ASIC3 plays a protective role in delayed-onset muscle soreness (DOMS) through muscle acid sensation during exercise.

Immediate exercise-induced pain (IEIP) and DOMS are two types of exercise-induced muscle pain and can act as barriers to exercise. The burning sensation of IEIP occurs during and immediately after intensive exercise, whereas the soreness of DOMS occurs later. Acid-sensing ion channels (ASICs) within muscle afferents are activated by H+ and other chemicals and have been shown to play a role in various chronic muscle pain conditions. Here, we further defined the role of ASICs in IEIP, and also tested if ASIC3 is required for DOMS. After undergoing exhaustive treadmill exercise, exercise-induced muscle pain was assessed in wild-type (WT) and ASIC3-/- mice at baseline via muscle withdrawal threshold (MWT), immediately, and 24 h after exercise. Locomotor movement, grip strength, and repeat exercise performance were tested at baseline and 24 h after exercise to evaluate DOMS. We found that ASIC3-/- had similar baseline muscle pain, locomotor activity, grip strength, and exercise performance as WT mice. WT showed diminished MWT immediately after exercise indicating they developed IEIP, but ASIC3-/- mice did not. At 24 h after baseline exercise, both ASIC3-/- and WT had similarly lower MWT and grip strength, however, ASIC3-/- displayed significantly lower locomotor activity and repeat exercise performance at 24 h time points compared to WT. In addition, ASIC3-/- mice had higher muscle injury as measured by serum lactate dehydrogenase and creatine kinase levels at 24 h after exercise. These results show that ASIC3 is required for IEIP, but not DOMS, and in fact might play a protective role to prevent muscle injury associated with strenuous exercise.

Mitral Valve Echodensities in a Young-Adult Female with Relapsing Polychondritis, Transiently Positive Lupus Anticoagulant, and Systemic Embolism.

Background: Valvular strands seen on echocardiography carry a wide differential diagnosis and may not always have a clear etiology despite taking clinical context into account. The decision of whether to provide anticoagulation for these lesions can be challenging. Case Presentation. A young adult female with an extensive rheumatologic history involving relapsing polychondritis and positive lupus anticoagulant presents to the emergency department with a discolored and painful right toe, as well as right auricular pain and swelling. Initial work-up revealed a possible splenic infarct, vasculitis of the right lower extremity, and mitral valve echodensities on echocardiography, without evidence of infective endocarditis. Due to concern that nonbacterial thrombotic endocarditis may be the cause of the patient's thromboembolic event, her valvular lesions were treated with low molecular weight heparin while awaiting serial imaging. When follow-up echocardiography showed no change in the size of her mitral valve lesions, which would be most consistent with Lambl's excrescences, the care team still faced a decision about which long-term anticoagulation to prescribe. This patient of childbearing age wished to avoid the teratogenicity and long-term monitoring associated with warfarin therapy. Although warfarin was the preferred agent for the patient's rheumatologic comorbidities, she elected to receive enoxaparin therapy for long-term thromboembolism prophylaxis. Conclusions: Even when accounting for clinical context, valvular lesions seen on echocardiography often have uncertain etiology and may require time and serial imaging to determine which treatment to pursue. When long-term anticoagulation is provided for females of childbearing age, shared decision-making with consideration of the patient's personal priorities and comorbidities is essential.

Sleep, circadian rhythm characteristics, and melatonin levels in later life adults with and without coronary artery disease.

STUDY OBJECTIVES: The purpose of this study was to conduct a comprehensive assessment of sleep and circadian rhythms in individuals with and without coronary artery disease (CAD). METHODS: This was a cross-sectional study. Participants were 32 individuals, mean age = 70.9, female 46.9%, 19 with CAD, and 13 without CAD. We assessed sleep quality and 24-hour rest-activity rhythms for 14 days using wrist actigraphy and self-report measures, and circadian rhythm using dim light melatonin onset. RESULTS: Melatonin levels prior to habitual bedtime were significantly lower in individuals with CAD than in those without CAD (median area under the curve = 12.88 vs 26.33 pg/ml × h, P = .049). The median circadian timing measured by dim light melatonin onset was the same for the 2 groups with 20:26 [hours:minutes] for individuals with CAD and 19:53 for the control group (P = .64, r = .14). Compared to the control group, the CAD group had significantly lower amplitude (P = .03, r =-.48), and lower overall rhythmicity (pseudo-F-statistic P = .004, r = -.65) in their 24-hour rest-activity rhythms. CONCLUSIONS: This is one of the first studies to comprehensively assess both sleep and circadian rhythm in individuals with CAD. Compared to non-CAD controls, individuals with CAD had lower levels of melatonin prior to habitual bedtime and a lower 24-hour rest-activity rhythm amplitude and overall rhythmicity. Future studies using larger sample sizes should further investigate the possibility of suppressed circadian rhythmicity in individuals with CAD. CITATION: Moon C, Benson CJ, Albashayreh A, Perkhounkova Y, Burgess HJ. Sleep, circadian rhythm characteristics, and melatonin levels in later life adults with and without coronary artery disease. J Clin Sleep Med. 2023;19(2):283-292.

Paradoxical Potentiation of Acid-Sensing Ion Channel 3 (ASIC3) by Amiloride via Multiple Mechanisms and Sites Within the Channel.

Acid-Sensing Ion Channels (ASICs) are proton-gated sodium-selective cation channels that have emerged as metabolic and pain sensors in peripheral sensory neurons and contribute to neurotransmission in the CNS. These channels and their related degenerin/epithelial sodium channel (DEG/ENaC) family are often characterized by their sensitivity to amiloride inhibition. However, amiloride can also cause paradoxical potentiation of ASIC currents under certain conditions. Here we characterized and investigated the determinants of paradoxical potentiation by amiloride on ASIC3 channels. While inhibiting currents induced by acidic pH, amiloride potentiated sustained currents at neutral pH activation. These effects were accompanied by alterations in gating properties including (1) an alkaline shift of pH-dependent activation, (2) inhibition of pH-dependent steady-state desensitization (SSD), (3) prolongation of desensitization kinetics, and (4) speeding of recovery from desensitization. Interestingly, extracellular Ca2+ was required for paradoxical potentiation and it diminishes the amiloride-induced inhibition of SSD. Site-directed mutagenesis within the extracellular non-proton ligand-sensing domain (E79A, E423A) demonstrated that these residues were critical in mediating the amiloride-induced inhibition of SSD. However, disruption of the purported amiloride binding site (G445C) within the channel pore blunted both the inhibition and potentiation of amiloride. Together, our results suggest that the myriad of modulatory and blocking effects of amiloride are the result of a complex competitive interaction between amiloride, Ca2+, and protons at probably more than one site in the channel.

Comparison of murine behavioural and physiological responses after forced exercise by electrical shock versus manual prodding.

NEW FINDINGS: What is the central question of this study? Forced treadmill exercise using electrical shock is the most common technique in rodent exercise studies. Here, we examined how the use of electrical shock during forced treadmill exercise affects behavioural and physiological responses in comparison to a novel non-electrical shock technique. What is the main finding and its importance? In comparison to mice that underwent traditional treadmill running induced by electrical shock, mice that underwent forced running using a novel technique involving gentle prodding to induce running showed: (i) higher locomotor activity; (ii) less anxiety-like behaviour; and (iii) altered exercise-induced muscle pain immediately after exercise. ABSTRACT: Animal models of exercise have been useful to understand underlying cellular and molecular mechanisms. Many studies have used methods of exercise that are unduly stressful (e.g., electrical shock to force running), potentially skewing results. Here, we compared physiological and behavioural responses of mice after exercise induced using a prodding technique that avoids electrical shock versus a traditional protocol using electrical shock. We found that exercise performance was similar for both techniques; however, the shock group demonstrated significantly lower locomotor activity and higher anxiety-like behaviour. We also observed divergent effects on muscle pain; the prodding group showed hyperalgesia immediately after exercise, whereas the shock group showed hypoalgesia. Corticosterone concentrations were elevated to a similar extent for both groups. In conclusion, mice that were exercised without shock generated similar maximal exercise performance, but postexercise these mice showed an increase in locomotor activity, less anxiety-like behaviour and altered muscle pain in comparison to mice that exercised with shock. Our data suggest that running of mice without the use of electrical shock is potentially less stressful and might be a better technique to study the physiological and behavioural responses to exercise.

ASICs are required for immediate exercise-induced muscle pain and are downregulated in sensory neurons by exercise training.

Exercise training is an effective therapy for many pain-related conditions, and trained athletes have lower pain perception compared with unconditioned people. Some painful conditions, including strenuous exercise, are associated with elevated levels of protons, metabolites, and inflammatory factors, which may activate receptors and/or ion channels, including acid-sensing ion channels (ASICs), on nociceptive sensory neurons. We hypothesized that ASICs are required for immediate exercise-induced muscle pain (IEIP) and that exercise training diminishes IEIP by modulating ASICs within muscle afferents. We found high-intensity interval training (HIIT) reduced IEIP in C57BL/6 mice and diminished ASIC mRNA levels in lumber dorsal root ganglia, and this downregulation of ASICs correlated with improved exercise capacity. Additionally, we found that ASIC3 -/- mice did not develop IEIP; however, the exercise capacity of ASIC3 -/- was similar to wild-type mice. These results suggest that ASICs are required for IEIP and that diminishment of IEIP after exercise training correlates with downregulation of ASICs in sensory neurons.NEW & NOTEWORTHY Exercise performance can be limited by the sensations of muscle fatigue and pain transmitted by muscle afferents. It has been proposed that exercise training abrogates these negative feedback signals. We found that acid-sensing ion channels (ASICs) are required for immediate exercise-induced muscle pain (IEIP). Moreover, exercise training prevented IEIP and was correlated with downregulation of ASICs in sensory neurons.

TMEM16B determines cholecystokinin sensitivity of intestinal vagal afferents of nodose neurons.

The satiety effects and metabolic actions of cholecystokinin (CCK) have been recognized as potential therapeutic targets in obesity for decades. We identified a potentially novel Ca2+-activated chloride (Cl-) current (CaCC) that is induced by CCK in intestinal vagal afferents of nodose neurons. The CaCC subunit Anoctamin 2 (Ano2/TMEM16B) is the dominant contributor to this current. Its expression is reduced, as is CCK current activity in obese mice on a high-fat diet (HFD). Reduced expression of TMEM16B in the heterozygote KO of the channel in sensory neurons results in an obese phenotype with a loss of CCK sensitivity in intestinal nodose neurons, a loss of CCK-induced satiety, and metabolic changes, including decreased energy expenditure. The effect on energy expenditure is further supported by evidence in rats showing that CCK enhances sympathetic nerve activity and thermogenesis in brown adipose tissue, and these effects are abrogated by a HFD and vagotomy. Our findings reveal that Ano2/TMEM16B is a Ca2+-activated chloride channel in vagal afferents of nodose neurons and a major determinant of CCK-induced satiety, body weight control, and energy expenditure, making it a potential therapeutic target in obesity.

Acid Sensing Ion Channel 1a (ASIC1a) Mediates Activity-induced Pain by Modulation of Heteromeric ASIC Channel Kinetics.

Chronic muscle pain is acutely worsened by exercise. Acid sensing ion channels (ASIC) are heteromeric channels expressed in muscle sensory neurons that detect decreases in pH. We have previously shown ASIC3 is important in activity-induced hyperalgesia. However, ASICs form heteromers with ASIC1a being a key component in sensory neurons. Therefore, we studied the role of ASIC1a in mice using behavioral pharmacology and genetic deletion in a model of activity-induced hyperalgesia. We found ASIC1a-/- mice developed mechanical hyperalgesia similar to wild-type mice, but antagonism of ASIC1a, with psalmotoxin, prevented development of mechanical hyperalgesia in wild-type mice, but not in ASIC1a-/- mice. To explain this discrepancy, we then performed electrophysiology studies of ASICs and examined the effects of psalmotoxin on ASIC heteromers. We expressed ASIC1a, 2 and 3 heteromers or ASIC1 and 3 heteromers in CHO cells, and examined the effects of psalmotoxin on pH sensitivity. Psalmotoxin significantly altered the properties of ASIC hetomeric channels. Specifically, in ASIC1a/2/3 heteromers, psalmotoxin slowed the kinetics of desensitization, slowed the recovery from desensitization, and inhibited pH-dependent steady-state desensitization, but had no effect on pH-evoked current amplitudes. We found a different pattern in ASIC1a/3 heteromers. There was a significant leftward shift in the pH dose response of steady-state desensitization and decrease in pH-evoked current amplitudes. These results suggest that blockade of ASIC1a modulates the kinetics of heteromeric ASICs to prevent development of activity-induced hyperalgesia. These data suggest ASIC1a is a key subunit in heteromeric ASICs and may be a pharmacological target for treatment of musculoskeletal pain.

MG53 is dispensable for T-tubule maturation but critical for maintaining T-tubule integrity following cardiac stress.

The cardiac transverse (T)-tubule membrane system is the safeguard for cardiac function and undergoes dramatic remodeling in response to cardiac stress. However, the mechanism by which cardiomyocytes repair damaged T-tubule network remains unclear. In the present study, we tested the hypothesis that MG53, a muscle-specific membrane repair protein, antagonizes T-tubule damage to protect against maladaptive remodeling and thereby loss of excitation-contraction coupling and cardiac function. Using MG53-knockout (MG53-KO) mice, we first established that deficiency of MG53 had no impact on maturation of the T-tubule network in developing hearts. Additionally, MG53 ablation did not influence T-tubule integrity in unstressed adult hearts as late as 10months of age. Following left ventricular pressure overload-induced cardiac stress, MG53 protein levels were increased by approximately three-fold in wild-type mice, indicating that pathological stress induces a significant upregulation of MG53. MG53-deficient mice had worsened T-tubule disruption and pronounced dysregulation of Ca2+ handling properties, including decreased Ca2+ transient amplitude and prolonged time to peak and decay. Moreover, MG53 deficiency exacerbated cardiac hypertrophy and dysfunction and decreased survival following cardiac stress. Our data suggest MG53 is not required for T-tubule development and maintenance in normal physiology. However, MG53 is essential to preserve T-tubule integrity and thereby Ca2+ handling properties and cardiac function under pathological cardiac stress.

The volume-regulated anion channel (LRRC8) in nodose neurons is sensitive to acidic pH.

The leucine rich repeat containing protein 8A (LRRC8A), or SWELL1, is an essential component of the volume-regulated anion channel (VRAC) that is activated by cell swelling and ionic strength. We report here for the first time to our knowledge its expression in a primary cell culture of nodose ganglia neurons and its localization in the soma, neurites, and neuronal membrane. We show that this neuronal VRAC/SWELL1 senses low external pH (pHo) in addition to hypoosmolarity. A robust sustained chloride current is seen in 77% of isolated nodose neurons following brief exposures to extracellular acid pH. Its activation involves proton efflux, intracellular alkalinity, and an increase in NOX-derived H2O2. The molecular identity of both the hypoosmolarity-induced and acid pHo-conditioned VRAC as LRRC8A (SWELL1) was confirmed by Cre-flox-mediated KO, shRNA-mediated knockdown, and CRISPR/Cas9-mediated LRRC8A deletion in HEK cells and in primary nodose neuronal cultures. Activation of VRAC by low pHo reduces neuronal injury during simulated ischemia and N-methyl-D-aspartate-induced (NMDA-induced) apoptosis. These results identify the VRAC (LRRC8A) as a dual sensor of hypoosmolarity and low pHo in vagal afferent neurons and define the mechanisms of its activation and its neuroprotective potential.

Heart failure induces changes in acid-sensing ion channels in sensory neurons innervating skeletal muscle.

Heart failure is associated with diminished exercise capacity, which is driven, in part, by alterations in exercise-induced autonomic reflexes triggered by skeletal muscle sensory neurons (afferents). These overactive reflexes may also contribute to the chronic state of sympathetic excitation, which is a major contributor to the morbidity and mortality of heart failure. Acid-sensing ion channels (ASICs) are highly expressed in muscle afferents where they sense metabolic changes associated with ischaemia and exercise, and contribute to the metabolic component of these reflexes. Therefore, we tested if ASICs within muscle afferents are altered in heart failure. We used whole-cell patch clamp to study the electrophysiological properties of acid-evoked currents in isolated, labelled muscle afferent neurons from control and heart failure (induced by myocardial infarction) mice. We found that the percentage of muscle afferents that displayed ASIC-like currents, the current amplitudes, and the pH dose-response relationships were not altered in mice with heart failure. On the other hand, the biophysical properties of ASIC-like currents were significantly different in a subpopulation of cells (40%) from heart failure mice. This population displayed diminished pH sensitivity, altered desensitization kinetics, and very fast recovery from desensitization. These unique properties define these channels within this subpopulation of muscle afferents as being heteromeric channels composed of ASIC2a and -3 subunits. Heart failure induced a shift in the subunit composition of ASICs within muscle afferents, which significantly altered their pH sensing characteristics. These results might, in part, contribute to the changes in exercise-mediated reflexes that are associated with heart failure.

ASICs and cardiovascular homeostasis.

In this review we address primarily the role of ASICs in determining sensory signals from arterial baroreceptors, peripheral chemoreceptors, and cardiopulmonary and somatic afferents. Alterations in these sensory signals during acute cardiovascular stresses result in changes in sympathetic and parasympathetic activities that restore cardiovascular homeostasis. In pathological states, however, chronic dysfunctions of these afferents result in serious sympatho-vagal imbalances with significant increases in mortality and morbidity. We identified a role for ASIC2 in the mechano-sensitivity of aortic baroreceptors and of ASIC3 in the pH sensitivity of carotid bodies. In spontaneously hypertensive rats, we reported decreased expression of ASIC2 in nodose ganglia neurons and overexpression of ASIC3 in carotid bodies. This reciprocal expression of ASIC2 and ASIC3 results in reciprocal changes in sensory sensitivity of baro- and chemoreceptors and a consequential synergistic exaggeration sympathetic nerve activity. A similar reciprocal sensory dysautonomia prevails in heart failure and increases the risk of mortality. There is also evidence that ASIC heteromers in skeletal muscle afferents contribute significantly to the exercise pressor reflex. In cardiac muscle afferents of the dorsal root ganglia, they contribute to nociception and to the detrimental sympathetic activation during ischemia. Finally, we report that an inhibitory influence of ASIC2-mediated baroreceptor activity suppresses the sympatho-excitatory reflexes of the chemoreceptors and skeletal muscle afferents, as well as the ASIC1a-mediated excitation of central neurons during fear, threat, or panic. The translational potential of activation of ASIC2 in cardiovascular disease states may be a beneficial sympatho-inhibition and parasympathetic activation. This article is part of the Special Issue entitled 'Acid-Sensing Ion Channels in the Nervous System'.

Intersubunit conformational changes mediate epithelial sodium channel gating.

The epithelial Na(+) channel (ENaC) functions as a pathway for Na(+) absorption in the kidney and lung, where it is crucial for Na(+) homeostasis and blood pressure regulation. However, the basic mechanisms that control ENaC gating are poorly understood. Here we define a role in gating for residues forming interfaces between the extracellular domains of the three ENaC subunits. Using cysteine substitution combined with chemical cross-linking, we determined that residues located at equivalent positions in the three subunits (αK477, ßE446, and γE455) form interfaces with residues in adjacent subunits (ßV85, γV87, and αL120, respectively). Cross-linking of these residues altered ENaC activity in a length-dependent manner; long cross-linkers increased ENaC current by increasing its open probability, whereas short cross-linkers reduced ENaC open probability. Cross-linking also disrupted ENaC gating responses to extracellular pH and Na(+), signals which modulate ENaC activity during shifts in volume status. Introduction of charged side chains at the interfacing residues altered ENaC activity in a charge-dependent manner. Current increased when like charges were present at both interfacing residues, whereas opposing charges reduced current. Together, these data indicate that conformational changes at intersubunit interfaces participate in ENaC transitions between the open and closed states; movements that increase intersubunit distance favor the open state, whereas the closed state is favored when the distance is reduced. This provides a mechanism to modulate ENaC gating in response to changing extracellular conditions that threaten Na(+) homeostasis.

ASIC2 subunits facilitate expression at the cell surface and confer regulation by PSD-95.

Acid-sensing ion channels (ASICs) are Na+ channels activated by changes in pH within the peripheral and central nervous systems. Several different isoforms of ASICs combine to form trimeric channels, and their properties are determined by their subunit composition. ASIC2 subunits are widely expressed throughout the brain, where they heteromultimerize with their partnering subunit, ASIC1a. However, ASIC2 contributes little to the pH sensitivity of the channels, and so its function is not well understood. We found that ASIC2 increased cell surface levels of the channel when it is coexpressed with ASIC1a, and genetic deletion of ASIC2 reduced acid-evoked current amplitude in mouse hippocampal neurons. Additionally, ASIC2a interacted with the neuronal synaptic scaffolding protein PSD-95, and PSD-95 reduced cell surface expression and current amplitude in ASICs that contain ASIC2a. Overexpression of PSD-95 also reduced acid-evoked current amplitude in hippocampal neurons. This result was dependent upon ASIC2 since the effect of PSD-95 was abolished in ASIC2-/- neurons. These results lend support to an emerging role of ASIC2 in the targeting of ASICs to surface membranes, and allows for interaction with PSD-95 to regulate these processes.

Effect of deep tissue incision on pH responses of afferent fibers and dorsal root ganglia innervating muscle.

BACKGROUND: Understanding the mechanisms underlying deep tissue pain in the postoperative period is critical to improve therapies. Using the in vitro plantar flexor digitorum brevis muscle-nerve preparation and patch clamp recordings from cultured dorsal root ganglia neurons innervating incised and unincised muscle, the authors investigated responses to various pH changes. METHODS: Incision including the plantar flexor digitorum brevis muscle or sham operation was made in the rat hind paw. On postoperative day 1, in vitro single-fiber recording was undertaken. On the basis of previous studies, the authors recorded from at least 40 fibers per group. Also DiI-labeled dorsal root ganglia innervating muscle from rats undergoing incision and a sham operation were cultured and tested for acid responses, using whole cell patch clamp recordings. RESULTS: The prevalence of responsive group IV afferents to lactic acid pH 6.5 in the incision group (15 of 67; 22.3%) was greater than that in the control group (2 of 35; 5.7%; P=0.022). In dorsal root ganglia neurons innervating muscle, incision increased mean current amplitudes of acid-evoked currents; the acid-sensing ion channel blocker, amiloride 300 µM, inhibited more than 75% of the acid-evoked current, whereas, the transient receptor vanilloid receptor 1 blocker (AMG9810 1 µM) did not cause significant inhibition. CONCLUSION: The authors' experiments demonstrated that incision increases the responses of flexor digitorum brevis muscle afferent fibers to weak acid solutions, and increased acid-evoked currents in dorsal root ganglia innervating muscle. The authors' data suggest that up-regulation of acid-sensing ion channels might underlie this increased chemosensitivity caused by surgery.

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.

Acid-sensing ion channels (ASICs) are differentially modulated by anions dependent on their subunit composition.

Acid-sensing ion channels (ASICs) are sodium channels gated by extracellular protons. ASIC1a channels possess intersubunit Cl(-)-binding sites in the extracellular domain, which are highly conserved between ASIC subunits. We previously found that anions modulate ASIC1a gating via these sites. Here we investigated the effect of anion substitution on native ASICs in rat sensory neurons and heterologously expressed ASIC2a and ASIC3 channels by whole cell patch clamp. Similar to ASIC1a, anions modulated the kinetics of desensitization of other ASIC channels. However, unlike ASIC1a, anions also modulated the pH dependence of activation. Moreover, the order of efficacy of different anions to modulate ASIC2a and -3 was very different from that of ASIC1a. More surprising, mutations of conserved residues that form an intersubunit Cl(-)-binding site in ASIC1a only partially abrogated the effects of anion modulation of ASIC2a and had no effect on anion modulation of ASIC3. The effects of anions on native ASICs in rat dorsal root ganglion neurons mimicked those in heterologously expressed ASIC1a/3 heteromeric channels. Our data show that anions modulate a variety of ASIC properties and are dependent on the subunit composition, and the mechanism of modulation for ASIC2a and -3 is distinct from that of ASIC1a. We speculate that modulation of ASIC gating by Cl(-) is a novel mechanism to sense shifts in extracellular fluid composition.

Identification of extracellular domain residues required for epithelial Na+ channel activation by acidic pH.

A growing body of evidence suggests that the extracellular domain of the epithelial Na(+) channel (ENaC) functions as a sensor that fine tunes channel activity in response to changes in the extracellular environment. We previously found that acidic pH increases the activity of human ENaC, which results from a decrease in Na(+) self-inhibition. In the current work, we identified extracellular domain residues responsible for this regulation. We found that rat ENaC is less sensitive to pH than human ENaC, an effect mediated in part by the γ subunit. We identified a group of seven residues in the extracellular domain of γENaC (Asp-164, Gln-165, Asp-166, Glu-292, Asp-335, His-439, and Glu-455) that, when individually mutated to Ala, decreased proton activation of ENaC. γ(E455) is conserved in ßENaC (Glu-446); mutation of this residue to neutral amino acids (Ala, Cys) reduced ENaC stimulation by acidic pH, whereas reintroduction of a negative charge (by MTSES modification of Cys) restored pH regulation. Combination of the seven γENaC mutations with ß(E446A) generated a channel that was not activated by acidic pH, but inhibition by alkaline pH was intact. Moreover, these mutations reduced the effect of pH on Na(+) self-inhibition. Together, the data identify eight extracellular domain residues in human ß- and γENaC that are required for regulation by acidic pH.

Coxsackievirus and adenovirus receptor (CAR) mediates trafficking of acid sensing ion channel 3 (ASIC3) via PSD-95.

We have previously shown that the Coxsackievirus and adenovirus receptor (CAR) can interact with post-synaptic density 95 (PSD-95) and localize PSD-95 to cell-cell junctions. We have also shown that activity of the acid sensing ion channel (ASIC3), a H(+)-gated cation channel that plays a role in mechanosensation and pain signaling, is negatively modulated by PSD-95 through a PDZ-based interaction. We asked whether CAR and ASIC3 simultaneously interact with PSD-95, and if so, whether co-expression of these proteins alters their cellular distribution and localization. Results indicate that CAR and ASIC3 co-immunoprecipitate only when co-expressed with PSD-95. CAR also brings both PSD-95 and ASIC3 to the junctions of heterologous cells. Moreover, CAR rescues PSD-95-mediated inhibition of ASIC3 currents. These data suggest that, in addition to activity as a viral receptor and adhesion molecule, CAR can play a role in trafficking proteins, including ion channels, in a PDZ-based scaffolding complex.

Simultaneous disruption of mouse ASIC1a, ASIC2 and ASIC3 genes enhances cutaneous mechanosensitivity.

Three observations have suggested that acid-sensing ion channels (ASICs) might be mammalian cutaneous mechanoreceptors; they are structurally related to Caenorhabditis elegans mechanoreceptors, they are localized in specialized cutaneous mechanosensory structures, and mechanical displacement generates an ASIC-dependent depolarization in some neurons. However, previous studies of mice bearing a single disrupted ASIC gene showed only subtle or no alterations in cutaneous mechanosensitivity. Because functional redundancy of ASIC subunits might explain limited phenotypic alterations, we hypothesized that disrupting multiple ASIC genes would markedly impair cutaneous mechanosensation. We found the opposite. In behavioral studies, mice with simultaneous disruptions of ASIC1a, -2 and -3 genes (triple-knockouts, TKOs) showed increased paw withdrawal frequencies when mechanically stimulated with von Frey filaments. Moreover, in single-fiber nerve recordings of cutaneous afferents, mechanical stimulation generated enhanced activity in A-mechanonociceptors of ASIC TKOs compared to wild-type mice. Responses of all other fiber types did not differ between the two genotypes. These data indicate that ASIC subunits influence cutaneous mechanosensitivity. However, it is unlikely that ASICs directly transduce mechanical stimuli. We speculate that physical and/or functional association of ASICs with other components of the mechanosensory transduction apparatus contributes to normal cutaneous mechanosensation.