the brain 7 days following controlled cortical impact model of traumatic brain injury (TBI) to the parietal cortex in male adult mice. We investigated if TRPC1/TRPC4/TRPC5 channel expression is upregulated in excitatory neurons after TBI in contribution to epileptogenic hyperexcitability in key hippocampal and cortical circuits that have substantial cholinergic innervation. This was tested by measuring TRPC1/TRPC4/TRPC5 protein and messenger RNA (mRNA) expression, assays of cholinergic function, neuronal Ca2+ imaging in brain slices, and seizure susceptibility after TBI. We found region-specific increases in expression of TRPC1, TRPC4, and TRPC5 subunits in the hippocampus and cortex following TBI. The dentate gyrus, CA3 region, and cortex all exhibited robust upregulation of TRPC4 mRNA and protein. TBI increased cFos activity in dentate gyrus granule cells (DGGCs) and layer 5 pyramidal neurons both at the time of TBI and 7 days post-TBI. DGGCs displayed greater magnitude and duration of acetylcholine-induced rises in intracellular Ca2+ in brain slices from mice subjected to TBI. The TBI mice also exhibited greater seizure susceptibility in response to pentylenetetrazol-induced kindling. Blockade of TRPC4/TRPC5 channels with M084 reduced neuronal hyperexcitation and impeded epileptogenic progression of kindling. We observed that the time-dependent upregulation of TRPC4/TRPC5-containing channels alters cholinergic responses and activity of principal neurons acting to increase proexcitatory sensitivity. The underlying mechanism includes acutely decreased acetylcholinesterase function, resulting in greater Gq/11-coupled muscarinic receptor activation of TRPC channels. Overall, our evidence suggests that TBI-induced plasticity of TRPC channels strongly contributes to overt hyperexcitability and primes the hippocampus and cortex for seizures.

Activation of TRPC (Transient Receptor Potential Canonical) Channel Currents in Iron Overloaded Cardiac Myocytes.

Arrhythmias and heart failure are common cardiac complications leading to substantial morbidity and mortality in patients with hemochromatosis, yet mechanistic insights remain incomplete. We investigated the effects of iron (Fe) on electrophysiological properties and intracellular Ca2+ (Ca2+i) handling in mouse left ventricular cardiomyocytes. Cardiomyocytes were isolated from the left ventricle of mouse hearts and were superfused with Fe3+/8-hydroxyquinoline complex (5-100 μM). Membrane potential and ionic currents including TRPC (transient receptor potential canonical) were recorded using the patch-clamp technique. Ca2+i was evaluated by using Fluo-4. Cell contraction was measured with a video-based edge detection system. The role of TRPCs in the genesis of arrhythmias was also investigated by using a mathematical model of a mouse ventricular myocyte with the incorporation of the TRPC component. We observed prolongation of the action potential duration and induction of early and delayed afterdepolarizations in myocytes superfused with 15 µmol/L Fe3+/8-hydroxyquinoline complex. Iron treatment decreased the peak amplitude of the L-type Ca2+ current and total K+ current, altered Ca2+i dynamics, and decreased cell contractility. During the final phase of Fe treatment, sustained Ca2+i waves and repolarization failure occurred and ventricular cells became unexcitable. Gadolinium abolished Ca2+i waves and restored the resting membrane potential to the normal range. The involvement of TRPC activation was confirmed by TRPC channel current recordings in the absence or presence of functional TRPC channel antibodies. Computer modeling captured the same action potential and Ca2+i dynamics and provided additional mechanistic insights. We conclude that iron overload induces cardiac dysfunction that is associated with TRPC channel activation and alterations in membrane potential and Ca2+i dynamics.

Abstract 507: Activation of Transient Receptor Potential Canonical Channel Currents in Iron-Overloaded Cardiac Myocytes

Background: Iron (Fe) overload cardiomyopathy is the leading cause of death in hemochromatotic patients, yet the mechanistic insight is still incomplete and controversial. We investigated alterations of action potentials (APs), ionic currents, and intracellular Ca 2+ (Ca 2+ i ) in Fe-loaded mouse cardiomyocytes, as well as functional impacts of Fe overload on single-cell contraction and whole-heart arrhythmias. Methods: Cardiomyocytes were isolated from left ventricles of mouse hearts and were superfused with Fe 3+ /8-hydroxyquinoline complex (5-100 μM). APs, L-type Ca 2+ currents (I Ca,L ), total outward K + currents (I K ), and transient receptor potential canonical (TRPC) channel currents were recorded by the patch-clamp technique. Ca 2+ i was evaluated by using Fluo-4. Cell contraction was measured by a video-based edge detection system. Arrhythmias were evaluated in Langendorff-perfused hearts under S 1 -S 2 stimulation protocol. Results: Persistent Fe (15 μM) treatment prolonged AP duration at 90% repolarization (APD 90 : 46.8 ± 2.8 vs. 203.6 ± 63.4 ms, p<0.05), induced early and delayed afterdepolarizations (EADs: 0 % vs. 45.0 ± 15.0 %; DADs: 4.3 ± 1.4 vs. 27.0 ± 7.0 %, p<0.05, respectively) in mouse cardiomyocytes. Consistently, arrhythmia incidence was increased in Fe 3+ /8-HQ-perfused hearts. Fe treatment decreased peak I Ca,L (16.5 ± 1.7 vs.11.4 ± 1.3 pA/pF, p<0.01) and I K (59.2 ± 3.3 vs. 50.4 ± 3.0 pA/pF, p<0.01), altered Ca 2+ i transient patterns and decreased contractility (4.8 ± 0.5 vs. 3.5 ± 0.4%, p<0.01). During the late phase of Fe treatment, fast Ca 2+ waves and sustained depolarization were induced to generate a secondary (shallow) resting membrane potential (RMP: from -68.8 ± 0.6 to -25.0 ± 3.7 mV) where the myocytes became unexcitable. Gadolinium, a TRPC channel blocker, abolished fast Ca 2+ waves and reversed RMP to the deep level (-62.9 ± 3.5 mV). The involvement of TRPC activation was determined for the first time by recording TRPC current and assessing the effect of functional TRPC channel antibodies. Conclusions: In mouse cardiomyocytes, Fe overload induced arrhythmogenic APD prolongation and EADs/DADs, aberrant Ca 2+ i dynamics, and impaired contractility. The activation of TRPC channels accounts for an important underlying mechanism.

Gq-Coupled Muscarinic Receptor Enhancement of KCNQ2/3 Channels and Activation of TRPC Channels in Multimodal Control of Excitability in Dentate Gyrus Granule Cells.

KCNQ (Kv7, "M-type") K+ channels and TRPC (transient receptor potential, "canonical") cation channels are coupled to neuronal discharge properties and are regulated via Gq/11-protein-mediated signals. Stimulation of Gq/11-coupled receptors both consumes phosphatidylinositol 4,5-bisphosphate (PIP2) via phosphalipase Cβ hydrolysis and stimulates PIP2 synthesis via rises in Ca2+i and other signals. Using brain-slice electrophysiology and Ca2+ imaging from male and female mice, we characterized threshold K+ currents in dentate gyrus granule cells (DGGCs) and CA1 pyramidal cells, the effects of Gq/11-coupled muscarinic M1 acetylcholine (M1R) stimulation on M current and on neuronal discharge properties, and elucidated the intracellular signaling mechanisms involved. We observed disparate signaling cascades between DGGCs and CA1 neurons. DGGCs displayed M1R enhancement of M-current, rather than suppression, due to stimulation of PIP2 synthesis, which was paralleled by increased PIP2-gated G-protein coupled inwardly rectifying K+ currents as well. Deficiency of KCNQ2-containing M-channels ablated the M1R-induced enhancement of M-current in DGGCs. Simultaneously, M1R stimulation in DGGCs induced robust increases in [Ca2+]i, mostly due to TRPC currents, consistent with, and contributing to, neuronal depolarization and hyperexcitability. CA1 neurons did not display such multimodal signaling, but rather M current was suppressed by M1R stimulation in these cells, similar to the previously described actions of M1R stimulation on M-current in peripheral ganglia that mostly involves PIP2 depletion. Therefore, these results point to a pleiotropic network of cholinergic signals that direct cell-type-specific, precise control of hippocampal function with strong implications for hyperexcitability and epilepsy.SIGNIFICANCE STATEMENT At the neuronal membrane, protein signaling cascades consisting of ion channels and metabotropic receptors govern the electrical properties and neurotransmission of neuronal networks. Muscarinic acetylcholine receptors are G-protein-coupled metabotropic receptors that control the excitability of neurons through regulating ion channels, intracellular Ca2+ signals, and other second-messenger cascades. We have illuminated previously unknown actions of muscarinic stimulation on the excitability of hippocampal principal neurons that include M channels, TRPC (transient receptor potential, "canonical") cation channels, and powerful regulation of lipid metabolism. Our results show that these signaling pathways, and mechanisms of excitability, are starkly distinct between peripheral ganglia and brain, and even between different principal neurons in the hippocampus.

New Aspects of the Contribution of ER to SOCE Regulation: TRPC Proteins as a Link Between Plasma Membrane Ion Transport and Intracellular Ca2+ Stores.

Transient receptor potential canonical (TRPC) proteins were identified as molecular candidates of receptor- and/or store-operated channels because of their close homology to the Drosophila TRP and TRPL. Functional studies have revealed that TRPC channels play an integrated part of phospholipase C-transduced cell signaling, mediating the influx of both Ca2+ and Na+ into cells. As a consequence, the TRPC channels have diverse functional roles in different cell types, including metabotropic receptor-evoked membrane depolarization and intracellular Ca2+ concentration elevation. Depending on the cellular environment and the protein partners present in the channel complex, the TRPC channels display different biophysical properties and mechanisms of regulation, including but not limited to the Ca2+ filling state of the endoplasmic reticulum. Despite the overwhelming focus on STIM-regulated Orai channels for store-operated Ca2+ entry, evidence is growing for STIM-operated TRPC channel activities in various cell types, demonstrating both store-dependent and store-independent mechanisms of TRPC channel gating. The existence of physical and functional interactions between plasma membrane-localized TRPC channels and other proteins involved in sensing and regulating the intracellular Ca2+ store contents, such as inositol trisphosphate receptors, Junctate, and Homer, further argues for the role of TRPC proteins in linking plasma membrane ion transport with intracellular Ca2+ stores. The interplay among these proteins will likely define the functional significance of TRPC channel activation in different cellular contexts and under different modes of stimulations.

Screening of Transient Receptor Potential Canonical Channel Activators Identifies Novel Neurotrophic Piperazine Compounds.

Transient receptor potential canonical (TRPC) proteins form Ca(2+)-permeable cation channels activated upon stimulation of metabotropic receptors coupled to phospholipase C. Among the TRPC subfamily, TRPC3 and TRPC6 channels activated directly by diacylglycerol (DAG) play important roles in brain-derived neurotrophic factor (BDNF) signaling, promoting neuronal development and survival. In various disease models, BDNF restores neurologic deficits, but its therapeutic potential is limited by its poor pharmacokinetic profile. Elucidation of a framework for designing small molecules, which elicit BDNF-like activity via TRPC3 and TRPC6, establishes a solid basis to overcome this limitation. We discovered, through library screening, a group of piperazine-derived compounds that activate DAG-activated TRPC3/TRPC6/TRPC7 channels. The compounds [4-(5-chloro-2-methylphenyl)piperazin-1-yl](3-fluorophenyl)methanone (PPZ1) and 2-[4-(2,3-dimethylphenyl)piperazin-1-yl]-N-(2-ethoxyphenyl)acetamide (PPZ2) activated, in a dose-dependent manner, recombinant TRPC3/TRPC6/TRPC7 channels, but not other TRPCs, in human embryonic kidney cells. PPZ2 activated native TRPC6-like channels in smooth muscle cells isolated from rabbit portal vein. Also, PPZ2 evoked cation currents and Ca(2+) influx in rat cultured central neurons. Strikingly, both compounds induced BDNF-like neurite growth and neuroprotection, which were abolished by a knockdown or inhibition of TRPC3/TRPC6/TRPC7 in cultured neurons. Inhibitors of Ca(2+) signaling pathways, except calcineurin, impaired neurite outgrowth promotion induced by PPZ compounds. PPZ2 increased activation of the Ca(2+)-dependent transcription factor, cAMP response element-binding protein. These findings suggest that Ca(2+) signaling mediated by activation of DAG-activated TRPC channels underlies neurotrophic effects of PPZ compounds. Thus, piperazine-derived activators of DAG-activated TRPC channels provide important insights for future development of a new class of synthetic neurotrophic drugs.

Podocyte injury in diabetic nephropathy: implications of angiotensin II-dependent activation of TRPC channels.

Injury to podocytes is considered a major contributor to diabetic kidney disease: their loss causes proteinuria and progressive glomerulosclerosis. Podocyte depletion may result from improper calcium handling due to abnormal activation of the calcium permeant TRPC (Transient Receptor Potential Canonical) channels. Angiotensin II (Ang II) levels are found to be elevated in diabetes; furthermore, it was reported that Ang II causes activation of TRPC6 in podocytes. We hypothesized here that Ang II-mediated calcium influx is aggravated in the podocytes under the conditions of type 1 diabetic nephropathy (DN). Diabetes was induced in the Dahl Salt-Sensitive rats by an injection of streptozotocin (STZ-SS). Eleven weeks post treatment was sufficient for the animals to develop hyperglycemia, excessive urination, weight loss, microalbuminuria, nephrinuria and display renal histological lesions typical for patients with DN. Patch-clamp electrophysiology performed on podocytes of the freshly isolated glomeruli showed enhanced basal TRPC channel activity in the STZ-SS rats, and increased response to Ang II; total calcium influx triggered by Ang II application was also augmented in podocytes of these rats. Our studies have a strong potential for advancing the understanding of TRPC-mediated effects on podocytopenia in DN initiation.

Enhanced leptin‐TRPC signaling in the hypothalamus contributes to the exaggerated sympatho‐excitation in type 2 diabetic rats (1130.9)

Accumulated evidence indicates that type 2 diabetes (T2D) is associated with enhanced sympathetic activation. Transient receptor potential canonical channels (TRPC) have been found in pro‐opiomelanocortin neurons and activated by leptin. The present study was conducted to investigate the role for leptin‐TRPC signaling in centrally mediated sympathetic response in rats with T2D. In high‐fat diet and low‐dose strepzotocin (30mg/kg)‐induced T2D rats, mRNA levels of TRPC subtype including TRPC1, 4, 5 and 6 were significantly increased in the paraventricular nucleus (PVN) and arcuate nucleus (ARCN) compared to control rats (n=8/group). Whole‐cell patch‐clamp was conducted in primary cultured hypothalamic neurons to record TPPC currents. Neurons from T2D rats had significant increased TRPC currents (about 2 folds to the control). In anaesthetized condition, microinjections of leptin (5ng~100ng) into the ARCN and PVN induced increases in renal sympathetic nerve activity and blood pressure. These effects were significantly blocked by pre‐microinjections of TRPC inhibitors (2APB and SKF96365). In cultured neuronal cell NG108, pre‐incubation with leptin (0.625~2.5μM) for 24hrs induced significant increases of TRPC mRNA expressions (TRPC1, 4 and 5). Taken together, these data suggest that within the hypothalamic nuclei, leptin‐induced sympatho‐excitation in T2D maybe mediated through an activation of TRPC. Supported by NIH grant DK82956.Grant Funding Source: NIH DK82956

Involvement of TRPC in the Slow Force Response Observed in Mouse Ventricular Myocytes

When cardiac muscle is stretched persistently, the contractile force slowly increases over several minutes. This phenomenon is termed by a slow force response (SFR). The SFR is due to a stretch-induced increase in Ca2+ influx, however, underlying mechanism remains unclear. The transient receptor potential, canonical (TRPC) channels are mechano-sensitive, non-selective cation channels. We hypothesized that activation of TRPC channels is involved in stretch-induced increase in Ca2+ influx in SFR. To test this hypothesis, mouse cardiac myocytes were isolated with collagenase, and a pair of computer-controlled piezo-positioned carbon fibers was attached to each cell-end. Cells were paced at 1 Hz and superfused in normal Tyrode solution. Passive and active forces were calculated from carbon fiber bending. Stretch was applied by moving carbon fiber positions to achieve 3-10 % increase in sarcomere length and maintained for 200 to 300 seconds to obtain SFR. At the plateu of SFR, the contractile force increased to 115.7 ± 3.7% (n = 16) of the value immediately after the stretch. Inhibition of TRPC channel with BTP-2 (10 μM) significantly reduced the magnitude of SFR (103.5 ± 1.0%, n = 8). It has been reported that activation of angiotensin II type 1 (AT1) receptor induces Ca2+ influx via TRPC channels. To reveal whether TRPC channels are activated secondarily by AT1 receptor activated by strech, we tested the effect of Olmesartan, an AT1 receptor inhibitor, in SFR. Olmesartan (10 μM) also reduced the magnitude of SFR (101.1 ± 2.4%, n = 8). These results suggest that stretch-induced activation of AT1 receptors leads to increase in Ca2+ influx via TRPC channels in SFR.

Transient Receptor Potential Canonical Channels Regulate the Induction of Cerebellar Long-Term Depression

In the cerebellum, synaptic strength at the synapses between parallel fibers and Purkinje cells is best known to be modulated via metabotropic glutamate receptor 1 (mGluR1)-dependent cerebellar long-term depression (LTD). An increase in intracellular calcium levels plays an important role in inducing mGluR1-dependent cerebellar LTD. Downstream of mGluR1, there are two major sources of calcium: transient receptor potential canonical (TRPC) channels and inositol trisphosphate receptors (IP(3)R). IP(3)R triggers a calcium release from the intracellular calcium store. Here, we show that TRPC channels mediate mGluR1-evoked slow currents to regulate cerebellar LTD in Sprague Dawley rats. We found that the inhibition of TRPC channels blocks the induction of cerebellar LTD. Moreover, we show that processes known to underlie cerebellar LTD induction, such as increases in intracellular calcium concentration, the activation of protein kinase C, and the internalization of GluR2, are also hindered by blocking TRPC. These results suggest that the mGluR1-evoked activation of TRPC channels is required for the induction of cerebellar LTD.

Metabotropic glutamate receptors (mGluR5) activate transient receptor potential canonical channels to improve the regularity of the respiratory rhythm generated by the pre‐Bötzinger complex in mice

Metabotropic glutamate receptors (mGluRs) are hypothesized to play a key role in generating the central respiratory rhythm and other rhythmic activities driven by central pattern generators (e.g. locomotion). However, the functional role of mGluRs in rhythmic respiratory activity and many motor patterns is very poorly understood. Here, we used mouse respiratory brain-slice preparations containing the pre-Bötzinger complex (pre-BötC) to identify the role of group I mGluRs (mGluR1 and mGluR5) in respiratory rhythm generation. We found that activation of mGluR1/5 is not required for the pre-BötC to generate a respiratory rhythm. However, our data suggest that mGluR1 and mGluR5 differentially modulate the respiratory rhythm. Blocking endogenous mGluR5 activity with 2-Methyl-6-(phenylethynyl)pyridine (MPEP) decreases the inspiratory burst duration, burst area and frequency, whereas it increases the irregularity of the fictive eupneic inspiratory rhythm generated by the pre-BötC. In contrast, blocking mGluR1 reduces the frequency. Moreover, the mGluR1/5 agonist 3,5-dihydroxyphenylglycine increases the frequency and decreases the irregularity of the respiratory rhythm. Based on previous studies, we hypothesized that mGluR signaling decreases the irregularity of the respiratory rhythm by activating transient receptor potential canonical (TRPC) channels, which carry a non-specific cation current (ICAN). Indeed, 3,5-dihydroxyphenylglycine (DHPG) application reduces cycle-by-cycle variability and subsequent application of the TRPC channel blocker 1-[2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl)propoxy]ethyl]imidazole (SKF-96365) hydrochloride reverses this effect. Our data suggest that mGluR5 activation of ICAN-carrying TRPC channels plays an important role in governing the cycle-by-cycle variability of the respiratory rhythm. These data suggest that modulation of TRPC channels may correct irregular respiratory rhythms in some central neuronal diseases.

Bromoenol Lactone Inhibits Voltage-Gated Ca2+and Transient Receptor Potential Canonical Channels

Circulating hormones stimulate the phospholipase Cβ (PLC)/Ca(2+) influx pathway to regulate numerous cell functions, including vascular tone. It was proposed previously that Ca(2+)-independent phospholipase A(2) (iPLA(2))-dependent store-operated Ca(2+) influx channels mediate hormone-induced contractions in isolated arteries, because bromoenol lactone (BEL), a potent irreversible inhibitor of iPLA(2), inhibited such contractions. However, the effects of BEL on other channels implicated in mediating hormone-induced vessel contractions, specifically voltage-gated Ca(2+) (Ca(V)1.2) and transient receptor potential canonical (TRPC) channels, have not been defined clearly. Using isometric tension measurements, we found that thapsigargin-induced contractions were ∼34% of those evoked by phenylephrine or KCl. BEL completely inhibited not only thapsigargin- but also phenylephrine- and KCl-induced ring contractions, suggesting that Ca(V)1.2 and receptor-operated TRPC channels also may be sensitive to BEL. Therefore, we investigated the effects of BEL on heterologously expressed Ca(V)1.2 and TRPC channels in human embryonic kidney cells, a model system that allows probing of individual protein function without interference from other signaling elements of native cells. We found that low micromolar concentrations of BEL inhibited Ca(V)1.2, TRPC5, TRPC6, and heteromeric TRPC1-TRPC5 channels in an iPLA(2)-independent manner. BEL also attenuated PLC activity, suggesting that the compound may inhibit TRPC channel activity in part by interfering with an initial PLC-dependent step required for TRPC channel activation. Conversely, BEL did not affect endogenous voltage-gated K(+) channels in human embryonic kidney cells. Our findings support the hypothesis that iPLA(2)-dependent store-operated Ca(2+) influx channels and iPLA(2)-independent hormone-operated TRPC channels can serve as smooth muscle depolarization triggers to activate Ca(V)1.2 channels and to regulate vascular tone.

STIM1-dependent and STIM1-independent Function of Transient Receptor Potential Canonical (TRPC) Channels Tunes Their Store-operated Mode

Ca(2+) influx by store-operated Ca(2+) channels is a key component of the receptor-evoked Ca(2+) signal. In all cells examined, transient receptor potential canonical (TRPC) channels mediate a significant portion of the receptor-stimulated Ca(2+) influx. Recent studies have revealed how STIM1 activates TRPC1 in response to store depletion; however, the role of STIM1 in TRPC channel activation by receptor stimulation is not fully understood. Here, we established mutants of TRPC channels that could not be activated by STIM1 but were activated by the "charge-swap" mutant STIM1(K684E,K685E). Significantly, WT but not mutant TRPC channels were inhibited by scavenging STIM1 with Orai1(R91W), indicating the STIM1 dependence and independence of WT and mutant TRPC channels, respectively. Importantly, mutant TRPC channels were robustly activated by receptor stimulation. Moreover, STIM1 and STIM1(K684E,K685E) reciprocally affected receptor-activated WT and mutant TRPC channels. Together, these findings indicate that TRPC channels can function as STIM1-dependent and STIM1-independent channels, which increases the versatility of TRPC channel function and their role in receptor-stimulated Ca(2+) influx.

Sensing the Right Path

During nervous system development, axonal growth cones find their way to their targets with the help of various chemoattractant and chemorepellant cues. Several neurite guidance signals modulate calcium influx into the growth cone, but the underlying mechanisms and the specific channels involved have been unclear (see Gomez). Now two groups, Li et al. and Wang and Poo, provide evidence implicating transient receptor potential canonical (TRPC) channels--a family of channels known for mediating sensory responses--in the response to the chemoattractants brain-derived neurotrophic factor (BDNF) and netrin-1. Wang and Poo performed perforated whole-cell patch clamp analysis of cultured Xenopus spinal neurons and found that netrin-1 elicited membrane depolarization that was inhibited by an antibody to its receptor, DDC. Netrin-induced current present during pharmacological blockade of most voltage-dependent ion channels was inhibited by SKF-96365, which blocks TRP channels, as was a similar current produced by BDNF. Further, growth cone turning in response to netrin-1 or BDNF was abolished by SKF-96365. Injection of morpholino oligonucleotides (MOs) that knocked down xTRPC1 expression inhibited the putative TRP currents and abolished growth cone turning in response to a netrin-1 gradient. Moreover, MO or SKF-96365 abolished the netrin-induced increase in growth cone calcium concentration. Li et al. investigated TRPC channels in BDNF-mediated guidance of cultured cerebellar granule cells, which was blocked by antibodies to the TrkB receptor, incubation with a membrane-permeable calcium chelator, or pharmacological depletion of intracellular calcium stores. Although pharmacological blockade of several voltage-sensitive sodium and calcium channels had no effect on growth cone turning elicited by BDNF, turning was abolished by SKF-96365, as well as by pharmacological inhibition of phospholipase C (PLC), phosphatidylinositol 3-kinase (PI3K), or blockade of the inositol 1,4,5-trisphosphate receptor (IP 3 R). In contrast, a PLC activator elicited turning, an effect that depended on extracellular calcium and was abolished by SKF-96365. BDNF increased growth cone calcium concentration, an effect that depended on both calcium influx and release from internal stores and that was reduced by SKF-96365, pharmacological inhibition of PLC, or IP 3 R blockade. BDNF-induced turning was abolished by siRNA directed against TRPC3 or by overexpression of dominant-negative mutant forms of TRPC3 or TRPC6. Thus, activation of TRPC channels appears to play a key role in calcium-dependent neurite pathfinding in response to both netrin-1 and BDNF. Y. Li, Y.-C. Jia, K. Cui, N. Li, Z.-Y. Zheng, Y.-Z. Wang, X.-B. Yuan, Essential role of TRPC channels in the guidance of nerve growth cones by brain-derived neurotrophic factor. Nature 434 , 894-898 (2005). [PubMed] G. X. Wang, M.-M. Poo, Requirement of TRPC channels in netrin-1-induced chemotropic turning of nerve growth cones. Nature 434 , 898-904 (2005). [PubMed] T. Gomez, Channels for pathfinding. Nature 434 , 835-838 (2005). [PubMed]