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.

Acid-Ion Sensing Channel 1a Deletion Reduces Chronic Brain Damage and Neurological Deficits after Experimental Traumatic Brain Injury.

Traumatic brain injury (TBI) causes long-lasting neurodegeneration and cognitive impairments; however, the underlying mechanisms of these processes are not fully understood. Acid-sensing ion channels 1a (ASIC1a) are voltage-gated Na+- and Ca2+-channels shown to be involved in neuronal cell death; however, their role for chronic post-traumatic brain damage is largely unknown. To address this issue, we used ASIC1a-deficient mice and investigated their outcome up to 6 months after TBI. ASIC1a-deficient mice and their wild-type (WT) littermates were subjected to controlled cortical impact (CCI) or sham surgery. Brain water content was analyzed 24 h and behavioral outcome up to 6 months after CCI. Lesion volume was assessed longitudinally by magnetic resonance imaging and 6 months after injury by histology. Brain water content was significantly reduced in ASIC1a-/- animals compared to WT controls. Over time, ASIC1a-/- mice showed significantly reduced lesion volume and reduced hippocampal damage. This translated into improved cognitive function and reduced depression-like behavior. Microglial activation was significantly reduced in ASIC1a-/- mice. In conclusion, ASIC1a deficiency resulted in reduced edema formation acutely after TBI and less brain damage, functional impairments, and neuroinflammation up to 6 months after injury. Hence, ASIC1a seems to be involved in chronic neurodegeneration after TBI.

Deletion of Acid-Sensing Ion Channel 3 Relieves the Late Phase of Neuropathic Pain by Preventing Neuron Degeneration and Promoting Neuron Repair.

Neuropathic pain is one type of chronic pain that occurs as a result of a lesion or disease to the somatosensory nervous system. Chronic excessive inflammatory response after nerve injury may contribute to the maintenance of persistent pain. Although the role of inflammatory mediators and cytokines in mediating allodynia and hyperalgesia has been extensively studied, the detailed mechanisms of persistent pain or whether the interactions between neurons, glia and immune cells are essential for maintenance of the chronic state have not been completely elucidated. ASIC3, a voltage-insensitive, proton-gated cation channel, is the most essential pH sensor for pain perception. ASIC3 gene expression is increased in dorsal root ganglion neurons after inflammation and nerve injury and ASIC3 is involved in macrophage maturation. ASIC currents are increased after nerve injury. However, whether prolonged hyperalgesia induced by the nerve injury requires ASIC3 and whether ASIC3 regulates neurons, immune cells or glial cells to modulate neuropathic pain remains unknown. We established a model of chronic constriction injury of the sciatic nerve (CCI) in mice. CCI mice showed long-lasting mechanical allodynia and thermal hyperalgesia. CCI also caused long-term inflammation at the sciatic nerve and primary sensory neuron degeneration as well as increased satellite glial expression and ATF3 expression. ASIC3 deficiency shortened mechanical allodynia and attenuated thermal hyperalgesia. ASIC3 gene deletion shifted ATF3 expression from large to small neurons and altered the M1/M2 macrophage ratio, thereby preventing small neuron degeneration and relieved pain.

Acid-Sensing Ion Channels: Novel Mediators of Cerebral Vascular Responses.

RATIONALE: Precise regulation of cerebral blood flow is critical for normal brain function. Insufficient cerebral blood flow contributes to brain dysfunction and neurodegeneration. Carbon dioxide (CO2), via effects on local acidosis, is one of the most potent regulators of cerebral blood flow. Although a role for nitric oxide in intermediate signaling has been implicated, mechanisms that initiate CO2-induced vasodilation remain unclear. OBJECTIVE: Acid-sensing ion channel-1A (ASIC1A) is a proton-gated cation channel that is activated by extracellular acidosis. Based on work that implicated ASIC1A in the amygdala and bed nucleus of the stria terminalis in CO2-evoked and acid-evoked behaviors, we hypothesized that ASIC1A might also mediate microvascular responses to CO2. METHODS AND RESULTS: To test this hypothesis, we genetically and pharmacologically manipulated ASIC1A and assessed effects on CO2-induced dilation of cerebral arterioles in vivo. Effects of inhalation of 5% or 10% CO2 on arteriolar diameter were greatly attenuated in mice with global deficiency in ASIC1A (Asic1a-/-) or by local treatment with the ASIC inhibitor, psalmotoxin. Vasodilator effects of acetylcholine, which acts via endothelial nitric oxide synthase were unaffected, suggesting a nonvascular source of nitric oxide may be key for CO2 responses. Thus, we tested whether neurons may be the cell type through which ASIC1A influences microvessels. Using mice in which Asic1a was specifically disrupted in neurons, we found effects of CO2 on arteriolar diameter were also attenuated. CONCLUSIONS: Together, these data are consistent with a model wherein activation of ASIC1A, particularly in neurons, is critical for CO2-induced nitric oxide production and vasodilation. With these findings, ASIC1A emerges as major regulator of microvascular tone.

Effect of knockout of the ASIC3 on cardiovascular reflexes arising from hindlimb muscle in decerebrated rats.

The exercise pressor reflex is initiated by the contraction-induced activation of group III and IV muscle afferents. The reflex is manifested by increases in arterial blood pressure and cardiac output, which, in turn, are generated by increases in the sympathetic outflow to the heart and vasculature and decreases in the vagal outflow to the heart. In previous experiments, we used a pharmacological approach to assess the role played by the acid-sensing ion channel 3 (ASIC3) on group III and IV afferents in evoking the exercise pressor reflex. In the present experiments, we used an alternative approach, namely functional knockout (KO) of the ASIC3 gene, to confirm and extend our previous finding that pharmacological blockade of the ASIC3 had only a small impact on the expression of the exercise pressor reflex when the arterial supply to the contracting hindlimb muscles of rats was patent. Using this alternative approach, we compared the magnitude of the exercise pressor reflex evoked in ASIC3 KO rats with that evoked in their wild-type (WT) counterparts. We found both WT and ASIC3 KO rats displayed similar pressor responses to static contraction (WT, n = 10, +12 ± 2 mmHg; KO, n = 9, +11 ± 2 mmHg) and calcaneal tendon stretch (WT, n = 9, +13 ± 2 mmHg; KO, n = 7, +11 ± 2 mmHg). Likewise, both WT and ASIC3 KO displayed similar pressor responses to intra-arterial injection of 12 mM lactic acid (WT, n = 9, +14 ± 3 mmHg; KO, n = 8, +18 ± 5 mmHg), 24 mM lactic acid (WT, n = 9,+24 ± 2 mmHg; KO, n = 8, +20 ± 5 mmHg), capsaicin (WT, n = 9,+27 ± 5 mmHg; KO, n = 10, +29 ± 5 mmHg), and diprotonated phosphate ([Formula: see text]; WT, n = 6,+22 ± 3 mmHg; KO, n = 6, +32 ± 6 mmHg). We conclude that redundant receptors are responsible for evoking the pressor reflexes arising from group III and IV afferents.

Role of acid-sensing ion channels in hypoxia- and hypercapnia-induced ventilatory responses.

Previous reports indicate roles for acid-sensing ion channels (ASICs) in both peripheral and central chemoreception, but the contributions of ASICs to ventilatory drive in conscious, unrestrained animals remain largely unknown. We tested the hypotheses that ASICs contribute to hypoxic- and hypercapnic-ventilatory responses. Blood samples taken from conscious, unrestrained mice chronically instrumented with femoral artery catheters were used to assess arterial O2, CO2, and pH levels during exposure to inspired gas mixtures designed to cause isocapnic hypoxemia or hypercapnia. Whole-body plethysmography was used to monitor ventilatory parameters in conscious, unrestrained ASIC1, ASIC2, or ASIC3 knockout (-/-) and wild-type (WT) mice at baseline, during isocapnic hypoxemia and during hypercapnia. Hypercapnia increased respiratory frequency, tidal volume, and minute ventilation in all groups of mice, but there were no differences between ASIC1-/-, ASIC2-/-, or ASIC3-/- and WT. Isocapnic hypoxemia also increased respiratory frequency, tidal volume, and minute ventilation in all groups of mice. Minute ventilation in ASIC2-/- mice during isocapnic hypoxemia was significantly lower compared to WT, but there were no differences in the responses to isocapnic hypoxemia between ASIC1-/- or ASIC3-/- compared to WT. Surprisingly, these findings show that loss of individual ASIC subunits does not substantially alter hypercapnic or hypoxic ventilatory responses.

RhoA increases ASIC1a plasma membrane localization and calcium influx in pulmonary arterial smooth muscle cells following chronic hypoxia.

Increases in pulmonary arterial smooth muscle cell (PASMC) intracellular Ca2+ levels and enhanced RhoA/Rho kinase-dependent Ca2+ sensitization are key determinants of PASMC contraction, migration, and proliferation accompanying the development of hypoxic pulmonary hypertension. We previously showed that acid-sensing ion channel 1a (ASIC1a)-mediated Ca2+ entry in PASMC is an important constituent of the active vasoconstriction, vascular remodeling, and right ventricular hypertrophy associated with hypoxic pulmonary hypertension. However, the enhanced ASIC1a-mediated store-operated Ca2+ entry in PASMC from pulmonary hypertensive animals is not dependent on an increase in ASIC1a protein expression, suggesting that chronic hypoxia (CH) stimulates ASIC1a function through other regulatory mechanism(s). RhoA is involved in ion channel trafficking, and levels of activated RhoA are increased following CH. Therefore, we hypothesize that activation of RhoA following CH increases ASIC1a-mediated Ca2+ entry by promoting ASIC1a plasma membrane localization. Consistent with our hypothesis, we found greater plasma membrane localization of ASIC1a following CH. Inhibition of RhoA decreased ASIC1a plasma membrane expression and largely diminished ASIC1a-mediated Ca2+ influx, whereas activation of RhoA had the opposite effect. A proximity ligation assay revealed that ASIC1a and RhoA colocalize in PASMC and that the activation state of RhoA modulates this interaction. Together, our findings show a novel interaction between RhoA and ASIC1a, such that activation of RhoA in PASMC, both pharmacologically and via CH, promotes ASIC1a plasma membrane localization and Ca2+ entry. In addition to enhanced RhoA-mediated Ca2+ sensitization following CH, RhoA can also activate a Ca2+ signal by facilitating ASIC1a plasma membrane localization and Ca2+ influx in pulmonary hypertension.

ASIC1a regulates insular long-term depression and is required for the extinction of conditioned taste aversion.

Acid-sensing ion channel 1a (ASIC1a) has been shown to play important roles in synaptic plasticity, learning and memory. Here we identify a crucial role for ASIC1a in long-term depression (LTD) at mouse insular synapses. Genetic ablation and pharmacological inhibition of ASIC1a reduced the induction probability of LTD without affecting that of long-term potentiation in the insular cortex. The disruption of ASIC1a also attenuated the extinction of established taste aversion memory without altering the initial associative taste learning or its long-term retention. Extinction of taste aversive memory led to the reduced insular synaptic efficacy, which precluded further LTD induction. The impaired LTD and extinction learning in ASIC1a null mice were restored by virus-mediated expression of wild-type ASIC1a, but not its ion-impermeable mutant, in the insular cortices. Our data demonstrate the involvement of an ASIC1a-mediated insular synaptic depression mechanism in extinction learning, which raises the possibility of targeting ASIC1a to manage adaptive behaviours.

ASIC1a Deficient Mice Show Unaltered Neurodegeneration in the Subacute MPTP Model of Parkinson Disease.

Inflammation contributes to the death of dopaminergic neurons in Parkinson disease and can be accompanied by acidification of extracellular pH, which may activate acid-sensing ion channels (ASIC). Accordingly, amiloride, a non-selective inhibitor of ASIC, was protective in an acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson disease. To complement these findings we determined MPTP toxicity in mice deficient for ASIC1a, the most common ASIC isoform in neurons. MPTP was applied i.p. in doses of 30 mg per kg on five consecutive days. We determined the number of dopaminergic neurons in the substantia nigra, assayed by stereological counting 14 days after the last MPTP injection, the number of Nissl positive neurons in the substantia nigra, and the concentration of catecholamines in the striatum. There was no difference between ASIC1a-deficient mice and wildtype controls. We are therefore not able to confirm that ASIC1a are involved in MPTP toxicity. The difference might relate to the subacute MPTP model we used, which more closely resembles the pathogenesis of Parkinson disease, or to further targets of amiloride.

Evidence for the involvement of ASIC3 in sensory mechanotransduction in proprioceptors.

Acid-sensing ion channel 3 (ASIC3) is involved in acid nociception, but its possible role in neurosensory mechanotransduction is disputed. We report here the generation of Asic3-knockout/eGFPf-knockin mice and subsequent characterization of heterogeneous expression of ASIC3 in the dorsal root ganglion (DRG). ASIC3 is expressed in parvalbumin (Pv+) proprioceptor axons innervating muscle spindles. We further generate a floxed allele of Asic3 (Asic3(f/f)) and probe the role of ASIC3 in mechanotransduction in neurite-bearing Pv+ DRG neurons through localized elastic matrix movements and electrophysiology. Targeted knockout of Asic3 disrupts spindle afferent sensitivity to dynamic stimuli and impairs mechanotransduction in Pv+ DRG neurons because of substrate deformation-induced neurite stretching, but not to direct neurite indentation. In behavioural tasks, global knockout (Asic3(-/-)) and Pv-Cre::Asic3(f/f) mice produce similar deficits in grid and balance beam walking tasks. We conclude that, at least in mouse, ASIC3 is a molecular determinant contributing to dynamic mechanosensitivity in proprioceptors.

Acid-sensing ion channels in trigeminal ganglion neurons innervating the orofacial region contribute to orofacial inflammatory pain.

Orofacial pain is a common clinical symptom that is accompanied by tooth pain, migraine and gingivitis. Accumulating evidence suggests that acid-sensing ion channels (ASICs), especially ASIC3, can profoundly affect the physiological properties of nociception in peripheral sensory neurons. The aim of this study is to examine the contribution of ASICs in trigeminal ganglion (TG) neurons to orofacial inflammatory pain. A Western blot (WB), immunofluorescence assay of labelled trigeminal ganglion neurons, orofacial formalin test, cell preparation and electrophysiological experiments are performed. This study demonstrated that ASIC1, ASIC2a and ASIC3 are highly expressed in TG neurons innervating the orofacial region of rats. The amplitude of ASIC currents in these neurons increased 119.72% (for ASIC1-like current) and 230.59% (for ASIC3-like current) in the formalin-induced orofacial inflammatory pain model. In addition, WB and immunofluorescence assay demonstrated a significantly augmented expression of ASICs in orofacial TG neurons during orofacial inflammation compared with the control group. The relative protein density of ASIC1, ASIC2a and ASIC3 also increased 58.82 ± 8.92%, 45.30 ± 11.42% and 55.32 ± 14.71%, respectively, compared with the control group. Furthermore, pharmacological blockade of ASICs and genetic deletion of ASIC1 attenuated the inflammation response. These findings indicate that peripheral inflammation can induce the upregulation of ASICs in TG neurons, causing orofacial inflammatory pain. Additionally, the specific inhibitor of ASICs may have a significant analgesic effect on orofacial inflammatory pain.

Altered myogenic vasoconstriction and regulation of whole kidney blood flow in the ASIC2 knockout mouse.

Previous studies from our laboratory have suggested that degenerin proteins contribute to myogenic constriction, a mechanism of blood flow regulation and protection against pressure-dependent organ injury, in renal vessels. The goal of the present study was to determine the importance of one family member, acid-sensing ion channel 2 (ASIC2), in myogenic constriction of renal interlobar arteries, myogenic regulation of whole kidney blood flow, renal injury, and blood pressure using ASIC2(+/+), ASIC2(+/-), and ASIC2(-/-) mice. Myogenic constriction in renal interlobar arteries was impaired in ASIC2(+/-) and ASIC2(-/-) mice, whereas constriction to KCl/phenylephrine was unchanged. Correction of whole kidney renal vascular resistance (RVR) during the first 5 s after a 10- to 20-mmHg step increase in perfusion pressure, a timeframe associated with myogenic-mediated correction of RVR, was slowed (4.2 ± 0.9, 0.3 ± 0.7, and 2.4 ± 0.3 resistance units/s in ASIC2(+/+), ASIC2(+/-), and ASIC2(-/-) mice). Although modest reductions in function were observed in ASIC2(-/-) mice, greater reductions were observed in ASIC2(+/-) mice, which may be explained by protein-protein interactions of ASIC2 with other degenerins. Isolated glomeruli from ASIC2(+/-) and ASIC2(-/-) mice had modest alterations in the expression of inflammation and injury markers (transforming growth factor-ß, mouse anti-target of antiproliferative antibody-1, and nephrin), whereas ASIC2(+/-) mice had an increase in the remodeling marker collagen type III. Consistent with a more severe loss of function, mean arterial pressure was increased in ASIC2(+/-) mice (131 ± 3 mmHg) but not in ASIC2(-/-) mice (122 ± 3 vs. 117 ± 2 mmHg in ASIC2(+/+) mice). These results suggest that ASIC2 contributes to transduction of the renal myogenic response and are consistent with the protective role of myogenic constriction against renal injury and hypertension.

The bed nucleus of the stria terminalis is critical for anxiety-related behavior evoked by CO2 and acidosis.

Carbon dioxide (CO2) inhalation lowers brain pH and induces anxiety, fear, and panic responses in humans. In mice, CO2 produces freezing and avoidance behavior that has been suggested to depend on the amygdala. However, a recent study in humans with bilateral amygdala lesions revealed that CO2 can trigger fear and panic even in the absence of amygdalae, suggesting the importance of extra-amygdalar brain structures. Because the bed nucleus of the stria terminalis (BNST) contributes to fear- and anxiety-related behaviors and expresses acid-sensing ion channel-1A (ASIC1A), we hypothesized that the BNST plays an important role in CO2-evoked fear-related behaviors in mice. We found that BNST lesions decreased both CO2-evoked freezing and CO2-conditioned place avoidance. In addition, we found that CO2 inhalation caused BNST acidosis and that acidosis was sufficient to depolarize BNST neurons and induce freezing behavior; both responses depended on ASIC1A. Finally, disrupting Asic1a specifically in the BNST reduced CO2-evoked freezing, whereas virus-vector-mediated expression of ASIC1A in the BNST of Asic1a(-/-) and Asic1a(+/+) mice increased CO2-evoked freezing. Together, these findings identify the BNST as an extra-amygdalar fear circuit structure important in CO2-evoked fear-related behavior.

Acid-sensing ion channels contribute to synaptic transmission and inhibit cocaine-evoked plasticity.

Acid-sensing ion channel 1A (ASIC1A) is abundant in the nucleus accumbens (NAc), a region known for its role in addiction. Because ASIC1A has been suggested to promote associative learning, we hypothesized that disrupting ASIC1A in the NAc would reduce drug-associated learning and memory. However, contrary to this hypothesis, we found that disrupting ASIC1A in the mouse NAc increased cocaine-conditioned place preference, suggesting an unexpected role for ASIC1A in addiction-related behavior. Moreover, overexpressing ASIC1A in rat NAc reduced cocaine self-administration. Investigating the underlying mechanisms, we identified a previously unknown postsynaptic current during neurotransmission that was mediated by ASIC1A and ASIC2 and thus well positioned to regulate synapse structure and function. Consistent with this possibility, disrupting ASIC1A altered dendritic spine density and glutamate receptor function, and increased cocaine-evoked plasticity, which resemble changes previously associated with cocaine-induced behavior. Together, these data suggest that ASIC1A inhibits the plasticity underlying addiction-related behavior and raise the possibility of developing therapies for drug addiction by targeting ASIC-dependent neurotransmission.

Knockdown of acid-sensing ion channel 1a (ASIC1a) suppresses disease phenotype in SCA1 mouse model.

The mutated ataxin-1 protein in spinocerebellar ataxia 1 (SCA1) targets Purkinje cells (PCs) of the cerebellum and causes progressive ataxia due to loss of PCs and neurons of the brainstem. The exact mechanism of this cellular loss is still not clear. Currently, there are no treatments for SCA1; however, understanding of the mechanisms that regulate SCA1 pathology is essential for devising new therapies for SCA1 patients. We previously established a connection between the loss of intracellular calcium-buffering and calcium-signalling proteins with initiation of neurodegeneration in SCA1 transgenic (Tg) mice. Recently, acid-sensing ion channel 1a (ASIC1a) have been implicated in calcium-mediated toxicity in many brain disorders. Here, we report generating SCA1 Tg mice in the ASIC1a knockout (KO) background and demonstrate that the deletion of ASIC1a gene expression causes suppression of the SCA1 disease phenotype. Loss of the ASIC1a channel in SCA1/ASIC1a KO mice resulted in the improvement of motor deficit and decreased PC degeneration. Interestingly, the expression of the ASIC1 variant, ASIC1b, was upregulated in the cerebellum of both SCA1/ASIC1a KO and ASIC1a KO animals as compared to the wild-type (WT) and SCA1 Tg mice. Further, these SCA1/ASIC1a KO mice exhibited translocation of PC calcium-binding protein calbindin-D28k from the nucleus to the cytosol in young animals, which otherwise have both cytosolic and nuclear localization. Furthermore, in addition to higher expression of calcium-buffering protein parvalbumin, PCs of the older SCA1/ASIC1a KO mice showed a decrease in morphologic abnormalities as compared to the age-matched SCA1 animals. Our data suggest that ASIC1a may be a mediator of SCA1 pathogenesis and targeting ASIC1a could be a novel approach to treat SCA1.

Acid-sensing ion channels 1a (ASIC1a) inhibit neuromuscular transmission in female mice.

Acid-sensing ion channels (ASIC) open in response to extracellular acidosis. ASIC1a, a particular subtype of these channels, has been described to have a postsynaptic distribution in the brain, being involved not only in ischemia and epilepsy, but also in fear and psychiatric pathologies. High-frequency stimulation of skeletal motor nerve terminals (MNTs) can induce presynaptic pH changes in combination with an acidification of the synaptic cleft, known to contribute to muscle fatigue. Here, we studied the role of ASIC1a channels on neuromuscular transmission. We combined a behavioral wire hanging test with electrophysiology, pharmacological, and immunofluorescence techniques to compare wild-type and ASIC1a lacking mice (ASIC1a (-/-) knockout). Our results showed that 1) ASIC1a (-/-) female mice were weaker than wild type, presenting shorter times during the wire hanging test; 2) spontaneous neurotransmitter release was reduced by ASIC1a activation, suggesting a presynaptic location of these channels at individual MNTs; 3) ASIC1a-mediated effects were emulated by extracellular local application of acid saline solutions (pH = 6.0; HEPES/MES-based solution); and 4) immunofluorescence techniques revealed the presence of ASIC1a antigens on MNTs. These results suggest that ASIC1a channels might be involved in controlling neuromuscular transmission, muscle contraction and fatigue in female mice.

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.

Acid-sensing ion channel 3 deficiency increases inflammation but decreases pain behavior in murine arthritis.

OBJECTIVE: Through its location on nociceptors, acid-sensing ion channel 3 (ASIC-3) is activated by decreases in pH and plays a significant role in musculoskeletal pain. We recently showed that decreases in pH activate ASIC-3 located on fibroblast-like synoviocytes (FLS), which are key cells in the inflammatory process. The purpose of this study was to test whether ASIC-3-deficient mice with arthritis have altered inflammation and pain relative to controls. METHODS: Collagen antibody-induced arthritis (CAIA) was generated by injection of an anti-type II collagen antibody cocktail. Inflammation and pain parameters in ASIC-3(-/-) and ASIC-3(+/+) mice were assessed. Disease severity was assessed by determining clinical arthritis scores, measuring joint diameters, analyzing joint histology, and assessing synovial gene expression by quantitative polymerase chain reaction analysis. Cell death was assessed with a Live/Dead assay of FLS in response to decreases in pH. Pain behaviors in the mice were measured by examining withdrawal thresholds in the joints and paws and by measuring their physical activity levels. RESULTS: Surprisingly, ASIC-3(-/-) mice with CAIA demonstrated significantly increased joint inflammation, joint destruction, and expression of interleukin-6 (IL-6), matrix metalloproteinase 3 (MMP-3), and MMP-13 in joint tissue as compared to ASIC-3(+/+) mice. ASIC-3(+/+) FLS showed enhanced cell death when exposed to pH 6.0 in the presence of IL-1ß, which was abolished in ASIC-3(-/-) FLS. Despite enhanced disease severity, ASIC-3(-/-) mice did not develop mechanical hypersensitivity of the paw and showed greater levels of physical activity. CONCLUSION: Our findings are consistent with the hypothesis that ASIC-3 plays a protective role in the inflammatory arthritides by limiting inflammation through enhanced synoviocyte cell death, which reduces disease severity, and through the production of pain, which reduces joint use.

Acid-sensing ion channels (ASICs) in mouse skeletal muscle afferents are heteromers composed of ASIC1a, ASIC2, and ASIC3 subunits.

Acid-sensing ion channels (ASICs) are expressed in skeletal muscle afferents, in which they sense extracellular acidosis and other metabolites released during ischemia and exercise. ASICs are formed as homotrimers or heterotrimers of several isoforms (ASIC1a, ASIC1b, ASIC2a, ASIC2b, and ASIC3), with each channel displaying distinct properties. To dissect the ASIC composition in muscle afferents, we used whole-cell patch-clamp recordings to study the properties of acid-evoked currents (amplitude, pH sensitivity, the kinetics of desensitization and recovery from desensitization, and pharmacological modulation) in isolated, labeled mouse muscle afferents from wild-type (C57BL/6J) and specific ASIC(-/-) mice. We found that ASIC-like currents in wild-type muscle afferents displayed fast desensitization, indicating that they are carried by heteromeric channels. Currents from ASIC1a(-/-) muscle afferents were less pH-sensitive and displayed faster recovery, currents from ASIC2(-/-) mice showed diminished potentiation by zinc, and currents from ASIC3(-/-) mice displayed slower desensitization than those from wild-type mice. Finally, ASIC-like currents were absent from triple-null mice lacking ASIC1a, ASIC2a, and ASIC3. We conclude that ASIC1a, ASIC2a, and ASIC3 heteromers are the principle channels in skeletal muscle afferents. These results will help us understand the role of ASICs in exercise physiology and provide a molecular target for potential drug therapies to treat muscle pain.

Asic3 is a neuronal mechanosensor for pressure-induced vasodilation that protects against pressure ulcers.

Pressure-induced vasodilation (PIV) delays the decrease in cutaneous blood flow produced by local application of low pressure to the skin, a physiologically appropriate adjustment of local vasomotor function. Individuals without a normal PIV response have a high risk of ulceration. Here we demonstrate that acid-sensing ion channel 3 (Asic3) is an essential neuronal sensor for the vasodilation response to direct pressure in both humans and rodents and for protecting against pressure ulcers in mice.