Glutamate acts on acid-sensing ion channels to worsen ischaemic brain injury.

Glutamate is traditionally viewed as the first messenger to activate NMDAR (N-methyl-D-aspartate receptor)-dependent cell death pathways in stroke1,2, but unsuccessful clinical trials with NMDAR antagonists implicate the engagement of other mechanisms3-7. Here we show that glutamate and its structural analogues, including NMDAR antagonist L-AP5 (also known as APV), robustly potentiate currents mediated by acid-sensing ion channels (ASICs) associated with acidosis-induced neurotoxicity in stroke4. Glutamate increases the affinity of ASICs for protons and their open probability, aggravating ischaemic neurotoxicity in both in vitro and in vivo models. Site-directed mutagenesis, structure-based modelling and functional assays reveal a bona fide glutamate-binding cavity in the extracellular domain of ASIC1a. Computational drug screening identified a small molecule, LK-2, that binds to this cavity and abolishes glutamate-dependent potentiation of ASIC currents but spares NMDARs. LK-2 reduces the infarct volume and improves sensorimotor recovery in a mouse model of ischaemic stroke, reminiscent of that seen in mice with Asic1a knockout or knockout of other cation channels4-7. We conclude that glutamate functions as a positive allosteric modulator for ASICs to exacerbate neurotoxicity, and preferential targeting of the glutamate-binding site on ASICs over that on NMDARs may be strategized for developing stroke therapeutics lacking the psychotic side effects of NMDAR antagonists.

EphB2-dependent prefrontal cortex activation promotes long-range social approach and partner responsiveness.

Social behavior starts with dynamic approach prior to the final consummation. The flexible processes ensure mutual feedback across social brains to transmit signals. However, how the brain responds to the initial social stimuli precisely to elicit timed behaviors remains elusive. Here, by using real-time calcium recording, we identify the abnormalities of EphB2 mutant with autism-associated Q858X mutation in processing long-range approach and accurate activity of prefrontal cortex (dmPFC). The EphB2-dependent dmPFC activation precedes the behavioral onset and is actively associated with subsequent social action with the partner. Furthermore, we find that partner dmPFC activity is responsive coordinately to the approaching WT mouse rather than Q858X mutant mouse, and the social defects caused by the mutation are rescued by synchro-optogenetic activation in dmPFC of paired social partners. These results thus reveal that EphB2 sustains neuronal activation in the dmPFC that is essential for the proactive modulation of social approach to initial social interaction.

Dynamic tripartite construct of interregional engram circuits underlies forgetting of extinction memory.

Fear extinction allows for adaptive control of learned fear responses but often fails, resulting in a renewal or spontaneous recovery of the extinguished fear, i.e., forgetting of the extinction memory readily occurs. Using an activity-dependent neuronal labeling strategy, we demonstrate that engram neurons for fear extinction memory are dynamically positioned in the medial prefrontal cortex (mPFC), basolateral amygdala (BLA), and ventral hippocampus (vHPC), which constitute an engram construct in the term of directional engram synaptic connectivity from the BLA or vHPC to mPFC, but not that in the opposite direction, for retrieval of extinction memory. Fear renewal or spontaneous recovery switches the extinction engram construct from an accessible to inaccessible state, whereas additional extinction learning or optogenetic induction of long-term potentiation restores the directional engram connectivity and prevents the return of fear. Thus, the plasticity of engram construct underlies forgetting of extinction memory.

Neurophysiological correlate of incubation of craving in individuals with methamphetamine use disorder.

Previous studies both in laboratory animals and humans have reported that abstinence induces incubation of cue-induced drug craving for nicotine, alcohol, cocaine, and methamphetamine. However, current experimental procedures utilized to study incubation of methamphetamine craving do not incorporate the temporal dynamics of neuropsychological measures and electrophysiological activities associated with this incubation process. This study utilized the high-density electroencephalogram (EEG) signals as a rapid, inexpensive, and noninvasive measure of cue-induced craving potential. A total of 156 male individuals with methamphetamine use disorder (MUD) enrolled in this multisite, cross-sectional study. Structured clinical interview data, self-report questionnaires (cued craving, quality of sleep, impulsivity, anxiety, and depression) and resting-state, eye-closed 128 high-density channel EEG signals were collected at 5 abstinence duration time points (<1, 1-3, 3-6, 6-12, and 12-24 months) to track the neuropsychological and neurophysiological signatures. Cue-induced craving was higher after 1-3 months than after the other time points. This incubation effect was also observed for sleep quality but not for anxiety, depression, and impulsivity symptoms, along with exhibited decreased power spectrum for theta (5.5-8 Hz) and alpha (8-13 Hz), and increased in beta (16.5-26.5 Hz) frequency band. Source reconstructed resting-state EEG analysis showed increased synchronization of medial prefrontal cortex (MPFC) for the beta frequency band in 1-3 months abstinent MUD group, and associated with the incubation of craving. Remarkably, the robust incubation-related abnormalities may be driven by beta-band source space connectivity between MPFC and bilateral orbital gyrus (ORB). Our findings suggest the enhancement of beta activity in the incubation period most likely originates from a dysfunction involving frontal brain regions. This neurophysiological signature of incubation of craving can be used to identify individuals who might be most susceptible to relapse, providing a potential insight into future therapeutic interventions for MUD via neuromodulation of beta activity.

Hippocampal Lnx1-NMDAR multiprotein complex mediates initial social memory.

Social interaction and communication are evolutionary conserved behaviours that are developed in mammals to establish partner cognition. Deficit in sociability has been represented in human patients and animal models of neurodevelopmental disorders, which are connected with genetic variants of synaptic glutamate receptors and associated PDZ-binding proteins. However, it remains elusive how these key proteins are specialized in the cellular level for the initial social behaviour during postnatal developmental stage. Here we identify a hippocampal CA3 specifically expressed PDZ scaffold protein Lnx1 required for initial social behaviour. Through gene targeting we find that Lnx1 deficiency led to a hippocampal subregional disorder in neuronal activity and social memory impairments for partner discrimination observed in juvenile mice which also show cognitive defects in adult stage. We further demonstrate that Lnx1 deletion causes NMDA receptor (NMDAR) hypofunction and this is attributable to decreased GluN2B expression in PSD compartment and disruption of the Lnx1-NMDAR-EphB2 complex. Specific restoration of Lnx1 or EphB2 protein in the CA3 area of Lnx1-/- mice rescues the defective synaptic function and social memory. These findings thus reveal crucial roles of postsynaptic NMDAR multiprotein complex that regulates the formation of initial social memory during the adolescent period.

Selection of an ASIC1a-blocking combinatorial antibody that protects cells from ischemic death.

Acid-sensing ion channels (ASICs) have emerged as important, albeit challenging therapeutic targets for pain, stroke, etc. One approach to developing therapeutic agents could involve the generation of functional antibodies against these channels. To select such antibodies, we used channels assembled in nanodiscs, such that the target ASIC1a has a configuration as close as possible to its natural state in the plasma membrane. This methodology allowed selection of functional antibodies that inhibit acid-induced opening of the channel in a dose-dependent way. In addition to regulation of pH, these antibodies block the transport of cations, including calcium, thereby preventing acid-induced cell death in vitro and in vivo. As proof of concept for the use of these antibodies to modulate ion channels in vivo, we showed that they potently protect brain cells from death after an ischemic stroke. Thus, the methodology described here should be general, thereby allowing selection of antibodies to other important ASICs, such as those involved in pain, neurodegeneration, and other conditions.

Protein Kinase C Lambda Mediates Acid-Sensing Ion Channel 1a-Dependent Cortical Synaptic Plasticity and Pain Hypersensitivity.

Chronic pain is a serious debilitating disease for which effective treatment is still lacking. Acid-sensing ion channel 1a (ASIC1a) has been implicated in nociceptive processing at both peripheral and spinal neurons. However, whether ASIC1a also contributes to pain perception at the supraspinal level remains elusive. Here, we report that ASIC1a in ACC is required for thermal and mechanical hypersensitivity associated with chronic pain. ACC-specific genetic deletion or pharmacological blockade of ASIC1a reduced the probability of cortical LTP induction and attenuated inflammatory thermal hyperalgesia and mechanical allodynia in male mice. Using cell type-specific manipulations, we demonstrate that ASIC1a in excitatory neurons of ACC is a major player in cortical LTP and pain behavior. Mechanistically, we show that ASIC1a tuned pain-related cortical plasticity through protein kinase C λ-mediated increase of membrane trafficking of AMPAR subunit GluA1 in ACC. Importantly, postapplication of ASIC1a inhibitors in ACC reversed previously established nociceptive hypersensitivity in both chronic inflammatory pain and neuropathic pain models. These results suggest that ASIC1a critically contributes to a higher level of pain processing through synaptic potentiation in ACC, which may serve as a promising analgesic target for treatment of chronic pain.SIGNIFICANCE STATEMENT Chronic pain is a debilitating disease that still lacks effective therapy. Ion channels are good candidates for developing new analgesics. Here, we provide several lines of evidence to support an important role of cortically located ASIC1a channel in pain hypersensitivity through promoting long-term synaptic potentiation in the ACC. Our results indicate a promising translational potential of targeting ASIC1a to treat chronic pain.

ASIC1a channels regulate mitochondrial ion signaling and energy homeostasis in neurons.

Acid-sensing ion channel 1a (ASIC1a) is well-known to play a major pathophysiological role during brain ischemia linked to acute acidosis of ~pH 6, whereas its function during physiological brain activity, linked to much milder pH changes, is still poorly understood. Here, by performing live cell imaging utilizing Na+ and Ca2+ sensitive and spatially specific fluorescent dyes, we investigated the role of ASIC1a in cytosolic Na+ and Ca2+ signals elicited by a mild extracellular drop from pH 7.4 to 7.0 and how these affect mitochondrial Na+ and Ca2+ signaling or metabolic activity. We show that in mouse primary cortical neurons, this small extracellular pH change triggers cytosolic Na+ and Ca2+ waves that propagate to mitochondria. Inhibiting ASIC1a with Psalmotoxin 1 or ASIC1a gene knockout blocked not only the cytosolic but also the mitochondrial Na+ and Ca2+ signals. Moreover, physiological activation of ASIC1a by this pH shift enhances mitochondrial respiration and evokes mitochondrial Na+ signaling even in digitonin-permeabilized neurons. Altogether our results indicate that ASIC1a is critical in linking physiological extracellular pH stimuli to mitochondrial ion signaling and metabolic activity and thus is an important metabolic sensor.

Photocatalysis Enables Visible-Light Uncaging of Bioactive Molecules in Live Cells.

The photo-manipulation of bioactive molecules provides unique advantages due to the high temporal and spatial precision of light. The first visible-light uncaging reaction by photocatalytic deboronative hydroxylation in live cells is now demonstrated. Using Fluorescein and Rhodamine derivatives as photocatalysts and ascorbates as reductants, transient hydrogen peroxides were generated from molecular oxygen to uncage phenol, alcohol, and amine functional groups on bioactive molecules in bacteria and mammalian cells, including neurons. This effective visible-light uncaging reaction enabled the light-inducible protein expression, the photo-manipulation of membrane potentials, and the subcellular-specific photo-release of small molecules.

The nonproton ligand of acid-sensing ion channel 3 activates mollusk-specific FaNaC channels via a mechanism independent of the native FMRFamide peptide.

The degenerin/epithelial sodium channel (DEG/ENaC) superfamily of ion channels contains subfamilies with diverse functions that are fundamental to many physiological and pathological processes, ranging from synaptic transmission to epileptogenesis. The absence in mammals of some DEG/ENaCs subfamily orthologues such as FMRFamide peptide-activated sodium channels (FaNaCs), which have been identified only in mollusks, indicates that the various subfamilies diverged early in evolution. We recently reported that the nonproton agonist 2-guanidine-4-methylquinazoline (GMQ) activates acid-sensing ion channels (ASICs), a DEG/ENaC subfamily mainly in mammals, in the absence of acidosis. Here, we show that GMQ also could directly activate the mollusk-specific FaNaCs. Differences in ion selectivity and unitary conductance and effects of substitutions at key residues revealed that GMQ and FMRFamide activate FaNaCs via distinct mechanisms. The presence of two activation mechanisms in the FaNaC subfamily diverging early in the evolution of DEG/ENaCs suggested that dual gating is an ancient feature in this superfamily. Notably, the GMQ-gating mode is still preserved in the mammalian ASIC subfamily, whereas FMRFamide-mediated channel gating was lost during evolution. This implied that GMQ activation may be essential for the functions of mammalian DEG/ENaCs. Our findings provide new insights into the evolution of DEG/ENaCs and may facilitate the discovery and characterization of their endogenous agonists.

Nav1.7 is phosphorylated by Fyn tyrosine kinase which modulates channel expression and gating in a cell type-dependent manner.

Voltage-gated sodium channel Nav1.7 is a key molecule in nociception, and its dysfunction has been associated with various pain disorders. Here, we investigated the regulation of Nav1.7 biophysical properties by Fyn, an Src family tyrosine kinase. Nav1.7 was coexpressed with either constitutively active (FynCA) or dominant negative (FynDN) variants of Fyn kinase. FynCA elevated protein expression and tyrosine phosphorylation of Nav1.7 channels. Site-directed mutagenesis analysis identified two tyrosine residues (Y1470 and Y1471) located within the Nav1.7 DIII-DIV linker (L3) as phosphorylation sites of Fyn. Whole-cell recordings revealed that FynCA evoked larger changes in Nav1.7 biophysical properties when expressed in ND7/23 cells than in Human Embryonic Kidney (HEK) 293 cells, suggesting a cell type-specific modulation of Nav1.7 by Fyn kinase. In HEK 293 cells, substitution of both tyrosine residues with phenylalanine dramatically reduced current amplitude of mutant channels, which was partially rescued by expressing mutant channels in ND7/23 cells. Phenylalanine substitution showed little effect on FynCA-induced changes in Nav1.7 activation and inactivation, suggesting additional modifications in the channel or modulation by interaction with extrinsic factor(s). Our study demonstrates that Nav1.7 is a substrate for Fyn kinase, and the effect of the channel phosphorylation depends on the cell background. Fyn-mediated modulation of Nav1.7 may regulate DRG neuron excitability and contribute to pain perception. Whether this interaction could serve as a target for developing new pain therapeutics requires future study.

Acidosis counteracts itch tachyphylaxis to consecutive pruritogen exposure dependent on acid-sensing ion channel 3.

Tachyphylaxis of itch refers to a markedly reduced scratching response to consecutive exposures of a pruritogen, a process thought to protect against tissue damage by incessant scratching and to become disrupted in chronic itch. Here, we report that a strong stimulation of the Mas-related G-protein-coupled receptor C11 by its agonist, Ser-Leu-Ile-Gly-Arg-Leu-NH2 (SL-NH2) or bovine adrenal medulla 8-22 peptide, via subcutaneous injection in mice induces tachyphylaxis to the subsequent application of SL-NH2 to the same site. Notably, co-application of acid and SL-NH2 following the initial injection of the pruritogen alone counteracted itch tachyphylaxis by augmenting the scratching behaviors in wild-type but not in acid-sensing ion channel 3-null, animals. Using an activity-dependent silencing strategy, we identified that acid-sensing ion channel 3-mediated itch enhancement mainly occurred via the Mas-related G-protein-coupled receptor C11-responsive sensory neurons. Together, our results indicate that acid-sensing ion channel 3, activated by concomitant acid and certain pruritogens, constitute a novel signaling pathway that counteracts itch tachyphylaxis to successive pruritogenic stimulation, which likely contributes to chronic itch associated with tissue acidosis.

Distinct microbial communities in the active and permafrost layers on the Tibetan Plateau.

Permafrost represents an important understudied genetic resource. Soil microorganisms play important roles in regulating biogeochemical cycles and maintaining ecosystem function. However, our knowledge of patterns and drivers of permafrost microbial communities is limited over broad geographic scales. Using high-throughput Illumina sequencing, this study compared soil bacterial, archaeal and fungal communities between the active and permafrost layers on the Tibetan Plateau. Our results indicated that microbial alpha diversity was significantly higher in the active layer than in the permafrost layer with the exception of fungal Shannon-Wiener index and Simpson's diversity index, and microbial community structures were significantly different between the two layers. Our results also revealed that environmental factors such as soil fertility (soil organic carbon, dissolved organic carbon and total nitrogen contents) were the primary drivers of the beta diversity of bacterial, archaeal and fungal communities in the active layer. In contrast, environmental variables such as the mean annual precipitation and total phosphorus played dominant roles in driving the microbial beta diversity in the permafrost layer. Spatial distance was important for predicting the bacterial and archaeal beta diversity in both the active and permafrost layers, but not for fungal communities. Collectively, these results demonstrated different driving factors of microbial beta diversity between the active layer and permafrost layer, implying that the drivers of the microbial beta diversity observed in the active layer cannot be used to predict the biogeographic patterns of the microbial beta diversity in the permafrost layer.

K+ channel reorganization and homeostatic plasticity during postembryonic development: biophysical and genetic analyses in acutely dissociated Drosophila central neurons.

Intrinsic electric activities of neurons play important roles in establishing and refining neural circuits during development. However, how the underlying ionic currents undergo postembryonic reorganizations remains largely unknown. Using acutely dissociated neurons from larval, pupal, and adult Drosophila brains, we show drastic re-assemblies and compensatory regulations of voltage-gated (IKv) and Ca2+-activated (IK(Ca)) K+ currents during postembryonic development. Larval and adult neurons displayed prominent fast-inactivating IKv, mediated by the Shaker (Sh) channel to a large extent, while in the same neurons IK(Ca) was far smaller in amplitude. In contrast, pupal neurons were characterized by large sustained IKv and prominent IK(Ca), encoded predominantly by the slowpoke (slo) gene. Surprisingly, deletion of Sh in the ShM null mutant removed inactivating, transient IKv from large portions of neurons at all stages. Interestingly, elimination of Sh currents was accompanied by upregulation of non-Sh transient IKv. In comparison, the slo1 mutation abolished the vast majority of IK(Ca), particularly at the pupal stage. Strikingly, the deficiency of IK(Ca) in slo pupae was compensated by the transient component of IKv mediated by Sh channels. Thus, IK(Ca) appears to play critical roles in pupal development and its absence induces functional compensations from a specific transient IKv current. While mutants lacking either Sh or slo currents survived normally, Sh;;slo double mutants deficient in both failed to survive through pupal metamorphosis. Together, our data highlight significant reorganizations and homeostatic compensations of K+ currents during postembryonic development and uncover previously unrecognized roles for Sh and slo in this plastic process.

[Acid-sensing ion channels as a target for neuroprotection: acidotoxicity revisited].

Protons are widespread in cells and serve a variety of important functions. In certain pathological conditions, acid-base balance was disrupted and therefore excessive protons were generated and accumulated, which is termed acidosis and proved toxic to the organism. In the nervous system, it has been reported that acidosis was a common phenomenon and contributed to neuronal injury in various kinds of neurological diseases, such as ischemic stroke, multiple sclerosis and Huntington's disease. Acid-sensing ion channels (ASICs) is the key receptor of protons and mediates acidosis-induced neuronal injury, but the underlying mechanism remains unclear. Traditionally, Ca(2+) influx through homomeric ASIC1a channels has been considered to be the main cause of acidotoxicity. Recent research showed that extracellular protons trigger a novel form of necroptosis in neurons via ASIC1a-mediated serine/threonine kinase receptor interaction protein 1 (RIP1) activation, independent of ion-conducting function of ASIC1a. In addition, ASIC1a was found in mitochondria and regulated mitochondrial permeability transition-dependent neuronal death. In this article, we will review the recent progresses on the mechanisms underlying ASIC-mediated neuronal death and discuss ASIC modulators involved in this process.

SLC17A9 protein functions as a lysosomal ATP transporter and regulates cell viability.

Lysosomes contain abundant ATP, which is released through lysosomal exocytosis following exposure to various stimuli. However, the molecular mechanisms underlying lysosomal ATP accumulation remain unknown. The vesicular nucleotide transporter, also known as solute carrier family 17 member 9 (SLC17A9), has been shown to function in ATP transport across secretory vesicles/granules membrane in adrenal chromaffin cells, T cells, and pancreatic cells. Here, using mammalian cell lines, we report that SLC17A9 is highly enriched in lysosomes and functions as an ATP transporter in those organelles. SLC17A9 deficiency reduced lysosome ATP accumulation and compromised lysosome function, resulting in cell death. Our data suggest that SLC17A9 activity mediates lysosomal ATP accumulation and plays an important role in lysosomal physiology and cell viability.

Temporal dynamics of anxiety phenotypes in a dental pulp injury model.

BACKGROUND: Accumulating clinical and preclinical evidence indicates that chronic pain is often comorbid with persistent low mood and anxiety. However, the mechanisms underlying pain-induced anxiety, such as its causality, temporal progression, and relevant neural networks are poorly understood, impeding the development of efficacious therapeutic approaches. RESULTS: Here, we have identified the sequential emergence of anxiety phenotypes in mice subjected to dental pulp injury (DPI), a prototypical model of orofacial pain that correlates with human toothache. Compared with sham controls, mice subjected to DPI by mechanically exposing the pulp to the oral environment exhibited significant signs of anxiogenic effects, specifically, altered behaviors on the elevated plus maze (EPM), novelty-suppressed feeding (NSF) tests at 1 but not 3 days after the surgery. Notably, at 7 and 14 days, the DPI mice again avoided the open arm, center area, and novelty environment in the EPM, open field, and NSF tests, respectively. In particular, DPI-induced social phobia and increased repetitive grooming did not occur until 14 days after surgery, suggesting that DPI-induced social anxiety requires a long time. Moreover, oral administration of an anti-inflammatory drug, ibuprofen, or an analgesic agent, ProTx-II, which is a selective inhibitor of NaV1.7 sodium channels, both significantly alleviated DPI-induced avoidance in mice. Finally, to investigate the underlying central mechanisms, we pharmacologically blocked a popular form of synaptic plasticity with a GluA2-derived peptide, long-term depression, as that treatment significantly prevented the development of anxiety phenotype upon DPI. CONCLUSIONS: Together, these results suggest a temporally progressive causal relationship between orofacial pain and anxiety, calling for more in-depth mechanistic studies on concomitant pain and anxiety disorders.

Role of the potassium chloride cotransporter isoform 2-mediated spinal chloride homeostasis in a rat model of visceral hypersensitivity.

Visceral hypersensitivity represents an important hallmark in the pathophysiology of irritable bowel syndrome (IBS), of which the mechanisms remain elusive. The present study was designed to examine whether cation-chloride cotransporter (CCC)-mediated chloride (Cl(-)) homeostasis of the spinal cord is involved in chronic stress-induced visceral hypersensitivity. Chronic visceral hypersensitivity was induced by exposing male Wistar rats to water avoidance stress (WAS). RT-PCR, Western blotting, and immunohistochemistry were used to assess the expression of CCCs in the spinal cord. Patch-clamp recordings were performed on adult spinal cord slices to evaluate Cl(-) homeostasis and Cl(-) extrusion capacity of lamina I neurons. Visceral sensitivity was estimated by measuring the abdominal withdrawal reflex in response to colorectal distension (CRD). After 10 days of WAS exposure, levels of both total protein and the oligomeric form of the K(+)-Cl(-) cotransporter isoform 2 (KCC2), but not Na(+)-K(+)-2Cl(-) transporter isoform 1 (NKCC1), were significantly decreased in the dorsal horn of the lumbosacral spinal cord. The downregulation of KCC2 resulted in a depolarizing shifted equilibrium potential of GABAergic inhibitory postsynaptic current and impaired Cl(-) extrusion capacity in lamina I neurons of the lumbosacral spinal cord from WAS rats. Acute noxious CRD disrupted spinal KCC2 expression and function 2 h after the final distention in sham rats, but not in WAS rats. Pharmacological blockade of KCC2 activity by intrathecal injection of a KCC2 inhibitor [(dihydroindenyl)oxy] alkanoic acid enhanced visceral nociceptive sensitivity in sham rats, but not in WAS rats. These results suggest that KCC2 downregulation-mediated impairment of spinal cord Cl(-) homeostasis may play an important role in chronic stress-induced visceral hypersensitivity.

Serotonin facilitates peripheral pain sensitivity in a manner that depends on the nonproton ligand sensing domain of ASIC3 channel.

Tissue acidosis and inflammatory mediators play critical roles in inflammatory pain. Extracellular acidosis activates acid-sensing ion channels (ASICs), which have emerged as key sensors for extracellular protons in the central and peripheral nervous systems and play key roles in pain sensation and transmission. Additionally, inflammatory mediators, such as serotonin (5-HT), are known to enhance pain sensation. However, functional interactions among protons, inflammatory mediators, and ASICs in pain sensation are poorly understood. In the present study, we show that 5-HT, a classical pro-inflammatory mediator, specifically enhances the proton-evoked sustained, but not transient, currents mediated by homomeric ASIC3 channels and heteromeric ASIC3/1a and ASIC3/1b channels. Unexpectedly, the effect of 5-HT on ASIC3 channels does not involve activation of 5-HT receptors, but is mediated via a functional interaction between 5-HT and ASIC3 channels. We further show that the effect of 5-HT on ASIC3 channels depends on the newly identified nonproton ligand sensing domain. Finally, coapplication of 5-HT and acid significantly increased pain-related behaviors as assayed by the paw-licking test in mice, which was largely attenuated in ASIC3 knock-out mice, and inhibited by the nonselective ASIC inhibitor amiloride. Together, these data identify ASIC3 channels as an unexpected molecular target for acute actions of 5-HT in inflammatory pain sensation and reveal an important role of ASIC3 channels in regulating inflammatory pain via coincident detection of extracellular protons and inflammatory mediators.

Molecular mechanism of constitutive endocytosis of Acid-sensing ion channel 1a and its protective function in acidosis-induced neuronal death.

Acid-sensing ion channels (ASICs) are proton-gated cation channels widely expressed in the peripheral and CNSs, which critically contribute to a variety of pathophysiological conditions that involve tissue acidosis, such as ischemic stroke and epileptic seizures. However, the trafficking mechanisms of ASICs and the related proteins remain largely unknown. Here, we demonstrate that ASIC1a, the main ASIC subunit in the brain, undergoes constitutive endocytosis in a clathrin- and dynamin-dependent manner in both mouse cortical neurons and heterologous cell cultures. The endocytosis of ASIC1a was inhibited by either the small molecular inhibitor tyrphostin A23 or knockdown of the core subunit of adaptor protein 2 (AP2) µ2 using RNA interference, supporting a clathrin-dependent endocytosis of ASIC1a. In addition, the internalization of ASIC1a was blocked by dominant-negative dynamin1 mutation K44A and the small molecular inhibitor dynasore, suggesting that it is also dynamin-dependent. We show that the membrane-proximal residues (465)LCRRG(469) at the cytoplasmic C terminus of ASIC1a are critical for interaction with the endogenous adaptor protein complex and inhibition of ASIC1a internalization strongly exacerbated acidosis-induced death of cortical neurons from wild-type but not ASIC1a knock-out mice. Together, these results reveal the molecular mechanism of ASIC1a internalization and suggest the importance of endocytic pathway in functional regulation of ASIC1a channels as well as neuronal damages mediated by these channels during neurodegeneration.