Metabolic regulation of single synaptic vesicle exo- and endocytosis in hippocampal synapses.

Glucose has long been considered a primary energy source for synaptic function. However, it remains unclear to what extent alternative fuels, such as lactate/pyruvate, contribute to powering synaptic transmission. By detecting individual release events in hippocampal synapses, we find that mitochondrial ATP production regulates basal vesicle release probability and release location within the active zone (AZ), evoked by single action potentials. Mitochondrial inhibition shifts vesicle release closer to the AZ center and alters the efficiency of vesicle retrieval by increasing the occurrence of ultrafast endocytosis. Furthermore, we uncover that terminals can use oxidative fuels to maintain the vesicle cycle during trains of activity. Mitochondria are sparsely distributed along hippocampal axons, and we find that terminals containing mitochondria display enhanced vesicle release and reuptake during high-frequency trains. Our findings suggest that mitochondria not only regulate several fundamental features of synaptic transmission but may also contribute to modulation of short-term synaptic plasticity.

Mitochondrial pyruvate transport regulates presynaptic metabolism and neurotransmission.

Glucose has long been considered the primary fuel source for the brain. However, glucose levels fluctuate in the brain during sleep, intense circuit activity, or dietary restrictions, posing significant metabolic stress. Here, we demonstrate that the mammalian brain utilizes pyruvate as a fuel source, and pyruvate can support neuronal viability in the absence of glucose. Nerve terminals are sites of metabolic vulnerability within a neuron and we show that mitochondrial pyruvate uptake is a critical step in oxidative ATP production in hippocampal terminals. We find that the mitochondrial pyruvate carrier is post-translationally modified by lysine acetylation which in turn modulates mitochondrial pyruvate uptake. Importantly, our data reveal that the mitochondrial pyruvate carrier regulates distinct steps in synaptic transmission, namely, the spatiotemporal pattern of synaptic vesicle release and the efficiency of vesicle retrieval, functions that have profound implications for synaptic plasticity. In summary, we identify pyruvate as a potent neuronal fuel and mitochondrial pyruvate uptake as a critical node for the metabolic control of synaptic transmission in hippocampal terminals.

Metabolic Regulation of Single Synaptic Vesicle Exo- and Endocytosis in Hippocampal Synapses.

Glucose has long been considered a primary source of energy for synaptic function. However, it remains unclear under what conditions alternative fuels, such as lactate/pyruvate, contribute to powering synaptic transmission. By detecting individual release events in cultured hippocampal synapses, we found that mitochondrial ATP production from oxidation of lactate/pyruvate regulates basal vesicle release probability and release location within the active zone (AZ) evoked by single action potentials (APs). Mitochondrial inhibition shifted vesicle release closer to the AZ center, suggesting that the energetic barrier for vesicle release is lower in the AZ center that the periphery. Mitochondrial inhibition also altered the efficiency of single AP evoked vesicle retrieval by increasing occurrence of ultrafast endocytosis, while inhibition of glycolysis had no effect. Mitochondria are sparsely distributed along hippocampal axons and we found that nerve terminals containing mitochondria displayed enhanced vesicle release and reuptake during high-frequency trains, irrespective of whether neurons were supplied with glucose or lactate. Thus, synaptic terminals can entirely bypass glycolysis to robustly maintain the vesicle cycle using oxidative fuels in the absence of glucose. These observations further suggest that mitochondrial metabolic function not only regulates several fundamental features of synaptic transmission but may also contribute to modulation of short-term synaptic plasticity.

Two forms of asynchronous release with distinctive spatiotemporal dynamics in central synapses.

Asynchronous release is a ubiquitous form of neurotransmitter release that persists for tens to hundreds of milliseconds after an action potential. How asynchronous release is organized and regulated at the synaptic active zone (AZ) remains debatable. Using nanoscale-precision imaging of individual release events in rat hippocampal synapses, we observed two spatially distinct subpopulations of asynchronous events, ~75% of which occurred inside the AZ and with a bias towards the AZ center, while ~25% occurred outside of the functionally defined AZ, that is, ectopically. The two asynchronous event subpopulations also differed from each other in temporal properties, with ectopic events occurring at significantly longer time intervals from synchronous events than the asynchronous events inside the AZ. Both forms of asynchronous release did not, to a large extent, utilize the same release sites as synchronous events. The two asynchronous event subpopulations also differ from synchronous events in some aspects of exo-endocytosis coupling, particularly in the contribution from the fast calcium-dependent endocytosis. These results identify two subpopulations of asynchronous release events with distinctive organization and spatiotemporal dynamics.

Negative self-regulation of transient receptor potential canonical 4 by the specific interaction with phospholipase C-δ1.

Transient receptor potential canonical (TRPC) channels are non-selective calcium-permeable cation channels. It is suggested that TRPC4ß is regulated by phospholipase C (PLC) signaling and is especially maintained by phosphatidylinositol 4,5-bisphosphate (PIP2). In this study, we present the regulation mechanism of the TRPC4 channel with PIP2 hydrolysis which is mediated by a channel-bound PLCδ1 but not by the GqPCR signaling pathway. Our electrophysiological recordings demonstrate that the Ca2+ via an open TRPC4 channel activates PLCδ1 in the physiological range, and it causes the decrease of current amplitude. The existence of PLCδ1 accelerated PIP2 depletion when the channel was activated by an agonist. Interestingly, PLCδ1 mutants which have lost the ability to regulate PIP2 level failed to reduce the TRPC4 current amplitude. Our results demonstrate that TRPC4 self-regulates its activity by allowing Ca2+ ions into the cell and promoting the PIP2 hydrolyzing activity of PLCδ1.

Rapid astrocyte-dependent facilitation amplifies multi-vesicular release in hippocampal synapses.

Synaptic facilitation is a major form of short-term plasticity typically driven by an increase in residual presynaptic calcium. Using near-total internal reflection fluorescence (near-TIRF) imaging of single vesicle release in cultured hippocampal synapses, we demonstrate a distinctive, release-dependent form of facilitation in which probability of vesicle release is higher following a successful glutamate release event than following a failure. This phenomenon has an onset of ≤500 ms and lasts several seconds, resulting in clusters of successful release events. The release-dependent facilitation requires neuronal contact with astrocytes and astrocytic glutamate uptake by EAAT1. It is not observed in neurons grown alone or in the presence of astrocyte-conditioned media. This form of facilitation dynamically amplifies multi-vesicular release. Facilitation-evoked release events exhibit spatial clustering and have a preferential localization toward the active zone center. These results uncover a rapid astrocyte-dependent form of facilitation acting via modulation of multi-vesicular release and displaying distinctive spatiotemporal properties.

Biophysical physiology of phosphoinositide rapid dynamics and regulation in living cells.

Phosphoinositide membrane lipids are ubiquitous low-abundance signaling molecules. They direct many physiological processes that involve ion channels, membrane identification, fusion of membrane vesicles, and vesicular endocytosis. Pools of these lipids are continually broken down and refilled in living cells, and the rates of some of these reactions are strongly accelerated by physiological stimuli. Recent biophysical experiments described here measure and model the kinetics and regulation of these lipid signals in intact cells. Rapid on-line monitoring of phosphoinositide metabolism is made possible by optical tools and electrophysiology. The experiments reviewed here reveal that as for other cellular second messengers, the dynamic turnover and lifetimes of membrane phosphoinositides are measured in seconds, controlling and timing rapid physiological responses, and the signaling is under strong metabolic regulation. The underlying mechanisms of this metabolic regulation remain questions for the future.

Compartmentalization of phosphatidylinositol 4,5-bisphosphate metabolism into plasma membrane liquid-ordered/raft domains.

Possible segregation of plasma membrane (PM) phosphoinositide metabolism in membrane lipid domains is not fully understood. We exploited two differently lipidated peptide sequences, L10 and S15, to mark liquid-ordered, cholesterol-rich (Lo) and liquid-disordered, cholesterol-poor (Ld) domains of the PM, often called raft and nonraft domains, respectively. Imaging of the fluorescent labels verified that L10 segregated into cholesterol-rich Lo phases of cooled giant plasma-membrane vesicles (GPMVs), whereas S15 and the dye FAST DiI cosegregated into cholesterol-poor Ld phases. The fluorescent protein markers were used as Förster resonance energy transfer (FRET) pairs in intact cells. An increase of homologous FRET between L10 probes showed that depleting membrane cholesterol shrank Lo domains and enlarged Ld domains, whereas a decrease of L10 FRET showed that adding more cholesterol enlarged Lo and shrank Ld Heterologous FRET signals between the lipid domain probes and phosphoinositide marker proteins suggested that phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] and phosphatidylinositol 4-phosphate (PtdIns4P) are present in both Lo and Ld domains. In kinetic analysis, muscarinic-receptor-activated phospholipase C (PLC) depleted PtdIns(4,5)P2 and PtdIns4P more rapidly and produced diacylglycerol (DAG) more rapidly in Lo than in Ld Further, PtdIns(4,5)P2 was restored more rapidly in Lo than in Ld Thus destruction and restoration of PtdIns(4,5)P2 are faster in Lo than in Ld This suggests that Lo is enriched with both the receptor G protein/PLC pathway and the PtdIns/PI4-kinase/PtdIns4P pathway. The significant kinetic differences of lipid depletion and restoration also mean that exchange of lipids between these domains is much slower than free diffusion predicts.

Phosphatidylinositol 4,5-bisphosphate is regenerated by speeding of the PI 4-kinase pathway during long PLC activation.

The dynamic metabolism of membrane phosphoinositide lipids involves several cellular compartments including the ER, Golgi, and plasma membrane. There are cycles of phosphorylation and dephosphorylation and of synthesis, transfer, and breakdown. The simplified phosphoinositide cycle comprises synthesis of phosphatidylinositol in the ER, transport, and phosphorylation in the Golgi and plasma membranes to generate phosphatidylinositol 4,5-bisphosphate, followed by receptor-stimulated hydrolysis in the plasma membrane and return of the components to the ER for reassembly. Using probes for specific lipid species, we have followed and analyzed the kinetics of several of these events during stimulation of M1 muscarinic receptors coupled to the G-protein Gq. We show that during long continued agonist action, polyphosphorylated inositol lipids are initially depleted but then regenerate while agonist is still present. Experiments and kinetic modeling reveal that the regeneration results from gradual but massive up-regulation of PI 4-kinase pathways rather than from desensitization of receptors. Golgi pools of phosphatidylinositol 4-phosphate and the lipid kinase PI4KIIIα (PI4KA) contribute to this homeostatic regeneration. This powerful acceleration, which may be at the level of enzyme activity or of precursor and product delivery, reveals strong regulatory controls in the phosphoinositide cycle.

Identification of phospholipase C ß downstream effect on transient receptor potential canonical 1/4, transient receptor potential canonical 1/5 channels.

Gαq-coupled receptor stimulation was implied in the activation process of transient receptor potential canonical (TRPC)1/4 and TRPC1/5 heterotetrameric channels. The inactivation occurs due to phosphatidylinositol 4,5-biphosphate (PI(4,5)P2) depletion. When PI(4,5)P2 depletion was induced by muscarinic stimulation or inositol polyphosphate 5-phosphatase (Inp54p), however, the inactivation by muscarinic stimulation was greater compared to that by Inp54p. The aim of this study was to investigate the complete inactivation mechanism of the heteromeric channels upon Gαq-phospholipase C ß (Gαq-PLCß) activation. We evaluated the activity of heteromeric channels with electrophysiological recording in HEK293 cells expressing TRPC channels. TRPC1/4 and TRPC1/5 heteromers undergo further inhibition in PLCß activation and calcium/protein kinase C (PKC) signaling. Nevertheless, the key factors differ. For TRPC1/4, the inactivation process was facilitated by Ca2+ release from the endoplasmic reticulum, and for TRPC1/5, activation of PKC was concerned mostly. We conclude that the subsequent increase in cytoplasmic Ca2+ due to Ca2+ release from the endoplasmic reticulum and activation of PKC resulted in a second phase of channel inhibition following PI(4,5)P2 depletion.

TRPC1 as a negative regulator for TRPC4 and TRPC5 channels.

Transient receptor potential canonical (TRPC) channels are calcium permeable, non-selective cation channels with wide tissue-specific distribution. Among 7 TRPC channels, TRPC 1/4/5 and TRPC3/6/7 are subdivided based on amino acid sequence homology. TRPC4 and TRPC5 channels exhibit cationic current with homotetrameric form, but they also form heterotetrameric channel such as TRPC1/4 or TRPC1/5 once TRPC1 is incorporated. The expression of TRPC1 is ubiquitous whereas the expressions of TRPC4 and TRPC5 are rather focused in nervous system. With the help of conditional knock-out of TPRC1, 4 and/or 5 genes, TRPC channels made of these constituents are reported to be involved in various pathophysiological functions such as seizure, anxiety-like behaviour, fear, Huntington's disease, Parkinson's disease and many others. In heterologous expression system, many issues such as activation mechanism, stoichiometry and relative cation permeabilites of homomeric or heteromeric channels have been addressed. In this review, we discussed the role of TRPC1 channel per se in plasma membrane, role of TRPC1 in heterotetrameric conformation (TRPC1/4 or TRPC1/5) and relationship between TRPC1/4/5 channels, calcium influx and voltage-gated calcium channels.

Differential PI(4,5)P2 sensitivities of TRPC4, C5 homomeric and TRPC1/4, C1/5 heteromeric channels.

Transient receptor potential canonical (TRPC) 4 and TRPC5 channels are modulated by the Gαq-PLC pathway. Since phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) maintains TRPC4 and TRPC5 channel function, the Gαq-PLC pathway inhibits channel activity by depleting PI(4,5)P2. Here we investigated the difference in PI(4,5)P2 sensitivity between homomeric and heteromeric TRPC channels. First, by using a Danio rerio voltage-sensing phosphatase (DrVSP), we show that PI(4,5)P2 dephosphorylation robustly inhibits TRPC4α, TRPC4ß, and TRPC5 homotetramer currents and also TRPC1/4α, TRPC1/4ß, and TRPC1/5 heterotetramer currents. Secondly, sensitivity of channels to PI(4,5)P2 dephosphorylation was suggested through the usage of FRET in combination with patch clamping. The sensitivity increased in the sequence TRPC4ß < TRPC4α < TRPC5 in homotetramers, whereas when forming heterotetramers with TRPC1, the sensitivity was approximately equal between the channels. Thirdly, we determined putative PI(4,5)P2 binding sites based on a TRPC4 prediction model. By neutralization of basic residues, we identified putative PI(4,5)P2 binding sites because the mutations reduced FRET to a PI(4,5)P2 sensor and reduced the current amplitude. Therefore, one functional TRPC4 has 8 pockets with the two main binding regions; K419, K664/R511, K518, H630. We conclude that TRPC1 channel function as a regulator in setting PI(4,5)P2 affinity for TRPC4 and TRPC5 that changes PI(4,5)P2 sensitivity.

Dual action of the Gαq-PLCß-PI(4,5)P2 pathway on TRPC1/4 and TRPC1/5 heterotetramers.

The transient receptor potential canonical (TRPC) 1 channel is widely distributed in mammalian cells and is involved in many physiological processes. TRPC1 is primarily considered a regulatory subunit that forms heterotetrameric channels with either TRPC4 or TRPC5 subunits. Here, we suggest that the regulation of TRPC1/4 and TRPC1/5 heterotetrameric channels by the Gαq-PLCß pathway is self-limited and dynamically mediated by Gαq and PI(4,5)P2. We provide evidence indicating that Gαq protein directly interacts with either TRPC4 or TRPC5 of the heterotetrameric channels to permit activation. Simultaneously, Gαq-coupled PLCß activation leads to the breakdown of PI(4,5)P2, which inhibits activity of TRPC1/4 and 1/5 channels.

Cervical cancer is addicted to SIRT1 disarming the AIM2 antiviral defense.

Mammalian cells are equipped with antiviral innate immunity. To survive and grow, human papilloma virus (HPV)-infected cervical cancer cells must overcome this host defense system. However, the precise mechanism whereby cervical cancer cells evade the immunity is not fully understood. We noted that Sirtuin 1 (SIRT1) is overexpressed in HPV-infected cervical cancer cells and hypothesized that SIRT1 counteracts antiviral immunity. Here, we found that cervical cancer cells undergo massive death by SIRT1 knockdown, but this effect is reversed by SIRT1 restoration. SIRT1-knocked-down cells showed representative features of pyroptosis, as well as highly expressed absent in melanoma 2 (AIM2) and its downstream genes related to the inflammasome response. Mechanistically, SIRT1 repressed the NF-κB-driven transcription of the AIM2 gene by destabilizing the RELB mRNA. Interestingly, pyroptotic death signaling in SIRT1-knocked-down cells was transmitted to naïve cervical cancer cells, which was mediated by extracellular vesicles carrying AIM2 inflammasome proteins. Furthermore, the growth of cervical cancer xenografts was significantly inhibited by either SIRT1-targeting siRNAs or SIRT1-knockdown-derived extracellular vesicles. Immunohistochemical analyses showed that SIRT1 expression correlated with poor clinical outcomes in cervical cancer. In conclusion, SIRT1 enabled HPV-infected cervical cancer cells to continue growing by nullifying AIM2 inflammasome-mediated immunity. Without SIRT1, cervical cancer cells could no longer survive because of the derepression of the AIM2 inflammasome. SIRT1 could therefore be a target for the effective treatment of cervical cancer.

Gαi-mediated TRPC4 activation by polycystin-1 contributes to endothelial function via STAT1 activation.

Hypertension and aneurysm are frequently associated with autosomal dominant polycystic kidney disease (ADPKD) caused by polycystin-1 (PC1) mutations, which is closely related to endothelial dysfunction. PC1 is an atypical G-protein-coupled receptor that activates G-proteins by self-cleavage; currently, however, the molecular and cellular mechanisms of the associated intracellular signaling and ion channel activation remain poorly elucidated. Here, we report an activation mechanism of a calcium-permeable canonical transient receptor potential 4 (TRPC4) channel by PC1 and its endothelial function. We found that the inhibitory Gαi3 protein selectively bound to the G-protein-binding domain on the C-terminus of PC1. The dissociation of Gαi3 upon cleavage of PC1 increased TRPC4 activity. Calcium influx through TRPC4 activated the transcription factor STAT1 to regulate cell proliferation and death. The down-regulation of PC1/TRPC4/STAT1 disrupted migration of endothelial cell monolayers, leading to an increase in endothelial permeability. These findings contribute to greater understanding of the high risk of aneurysm in patients with ADPKD.

The regulation of transient receptor potential canonical 4 (TRPC4) channel by phosphodiesterase 5 inhibitor via the cyclic guanosine 3'5'-monophosphate.

The transient receptor potential (TRP) protein superfamily consists of a diverse group of cation channels that bear structural similarities to the fruit fly Drosophila TRP. The TRP superfamily is distinct from other groups of ion channels in displaying a large diversity in ion selectivity, modes of activation, and physiological functions. Classical TRP (transient receptor potential canonical (TRPC)) channels are activated by stimulation of Gq-PLC-coupled receptors and modulated by phosphorylation. The cyclic guanosine monophosphate (cGMP)-PKG pathway is involved in the regulation of TRPC3 and TRPC6 channels. Phosphodiesterase (PDE) 5 inhibitor induced muscle relaxation in corporal smooth muscle cells and was used to treat erectile dysfunction by inhibiting cGMP degradation. Here, we report the functional relationship between TRPC4 and cGMP. In human embryonic kidney (HEK) 293 cells overexpressing TRPC4, cGMP selectively activated TRPC4 channels and increased cytosolic calcium level through TRPC4 channel. We investigated phosphorylation sites in TRPC4 channels and identified S688 as an important phosphorylation site for the cGMP-PKG pathway. Cyclic GMP also activated TRPC4-like current with doubly rectifying current-voltage relationship in prostate smooth muscle cell lines. Taken together, these results show that TRPC4 is phosphorylated by the cGMP-PKG pathway and might be an important target for modulating prostate function by PDE5 inhibitors.

Calcium permeability of transient receptor potential canonical (TRPC) 4 channels measured by TRPC4-GCaMP6s.

Conflicting evidence has been obtained regarding whether transient receptor potential cation channels (TRPC) are store-operated channels (SOCs) or receptor-operated channels (ROCs). Moreover, the Ca/Na permeability ratio differs depending on whether the current-voltage (I-V) curve has a doubly rectifying shape or inward rectifying shape. To investigate the calcium permeability of TRPC4 channels, we attached GCaMP6s to TRPC4 and simultaneously measured the current and calcium signals. A TRPC4 specific activator, (-)-englerin A, induced both current and calcium fluorescence with the similar time course. Muscarinic receptor stimulator, carbachol, also induced both current and calcium fluorescence with the similar time course. By forming heteromers with TRPC4, TRPC1 significantly reduced the inward current with outward rectifying I-V curve, which also caused the decrease of calcium fluorescence intensity. These results suggest that GCaMP6s attached to TRPC4 can detect slight calcium changes near TRPC4 channels. Consequently, TRPC4-GCaMP6s can be a useful tool for testing the calcium permeability of TRPC4 channels.

Helix O modulates voltage dependency of CLC-1.

The chloride channel (CLC) family of proteins consists of channels and transporters that share similarities in architecture and play essential roles in physiological functions. Among the CLC family, CLC-1 channels have the representative homodimeric double-barreled structure carrying two gating processes. One is protopore gating that acts on each pore independently by glutamate residue (Eext). The other is common gating that closes both pores simultaneously in association with large conformational changes across each subunit. In skeletal muscle, CLC-1 is associated with maintaining normal sarcolemmal excitability, and a number of myotonic mutants were reported to modify the channel gating of CLC-1. In this study, we characterized highly conserved helix O as a key determinant of structural stability in CLC-1. Supporting this hypothesis, myotonic mutant (G523D) at N-terminal of helix O showed the activation at hyperpolarizing membrane potentials with a reversed voltage dependency. However, introducing glutamate at serine residue (S537) at the C-terminal of the helix O on G523D restored WT-like voltage dependency of the common gate and showed proton insensitive voltage dependency. To further validate this significant site, site-specific mutagenesis experiments was performed on V292 that is highly conserved as glutamate in antiporter and closely located to S537 and showed that this area is essential for channel function. Taken together, the results of our study suggest the importance of helix O as the main contributor for stable structure of evolutionary conserved CLC proteins and its key role in voltage dependency of the CLC-1. Furthermore, the C-terminal of the helix O can offer a clue for possible proton involvement in CLC-1 channel.

The interaction domains of transient receptor potential canonical (TRPC)1/4 and TRPC1/5 heteromultimeric channels.

Transient receptor potential canonical (TRPC) family contains a non-selective cation channel, and four TRPC subunits form a functional tetrameric channel. TRPC4/5 channels form not only the homotetrameric channel but also a heterotetrameric channel with TRPC1. We investigated the interaction domain required for TRPC1/4 or TRPC1/5 heteromultimeric channels using FRET and the patch-clamp technique. TRPC1 only localized at the plasma membrane (PM) when it was coexpressed with TRPC4 or TRPC5. The TRPC1/4 or TRPC1/5 heteromultimeric showed the typical outward rectifying I/V curve. When TRPC1 and TRPC4 form a heteromeric channel, the N-terminal coiled-coil domain (CCD) and C-terminal 725-745 region of TRPC1 interact with the N-terminal CCD and C-terminal 700-728 region of TRPC4. However, when TRPC1 and TRPC5 form a heteromeric channel, the N-terminal CCD and C-terminal 673-725 region of TRPC1 interact with the N-terminal CCD and C-terminal 707-735 region of TRPC5. In conclusion, the N-terminal CCD of TRPC channels is essential for the heteromultimeric structure of TRPC channels, whereas specific C-terminal regions are required for unique heteromerization between subgroups of TRPC channels.

Increased TRPC5 glutathionylation contributes to striatal neuron loss in Huntington's disease.

Aberrant glutathione or Ca(2+) homeostasis due to oxidative stress is associated with the pathogenesis of neurodegenerative disorders. The Ca(2+)-permeable transient receptor potential cation (TRPC) channel is predominantly expressed in the brain, which is sensitive to oxidative stress. However, the role of the TRPC channel in neurodegeneration is not known. Here, we report a mechanism of TRPC5 activation by oxidants and the effect of glutathionylated TRPC5 on striatal neurons in Huntington's disease. Intracellular oxidized glutathione leads to TRPC5 activation via TRPC5 S-glutathionylation at Cys176/Cys178 residues. The oxidized glutathione-activated TRPC5-like current results in a sustained increase in cytosolic Ca(2+), activated calmodulin-dependent protein kinase and the calpain-caspase pathway, ultimately inducing striatal neuronal cell death. We observed an abnormal glutathione pool indicative of an oxidized state in the striatum of Huntington's disease transgenic (YAC128) mice. Increased levels of endogenous TRPC5 S-glutathionylation were observed in the striatum in both transgenic mice and patients with Huntington's disease. Both knockdown and inhibition of TRPC5 significantly attenuated oxidation-induced striatal neuronal cell death. Moreover, a TRPC5 blocker improved rearing behaviour in Huntington's disease transgenic mice and motor behavioural symptoms in littermate control mice by increasing striatal neuron survival. Notably, low levels of TRPC1 increased the formation of TRPC5 homotetramer, a highly Ca(2+)-permeable channel, and stimulated Ca(2+)-dependent apoptosis in Huntington's disease cells (STHdh(Q111/111)). Taken together, these novel findings indicate that increased TRPC5 S-glutathionylation by oxidative stress and decreased TRPC1 expression contribute to neuronal damage in the striatum and may underlie neurodegeneration in Huntington's disease.