Acid-sensing ion channels in sensory signaling

Abstract

Acid-sensing ion channels (ASICs) are cation-permeable channels that in the periphery are primarily expressed in sensory neurons that innervate tissues and organs. Soon after the cloning of the ASIC subunits, almost 20 yr ago, investigators began to use genetically modified mice to assess the role of these channels in physiological processes. These studies provide critical insights about the participation of ASICs in sensory processes, including mechanotransduction, chemoreception, and nociception. Here, we provide an extensive assessment of these findings and discuss the current gaps in knowledge with regard to the functions of ASICs in the peripheral nervous system.

INTRODUCTION

Acid-sensing ion channels (ASICs) are trimeric cation-permeable channels that are expressed primarily in neurons of the peripheral and central nervous system. The purpose of this review is to provide a comprehensive summary of the function of ASICs in the former. In the first section, we provide a brief description of the family organization, subunit topology, channel structure, protomer assembly, and potential mechanisms of activation of these channels in vivo. We then summarize the knowledge gained from studies with genetically modified mice about the role of these channels in physiological process, including mechanosensation, chemosensation, and nociception. The reader will realize that there are fundamental questions regarding the mechanism of activation and function of these channels in various settings that remain to be addressed. In the final part of the review, we emphasize areas that need future exploration.

EPITHELIAL Na⁺ CHANNEL/DEGENERIN FAMILY

ASICs belong to the epithelial Na⁺ channel (ENaC)/degenerin (Deg) family of ion channels, a group of structurally related proteins expressed in animals from invertebrates to mammals. Members of this family have evolved to accomplish diverse functions with a similar structure. For instance, ENaCs mediate the rate-limiting step for Na⁺ reabsorption in the apical membrane of epithelial cells in the distal colon, airways, and principal cells of the distal nephron (70). The Degs MEC-4 and MEC-10 are pore-forming subunits of a mechanosensitive complex that facilitates gentle touch sensation in neurons of the worm Caenorhabditis elegans (18, 77, 92). Snails express the FMRFamide Na⁺ channel (FaNaCh), a ligand-gated Na⁺ channel activated by the peptide FMFRamide (Phe-Met-Arg-Phe-NH₂) (61, 86). ASICs are activated in vitro by sudden drops in extracellular pH, although fluctuations in extracellular pH of sufficient magnitude to activate these channels have not been registered in tissues outside the central nervous system. Significantly, and as discussed below, deletion of ASIC subunits in sensory neurons alters sensory modalities (e.g., mechanoreception and nociception) that do not appear to be mediated by or involved extracellular acidification. Therefore, future studies are needed to define the mechanisms of activation of ASICs in vivo.

ASIC GENES AND SPLICE VARIANTS

Acid-gated currents were first detected in sensory neurons in the early 1980s (49, 76, 78), but it was not until the late 1990s that ASIC subunits were cloned using homology-based approaches (1, 3, 7, 19, 26, 38, 47, 112, 142). Four genes have been identified in rodents (Asic1, Asic2, Asic3, and Asic4) that encode for six ASIC subunits and splice variants (ASIC1A, ASIC1B, ASIC2A, ASIC2B, ASIC3, and ASIC4) (1, 3, 7, 19, 26, 38, 47, 112, 142). ASIC1B results from alternative splicing of the Asic1 gene and differs from ASIC1A in the first 218 residues (7). Asic2 encodes two major splice variants, ASIC2A and ASIC2B, which differ in their first 237 NH₂-terminal residues (87). ASIC1A, ASIC2A, ASIC2B, and ASIC4 are expressed throughout the central and peripheral nervous system, whereas ASIC1B and ASIC3 are primarily expressed in sensory neurons (3, 19, 26, 141, 145, 147). Asic3 encodes a single variant in rodents but three splice variants in humans that differ in the COOH terminus [human (h)ASIC3A, hASIC3B, and hASIC3B]. Of these three transcripts, hASIC3A is the most abundant in the peripheral and central nervous systems and to a significantly lesser extent hASIC3C (27). hASIC3B expression appears to be negligible.

SUBUNIT ORGANIZATION AND CHANNEL ARCHITECTURE

ENaC/Deg family members share a common topology with two transmembrane domains (TM1 and TM2) connected by a large extracellular loop and intracellular NH₂- and COOH-terminal domains (Fig. 1) (14, 114, 118, 127). In the last decade, the structure of chicken (c)ASIC1 was resolved in a desensitized-like state (43, 58), a resting state (149), in complex with the tarantula Psalmopoeus cambridgei toxin psalmotoxin (5, 25), and in complex with the Texas coral snake toxin MitTx (4). The crystal structure of cASIC1 showed that subunits assemble to form trimers that resemble a chalice-like shape, with the extracellular region protruding from the plane of the membrane (Fig. 1). Consistent with structure-function studies (15, 68, 69, 84, 119, 121, 122, 128), residues in the second transmembrane region of cASIC1 define the conductive pathway (4, 5, 25, 43, 58). Residues in the first transmembrane region surround the ion permeation pathway and interact with the lipid bilayer (Fig. 1). The extracellular region of cASIC1 has well-defined domains that were termed in analogy to a hand holding a ball as the finger, thumb, β-ball, knuckle, and palm domain (Fig. 1) (58). Because the crystallization of cASIC1 required the removal of 13 residues from the NH₂ terminus and 64 residues from the COOH terminus (58), there is presently no structural information of the intracellular domains. More recently, the structure of ENaC was resolved showing a similar domain architecture, albeit with extended finger domains (103). A great deal of effort has been devoted to understanding the molecular mechanism by which pH controls ASIC function. Interested readers are referred to a recent review from Vullo and Kellenberger (137) for detailed information about the molecular mechanisms underlying the function of these channels.

Fig. 1.

Architecture of chicken acid-sensing ion channel 1 (cASIC1). A: schematic domain organization of a cASIC1 subunit in the desensitized state. Disulfide bridges are shown as cyan sticks. Cl⁻ bound to the thumb domain is shown in red. The first and second transmembrane regions (TM1 and TM2, respectively) are shown in black. B: organization of a cASIC1 trimer.

PROTOMER ASSEMBLY

ASIC subunits assemble in heterologous expression systems to form homo- and hetero-trimers with distinctive biophysical properties, including agonist affinity, single channel conductance, rate of desensitization, recovery after desensitization, and cation selectivity (9, 70, 112, 140, 141). For details about the biophysical properties of ASICs, see previous reviews (48, 71). Only ASIC1A, ASIC1B, and ASIC3 subunits form by themselves channels that respond to extracellular acidification in the physiological range (9). ASIC2A homotrimers respond to extracellular acidification, but at pHs unlikely to be found in vivo [pH of half-maximal activation (pH₅₀) is 4–4.9] (87). The consensus is that in sensory and most central nervous system neurons, proton-gated currents are mediated by a mix of assembled ASIC subunits (2, 6, 9, 40, 100, 136). For instance, a patch-clamp study (40) of mouse skeletal muscle afferents indicated that proton-gated currents in these cells are mediated by channels composed of ASIC1A, ASIC2, and ASIC3 subunits. Deletion of individual subunits from mouse skeletal muscle afferents does not eliminate proton-gated currents, but it changes their biophysical properties (40). Proton-gated currents were ablated only upon removal of ASIC1A, ASIC2, and ASIC3 (40). Likewise, loss of ASIC1A or ASIC3 does not eliminate proton-gated currents in sensory neurons innervating the skin, urinary bladder, or muscle, but it alters the desensitization and recovery from desensitization (40, 100, 111). In contrast, in hippocampal neurons, loss of ASIC1A abolished, to a large extent, currents evoked by a pulse of pH 5.0 (144, 150). However, the contribution of “electrically silent” subunits (i.e., ASIC2 and ASIC4) to channel function was not addressed in these studies.

PROTON SIGNALING AND ASIC FUNCTION

ASICs expressed in heterologous expression systems and in isolated neurons are activated by sudden changes in extracellular pH. However, whether transient pH fluctuations of sufficient magnitude to activate ASICs occur in vivo, particularly in the peripheral nervous system, is still uncertain. The pH of half-maximal activation of ASICs in isolated dorsal root ganglion (DRG) neurons is 6.2 (9). A previous patch-clamp study (9) revealed that a drop in extracellular pH from 7.4 to 7.0 evokes negligible desensitizing currents in isolated DRG neurons and COS cells expressing ASIC1a or ASIC3. Therefore, it is reasonable to assume that extracellular pH needs to drop below 7.0 to produce a sizable activation of ASICs in afferent fibers. To the best of our knowledge, there is no experimental evidence that such changes in extracellular pH take place in vivo. Of major significance is that Yagi et al. (148) reported that modest changes in extracellular pH (i.e., 0.2 units) activate a sustained current in Chinese hamster ovary (CHO) cells transfected with ASIC3 DNA and evoke action potential firing on isolated cardiac sensory neurons. Therefore, further studies are needed to assess whether these ASIC-mediated sustained currents contribute to sensory signaling in other organs.

As summarized below, the deletion of ASIC subunits (e.g., Asic1 and Asic3) alters somatic and visceral mechanoreception and nociception. Although one could argue that acidification of the extracellular fluid might occur in inflammatory processes that coexist with pain, there is currently no evidence to suggest that the application of mechanical forces to tissues evokes transient pH fluctuations that could activate ASICs. The reader should take into consideration that ASICs desensitize rapidly in an acidic environment, and the recovery from the inactive state (i.e., desensitized) takes from a few seconds to minutes at neutral pH, depending on the subunit composition of the channel complex (9). Therefore, in the continuous presence of an acidic milieu, ASICs will remain desensitized and unresponsive. Significantly, deletion of individual ASIC subunits does not cause dramatic changes in the biophysical properties of the proton-gated current in sensory neurons (9), which would be expected if the physiological changes seen in genetically modified mice result from loss of proton signaling. For instance, absence of ASIC3 in sensory neurons causes an increase in the magnitude of proton-evoked current and a decrease in the rate of current decay following activation (desensitization) (100, 111). However, it is worthwhile mentioning that we recently reported that bladder sensory neurons from Asic3 knockout (KO) mice do not discharge action potentials when exposed to pH transients (100). Although the capability to discharge action potential in response to extracellular acidification has not been examined in sensory neurons innervating organs other than the bladder, this finding supports the notion that ASICs might be bonafide proton sensors. If indeed ASICs are bonafide proton sensors, it will essential to understand in what context pH transients occur in vivo and whether they can be pharmacologically manipulated to tune ASIC function.

MECHANOTRANSDUCTION

Over the past quarter of the century, researchers have battled to understand the molecular basis of mechanotransduction in different systems. The first indication that ENaC/Deg family proteins were involved in mechanotransduction was provided by Driscoll and Chalfie (31) and Huang and Chalfie (54) with the identification of the Degs MEC-4 and MEC-10 as essential components of the mechanosensitive complex in touch-sensitive neurons of C. elegans. Further work from O’Hagan et al. (105) showed that mutations of a conserved Gly in the second transmembrane domain of MEC-4 or MEC-10 alter ion selectivity, reduce mechanoreceptor currents, and abolish touch sensation. These findings provide the first direct evidence that MEC-4 and MEC-10 form the pore of the complex responsible for converting mechanical force into ionic current in C. elegans touch-sensitive neurons. Of major significance is that MEC-4 and MEC-10 assemble to form a mechanosensitive channel that responds to changes in shear stress induced by fluid jet when expressed in Xenopus oocytes (123). Likewise, early studies revealed a positive correlation between intraluminal fluid flow rates with Na⁺ reabsorption and K⁺ secretion in the distal segments of the nephron (44, 72, 79, 91, 117). The rate-limiting step for Na⁺ reabsorption in this region of the nephron is mediated by amiloride-sensitive Na⁺ channels (i.e., ENaC). ENaCs when expressed in Xenopus oocytes respond to shear stress forces of magnitude similar to those found in vivo at the surface of collecting duct cells (16). In addition, Lin et al. (85) recently showed that Asic3 deletion from parvalbumin (Pv)⁺ DRG neurons ablates action potential firing evoked by substrate deformation. However, other studies have reported that ASIC2 and ASIC3 do not contribute to mechanically activated currents in sensory neurons (30). This discrepancy might be due to several factors, including the neuronal population studied, the mechanical stimulus applied, the presence of neurites, or the time of neurons in culture. It is worthwhile to mention that at present there is no evidence to support the notion that MEC-4/MEC-10, ENaC, or ASIC subunits are inherently mechanosensitive. The “mechanosensitivity” of ENaC/Deg channels might depend on the interaction with accessory proteins. For instance, studies in C. elegans indicate that touch sensation not only requires MEC-4 and MEC-10 but also MEC-2, a membrane-associated protein with a stomatin domain, and intracellular and extracellular tethers (18, 77, 92). Note that intrinsically mechanosensitive channels such as Piezo1 respond to mechanical perturbations within the membrane in cell-free systems (i.e., lipid bilayers) (133). Significantly, stomatin (STOM) and stomatin-like 3 (STOML3), which are mammalian homologs of MEC-2, interact with ASICs (12, 81, 113, 146). Stoml3 KO mice exhibit complete loss of mechanosensitivity in many cutaneous afferents and a concomitant loss of mechanosensitive currents in subsets of DRG neurons (146). STOML-null mice exhibit only mild effects on the sensitivity of rapidly adapting mechanoreceptors (D-hair) (93). Further studies are needed to determine whether a complex with similar architecture to the mechanosensitive complex found in C. elegans touch sensory neurons exists in mammals and to define the role of ASICs in mechanotransduction.

Cutaneous Mechanotransduction and Proprioception

Based on the premise that ASICs might resemble a mechanoreceptor in mammals, Price and colleagues (110, 111) examined cutaneous mechanotransduction in mice lacking ASIC2 or ASIC3 subunits. Consistent with a role in cutaneous mechanoreception, immunoreactivities for ASIC2 and ASIC3 were detected in putative sites of mechanoreception in the skin, the palisades of lanceolate nerve endings that run longitudinally to and surround the hair shaft, Merkel cell neurite complexes, and Meissner corpuscles (39, 110, 111). Loss of ASIC2 in mice reduces the sensitivity of rapidly and slowly adapting low-threshold mechanoreceptors; the former are considered to mediate the touch response (110). In contrast, skin mechanoreceptors in Asic3 KO mice exhibit increased sensitivity to light touch and reduced sensitivity to noxious pinch, whereas nociceptors exhibit an attenuated response to acid and noxious heat (111). Using an in vitro skin preparation, Moshourab et al. (101) showed that absence of ASIC3 increases the mechanosensitivity of rapidly adapting mechanoreceptors in the skin but decreases the mechanosensitivity of both Aδ-fibers and C-fiber nociceptors. No significant differences in cutaneous afferents (e.g., rapidly adapting mechanoreceptors, slowly adapting mechanoreceptors, slowly conducting myelinated mechanonociceptors, down-hair afferents, and C-fibers) were observed between ASIC1A-null mice and wild-type littermates (106).

To define the role of ASICs in mechanotransduction and nociception and to overcome the confounding effects associated with channel heteromultimerization in single subunit-null mice, Mogil et al. (99) generated a transgenic ASIC3 mouse carrying a dominant negative mutation (G439R) that eliminates all ASIC-like currents in neurons. ASIC3^G439R transgenic mice presented abnormal responses to mechanical as well as chemical and inflammatory mediators. In behavioral studies, at baseline these mice showed an enhanced mechanical sensitivity to von Frey filaments; following intramuscular hypotonic saline injections, they exhibited mechanical hypersensitivity (99). In good agreement with these findings, Kang et al. (64) reported that simultaneous loss of ASIC1a, ASIC2, and ASIC3 in mice enhances the response of cutaneous A-mechanonociceptors but not of rapidly adapting mechanoreceptors, slowly adapting mechanoreceptors, down-hair afferents, or C-fibers. Although deletion of all ASIC subunits affects the response of A-mechanonociceptors to mechanical stimuli, it does not seem to affect their sensitivity (e.g., force required to evoke 50% of the maximal response) (64). In behavioral studies, this enhanced sensitivity of A-mechanonociceptors translated in increased paw withdrawal frequency to von Frey filaments.

Recently, Lin et al. (85) generated a floxed allele Asic3 mouse line to evaluate the role of ASIC3 in mechanotransduction in proprioreceptors innervating muscle spindles. Using a reporter mouse line, the authors showed that ASIC3 is expressed in Pv⁺ proprioreceptor axons innervating muscle spindles. Significantly, loss of this subunit impairs mechanotransduction induced by substrate deformation in Pv⁺ DRG neurons (85). Surprisingly, deletion of Asic3 from Pv⁺ sensory neurons does not prevent afferent firing in response soleus muscle stretch, but it significantly affects the dynamic responses triggered by elongation. In behavioral assays, loss of ASIC3 from Pv⁺ DRG neurons resulted in deficits in proprioreception in grid and balance beam walking tasks (85). Based on these findings, Lin et al. (85) proposed that ASIC3 regulates the dynamic response of proprioreceptors in muscle spindles.

In summary, compelling evidence indicates that ASICs contribute to mechanotransduction and proprioreception. Although ASIC2 and ASIC3 are thought to be expressed in rapidly adapting mechanoreceptors and likely form heterotrimers, it is unclear why the independent removal of these subunits produces such strongly contrasting outcomes. Even more intriguing is the fact that the deletion of ASIC3 results in increased mechanosensitivity of distinct types of mechanoreceptors. In summary, future studies are needed to understand the role of ASICs in mechanotransduction.

Visceral Mechanotransduction

ASIC1a, ASIC2, and ASIC3 are expressed in gastric and colonic afferents, and disruption of individual ASIC subunits alters their mechanical sensitivity in distinctive manners (55, 106, 107). For instance, loss of ASIC1a increases the mechanosensitivity of gastroesophageal and mesenteric afferents and slows gastric emptying (106, 107). Disruption of ASIC2 reduces fecal pellet output, increases the mechanosensitivity of serosal colon afferents, and reduces the mechanosensitivity of gastroesophageal tension receptors (107). Loss of ASIC3 reduces the mechanosensitivity of serosal and mesenteric colon afferents and of tension gastroesophageal afferents (107).

About 25% of the sensory neurons innervating the urinary bladder exhibit ASIC-like currents (100). Deletion of Asic3 does not eliminate proton-evoked currents from bladder sensory neurons, but it slows the current decay (desensitization). Of major significance is that bladder sensory neurons with ASIC-like currents from ASIC3-null mice do not discharge action potentials in response to a drop in extracellular pH, supporting the notion that ASIC3 mediates proton signaling in a cluster of bladder afferents (100). In voiding assays, Asic3 KO mice exhibit a lower threshold to trigger voiding and greater void volume than wild-type mice (100).

The studies described above indicate that ASICs regulate mechanotransduction in the stomach, gut, and urinary bladder (55, 100, 106, 107). However, and similar to the investigations that examined the contribution of ASICs to cutaneous mechanoreception, it is unclear why the deletion of specific subunits results in such different outcomes. An important question that arises from these studies is whether the regulatory function of ASICs on visceral mechanotransduction involves proton sensing.

Blood Pressure Regulation

Baroreceptor neurons with nerve endings in the aortic arch and carotid sinus are crucial in detecting acute fluctuation in arterial blood pressure and signaling the central nervous. Early studies have shown that benzamil, a promiscuous ENaC/Deg blocker, inhibits baroreceptor nerve activity and baroreflex control of blood pressure (32). Using a combined physiological approach, Lu et al. (88) showed that ASIC2 is an important determinant of autonomic circulatory control and baroreceptor sensitivity. Loss of ASIC2 reduces baroreflex sensitivity and engagement, suppresses the tachycardic response to atropine, enhances the bradycardic response to propranolol, and increases mean arterial blood pressure in awake mice (88). Because loss of ASIC2 reduces and its overexpression enhances the response of nodose neurons to mechanical stimulus, Lu et al. (88) proposed that ASIC2 is a component of a mechanosensitive complex that senses changes in arterial blood pressure in the aortic arch and carotid sinus.

The organum vasculosum lamina terminalis (OVLT), a region in the central nervous system devoid of the blood-brain barrier, was recently proposed to be the site for Na⁺ concentration sensing that mediates sympathetic nerve activity and blood pressure responses to central hypernatremia (74, 75). Na_x (SCN7a) channels, which are expressed in specific glial cells in the OVLT (143) and are proposed to serve as Na⁺ sensors by monitoring increases in Na⁺ concentration in the blood and cerebrospinal fluid within the physiological range (53). Nomura et al. (102) recently reported that the activation of Na_x by subtle increases in Na⁺ concentration stimulates anaerobic glycolysis in OVLT glial cells, leading to the release of lactate via monocarboxylate transporters and protons, which activate ASIC1a in OVLT neurons projecting to the paraventricular nucleus, an area of the brain that regulates cardiovascular sympathetic outflow. In summary, this study argued that ASIC1a signaling is required for the salt-induced activation of the OVLT-paraventricular nucleus neural circuit, leading to blood pressure elevations.

Blood Volume Control

ASIC3 is expressed in nodose neurons innervating the venoatrial junction, an area where low-pressure baroreceptors involved in the regulation of blood volume homeostasis are located (82). Blood volume expansion in mice increases urinary flow and promotes activation of specific afferent pathways in nodose ganglia and at cervical (C8) and thoracic (T1−T4 and T9−T13) levels and the release of atrial natriuretic peptide (82). Deletion of Asic3 limits, to some extent, the responses evoked by blood volume expansion, supporting the involvement of this subunit in blood volume homeostasis (82). However, given the complexity and extent of visceral pathways activated by blood volume expansion, additional studies will need to be carried out to understand the mechanism by which ASIC3 mediates the changes.

Pressure-Induced Vasodilation

The application of low pressure to the skin induces a local increase in blood flow, which protects against pressure-induced microvascular dysfunction and ulcer formation. Fromy et al. (34) revealed that ASIC3 is required for the vasodilation response to low pressure and that loss of this subunit increases the incidence and extent of ischemic lesions caused by long-lasting compression in mice. Significantly, local administration of the ASIC inhibitors amiloride and diclofenac in humans abolished pressure-induced vasodilation. Because loss of ASIC3 did not affect endothelial or smooth muscle function, these authors proposed that ASIC3 serves as a mechanosensor in specialized sensory neurons innervating the skin responsible for the microvascular response to low pressure.

Myogenic Tone

ASIC2 and ENaC β- and γ-subunits are expressed in vascular smooth muscle cells and have been proposed to assemble in a mechanosensitive ion channel complex that regulates local blood flow in certain blood vessels (59, 60). Gannon and colleagues (35, 36) and Jernigan and Drummond (59, 60) showed that loss of ASIC2 in cerebral and renal interlobar arteries or ENaC β- or γ-subunits in renal interlobular arteries alters pressure-induced vascular tone. This mechanism, known as the myogenic response, contributes to local blood flow autoregulation and protection against systemic blood pressure-induced microvascular damage. Significantly, heterozygous and homozygous ASIC2-null mice exhibit weakened myogenic control of small renal interlobular arteries, increased renal blood flow correction after a step increase in mean arterial pressure, increased mean arterial blood pressure, and mild renal injury (35). As a whole, these studies suggest that ASIC2 and β- and γ-ENaC assemble to form a mechanosensitive complex in vascular smooth muscle cells that mediates the myogenic response.

CHEMOTRANSDUCTION

Chemoreceptor Reflex

Alterations in arterial O₂, CO₂, and pH are detected by peripheral chemoreceptors in glomus type I cells in the carotid and aortic bodies. Glomus type I cells reside in close proximity to afferent endings from the glossopharyngeal nerve, which relayed to the nucleus of the tractus solitarius in the medulla oblongata to mediate the chemoreceptor reflex (80, 108, 109). The activation of chemoreceptors in glomus type I cells leads to depolarization, an increase in intracellular Ca²⁺ concentration, mediator release (e.g., ATP and acetylcholine), and synaptic activation of glossopharyngeal nerve endings, triggering a reflex that elicit hyperventilation and sympathetic activation to restore homeostasis (33, 89, 104). Proton-gated currents with biophysical and pharmacological properties comparable with those of ASICs, as well as expression of ASIC1 and ASIC3 subunits, have been reported in glomus type I cells (134). Functional and morphological evidence suggest that different clusters of glomus type I cells mediate the response to pH and hypoxia (89). Deletion of Asic3 blunts the increase in intracellular Ca²⁺ evoked in response to extracellular acidification of glomus type I cells, but not to hypoxia (89). However, despite the fact that exposure of mice to increased levels of CO₂ (hypercapnia) leads to a drop in arterial pH, loss of ASIC1, ASIC2, or ASIC3 does not significantly alter hypercapnic or hypoxic ventilatory responses (28). The reasons for the discrepancy between early studies with isolated glomus type I cells and physiological studies with ASIC-null mice require further work. Finally, the enhanced ASIC-mediated pH sensitivity of peripheral chemoreceptors in prehypertensive spontaneously hypertensive rats was proposed to contribute to the exacerbated sympathetic activity present in this model (135).

Exercise Pressor Reflex

Skeletal muscle afferent fibers consist of thinly myelinated Aδ (type III) and nonmyelinated C (type IV) fibers. Aδ-fiber afferents are believed to respond primarily to mechanical stimulus, whereas C-fiber afferents are considered primarily metaboreceptors that respond to metabolic products (57, 66, 67). Functional studies have indicated that 40−80% of muscle sensory neurons exhibit proton-gated currents (29, 40, 42) mediated by channels assembled of ASIC1a, ASIC2a, and ASIC3 subunits (40). Physiological studies have indicated that in muscular afferents, ASICs play major roles in the exercise pressor reflex and muscle pain. Skeletal muscle contraction during physical activity evokes increases in arterial blood pressure, heart rate, and ventilation. This sympathetic response called “exercise pressor reflex” is activated by stimulation of receptors that respond to either mechanical distortion or the metabolic byproducts of muscle contraction (for a review, see Ref. 98). The metabolic component, but not the mechanical component, of the exercise pressor reflex has been shown to be attenuated by the systemic administration of the ASIC inhibitors amiloride, A-317567, and APETx2 (37, 51, 52, 83, 94, 95, 130). Although these studies support the notion that ASICs contribute to the metabolic component of the exercise pressor reflex, further studies with ASIC-null mice are needed to validate the conclusions.

NOCICEPTION

Myocardial Ischemia

The myocardium is innervated by sympathetic and parasympathetic afferent fibers that follow the sympathetic efferent cardiac and vagus nerves, respectively (8, 96). Cell bodies of sympathetic afferent fibers (referred to here as cardiac sensory neurons) reside in the lower cervical (C8) and thoracic (T1−T3) DRG, whereas vagus nerve afferents are located in the nodose ganglia. The pericardium is innervated by phrenic nerve afferents with cell bodies located in the upper cervical (C3–C5) DRG. Benson et al. (8) reported that acid-evoked (ASIC-like) currents were particularly large (mean: ∼8 nA) in rat cardiac sensory neurons compared with nodose heart and other sensory neuron populations. The transient current evoked by acidification in rat cardiac sensory neurons was selective for Na⁺ but Ca²⁺ permeable (8). Because rat cardiac sensory neurons responded to pH in the range produced by myocardial ischemia, Benson et al. (8) proposed protons as potential mediators of angina chest pain, a condition that occurs when blood flow to the vessel irrigating the myocardium is partial or completed obstructed. An extensive functional characterization of the acid-evoked currents in cardiac sensory neurons and comparison with properties of ASIC subunits expressed in heterologous expression systems led Sutherland et al. (132) to propose ASIC3 as a major sensor of myocardial acidity. Further analysis of acid-evoked currents in cardiac sensory neurons from wild-type, Asic2 KO, and Asic3 KO mice, as well as CHO cells expressing different combinations of ASIC subunits, established that the proton sensor in cardiac sympathetic afferents is a heteromer made of ASIC2A and ASIC3 subunits (50). Of note is that lactate, a product of anaerobic metabolism, increases the sensitivity of ASICs to protons in cardiac sensory neurons by shifting the threshold of activation (56). It has been hypothesized that the potentiating effect of lactate permits cardiac sensory neurons to sense myocardial ischemia and generate pain sensation (56).

The evidence presented above indicates that cardiac sensory neurons sense changes in extracellular pH and serve as pain sensors during cardiac ischemia and infarction. Activation of cardiac sensory neurons triggers sympathetic activation during cardiovascular disease states (90, 97), which was proposed to elicit multifarious cardioprotective signals to limit the severity of the injury (11). In support of this notion, Cheng et al. (24) showed that ASIC3-null mice present normal baseline heart rate under basal conditions but abnormal autonomic regulation of cardiac activity in response to isoproterenol-induced myocardial ischemia. In addition, ASIC3-null mice have significative lower systolic and diastolic arterial blood pressure under stress than wild-type littermates (24). In support of the notion that ASIC3 plays a protective role in cardiac ischemia, histological studies revealed that Asic3 KO mice exhibit more severe left ventricular fibrosis and monocyte infiltration 7 days after isoproterenol-induced myocardial ischemia than wild-type littermates (23).

Musculoskeletal Pain

The contribution of ASICs to nociception in the musculoskeletal system has been investigated in several models of inflammatory and noninflammatory pain. Early studies showed that a single injection of an acidic solution in skeletal muscle produces transient hyperalgesia, but repeated administration primes muscle nociceptors, resulting in long-lasting bilateral mechanical hyperalgesia (120, 124). Deletion of Asic3, but not Asic1, prevents the development of mechanical hyperalgesia induced by repeated intramuscular injections of acidic solutions (125). Pharmacological studies using amiloride, APETx2, or A-317567 supported the notion that ASIC3 is required for the induction of primary hyperalgesia and the priming of nociceptors and for the development, but not the maintenance, of musculoskeletal chronic pain (21, 22, 41, 65, 125). No changes in the biophysical properties of ASIC-like currents in muscle sensory neurons were observed after the long-lasting hyperalgesia has already developed, suggesting that chronic musculoskeletal pain is not associated with changes in the expression of ASIC subunits (41). However, dorsal horn neurons exhibit increased responses to noxious stimuli bilaterally, supporting the notion that activation of muscle afferents that produce widespread hyperalgesia is mediated by changes in the central nervous system (central sensitization) (125). Consistent with this, dorsal horn neurons from mice lacking ASIC3 did not sensitize following repetitive acid injections (125). Together, these data suggest that ASIC3 is required in primary muscle afferents for the induction of long-lasting hyperalgesia and for dorsal horn neuron sensitization by intramuscular acid solutions, but it is not required to maintain chronic hyperalgesia once developed.

The administration of carrageenan in the left gastrocnemius muscle induces an inflammatory process characterized by the development of (primary) hyperalgesia in the ipsilateral inflamed muscle but also secondary (cutaneous) hyperalgesia in the contralateral side. Muscle inflammation causes bilateral increases in mRNA levels of ASIC2A and ASIC3 in L4, L5, and L6 DRG (139). Consistent with this finding, Gautam et al. (42) reported a significant increase in the magnitude of proton-gated currents in sensory neurons from inflamed mice compared with controls. Significantly, ASIC1-null mice do not develop primary muscle hyperalgesia following carrageenan injection in the gastrocnemius muscle but experience secondary hyperalgesia (139). In contrast, ASIC3-null mice developed primary hyperalgesia at the site of injection but did not develop secondary hyperalgesia (126, 139). Muscle injection of a recombinant herpes simplex virus encoding for Asic3, but not in the skin, of ASIC3-null mice treated with carrageenan resulted in cutaneous hyperalgesia similar to that developed by wild-type mice (126). In summary, these studies showed that in a model of muscle inflammation induced by carrageenan administration, ASIC3 actions in the periphery are critical for the development of secondary cutaneous hyperalgesia at the contralateral site, whereas the generation of primary hyperalgesia requires the action of ASIC1a at the central level. Interestingly, miRNA-mediated downregulation of ASIC3 in muscle primary afferent neurons in vivo prevents the development of primary and secondary carrageenan-induced hyperalgesia (138).

The development of muscle fatigue in response to exercise has been shown to be sex and task dependent and modulated by testosterone in mice (13, 46). Asic3 KO mice showed more muscle fatigue in three 1-h run tests and lower testosterone plasma levels than wild-type littermates. Ovariectomized female mice administered testosterone behaved similarly to male mice and developed less muscle fatigue during activity than nonovariectomized female mice. Significantly, testosterone administration did not rescue the muscle fatigue responses in ovariectomized Asic3 KO mice. Together, these studies indicate ASIC3 and testosterone prevent against muscle fatigue during intense exercise (13). A question that remains to be answered is why ASIC3-null mice have lower testosterone plasma levels than wild-type littermates.

The role of ASIC1A in the development of noninflammatory pain in the musculoskeletal system is less clear. Deletion of Asic1 has no effect on the development of activity-induced mechanical hyperalgesia. However, the intramuscular injection of the spider toxin psalmotoxin-1 prevents activity-induced mechanical hyperalgesia in wild-type mice but not in ASIC1-null mice (45). Although the mechanism by which psalmotoxin-1 prevents the development of mechanical hyperalgesia in wild-type remains unclear, the study by Gregory et al. (45) implies that pharmacological modulation of ASICs could be potentially employed to treat activity-induced muscle hyperalgesia.

Muscular Ischemia and Reperfusion

ASIC3 has been proposed to contribute to sensory neuron sensitization and myalgia in a mouse model of muscular ischemia-reperfusion injury (115). Ligation of the brachial artery of the right forelimb in mouse increases the expression of the IL-1β receptor in macrophages and neutrophils present in injured muscles and in DRG neurons innervating the muscle as well as ASIC3 in group III and IV muscle afferents (115). siRNA-mediated knockdown of IL-1β receptors in DRG was shown to prevent pain-related behaviors triggered by ischemia-reperfusion injury and to attenuate the increase in ASIC3 expression in muscle sensory neurons. Likewise, nerve-specific siRNA knockdown of ASIC3 attenuated behavioral effects of ischemia-reperfusion injury (115). In summary, this study indicates that during muscular ischemia-reperfusion, activation of IL-1β signaling upregulates ASIC3 in primary afferents, which contributes to afferent sensitization and pain-related behaviors.

Visceral Pain

To assess whether ASIC3 participates in the detection of noxious mechanical stimuli in the colon, Jones et al. (62) examined the functional properties of mechanosensitive colon afferents and the visceromotor response to colon distension in Asic3 KO mice and wild-type littermates. The reflex contraction of the abdominal musculature to colon distension (i.e., visceromotor response) constitutes a well-established method to evaluate mechanical sensation and pain arising from visceral hollow organs such as the colon and urinary bladder (10, 17, 62, 63, 116, 131). Consistent with the findings reported by Page et al. (107), muscular and muscular-mucosal afferents from Asic3 KO mice were less sensitive to circumferential stretch than those from wild-type littermates (62). In good agreement with this finding, the visceromotor response to colon distension was partially impaired in Asic3 KO mice (62). In addition, these authors reported that ASIC3 was required for the sensitization of colon afferents by an acidic inflammatory soup in vitro (62). In summary, this study indicates that ASIC3 contributes to mechanosensation in the colon and is an important mediator of mechanical hypersensitivity induced by noxious chemical insult.

Other Pain Models

Chen et al. (20) and Staniland and McMahon (129) reported that ASIC3-null mice exhibit reduced latency to the onset of pain responses and enhanced pain behaviors following the intraperitoneal administration of 0.6% acetic acid solutions, in the hot plate test, and in tests for mechanical induced pain. Asic1a and Asic2, but not Asic3, KO mice exhibited enhanced pain behaviors following a subcutaneous injection of formalin in the plantar surface of the paw (129). However, in a chronic model of inflammation induced by complete Freund’s adjuvant injection in the paw loss of ASIC1a, ASIC2 or ASIC3 did not alter the response to mechanical stimuli (allodynia) or thermal hyperalgesia (129). The different outcomes from these studies might rely on differences in the pathways that contribute to pain generation in these models or the time spanned between the insult and behavioral evaluation (acute vs. chronic).

Finally, Deval et al. (29) revealed that ASICs contribute to postoperative pain in a plantar incisional model. Muscle incision was reported to cause increases in ASIC3 transcripts and protein expression (29), as well as proton-gated currents, in sensory neurons innervating the injured muscle (73). Postoperative spontaneous, thermal, and postural pain behaviors were significantly reduced in mice that received the ASIC3 inhibitor APETx2 at the site of injury or intrathecal injections of siRNA targeting ASIC3 before the surgery (29).

SUMMARY

The purpose of this review was to provide an objective assessment of the function of ASICs in the periphery. The studies discussed in the review offer overwhelming evidence that ASICs contribute to the transduction of mechanical, chemical, and nociceptive information in sensory neurons and other cell types (Tables 1 and 2). However, the impact that deletion of distinct ASIC subunits has on mechanotransduction and nociception is not easy to comprehend. There is not clear understanding of the stimulus that ASICs are sensing and how their function contributes to these processes. Even more confusing is the increased sensitivity of mechanoreceptors or enhanced pain behaviors reported in certain ASIC subunit-null mice. Readers likely realize that whereas ASICs are generally considered to be bonafide proton sensors, currently there is no evidence that pH transients of enough magnitude to activate these channels occur in tissues. Therefore, additional studies should be carried out to determine in what context pH transients occur in vivo and whether they can be altered to tune ASIC function. In addition, there is a pressing need to apply new tools and approaches to study the function of these channels in its native environment. These studies could help us to understand the mechanism by which ASICs modulate mechanotransduction, what kind of stimuli they sense, and how accessory proteins (i.e., STOML3) contribute to this process. Because most of the studies discussed here were performed with global KO mice and because ASICs are expressed in regions of the central nervous system that process sensory information, it is not possible to exclude a central contribution of ASICs to the outcomes seen in behavioral studies. Therefore, there is great need for the use of conditional null mice in the field. In addition, consideration should be also given to the differences in subunit expression among species, particularly ASIC3, which is expressed in rodents primarily in the periphery but in both peripheral and central neuronal tissue in humans (27). Taken together, the studies discussed in this review highlight the importance of ASICs in sensory signaling and the need for future research in the field.

Table 1.

ASIC subunits in mechanotransduction and chemotransduction

Subunit (System)	Genetic Manipulation	Effect	Physiological Outcome	Reference(s)
ASIC1a
Cutaneous MT	KO	No change		106
Visceral MT	KO	↑MS of gastroesophageal tension receptors and mesenteric afferents	Slows gastric emptying	106, 107
ASIC2
Cutaneous MT	KO	↓Sensitivity of rapidly and slowly adapting low-threshold MRs		110
Visceral MT	KO	↑MS of serosal colon afferents ↓MS of gastroesophageal tension receptors	↓Fecal pellet output	107
Blood pressure regulation	KO	↓MS of nodose neurons	↓Baroreflex sensitivity and engagement Suppressed the tachycardic response to atropine ↑Bradycardic response to propranolol ↑Mean arterial blood pressure in awake mice	88
Myogenic response	KO	Impaired pressure-induced vascular tone of middle cerebral and renal interlobar arteries	↓Myogenic tone of middle cerebral and small renal interlobular arteries ↑Renal blood flow correction after a step increase in mean arterial pressure	35, 36
ASIC3
Muscle MT	KO	Disrupts spindle afferent sensitivity to dynamic stimuli and impairs MT induced by substrate deformation in parvalbumin+ proprioreceptors	Deficits in proprioreception in grid and balance beam walking tasks	85
Cutaneous MT	KO	↑MS rapidly adapting skin MRs ↓MS of MRs detecting noxious pinch-attenuated response to acid and noxious heat in nociceptors		101, 111
Cutaneous MT	ASIC3 mice carrying a dominant negative mutation (G439R)	Abnormal response to mechanical as well as chemical and inflammatory mediators	↑Mechanical sensitivity ↑Chemical/inflammatory sensitivity	99
Visceral MT	KO	Ablates H+-induced action potential firing in bladder sensory neurons	↓Threshold to trigger voiding ↑Void volume	100
Visceral MT		↓MS gastroesophageal tension receptors ↓MS serosal and mesenteric afferents		107
Vasodilation response to low pressure	KO		↑Incidence and extent of ischemic lesion caused by long-lasting compression	34
Chemotransduction	KO	Blunt the increase in intracellular Ca2+ evoked in response to extracellular acidification in glomus type I cells of the carotid and aortic bodies		88
ASIC1a, ASIC2, and ASIC3
Cutaneous MT	KO	↑Response of cutaneous A-MRs to force stimuli	↑Withdrawal frequency to von Frey filaments applied to the paw	64

ASIC, acid-sensing ion channel; KO, knockout; MRs, mechanoreceptors; MS, mechanosensitivity; MT, mechanotransduction; ↑, increase; ↓, decrease.

Table 2.

ASIC subunits in nociception

Subunit (Treatment)	Genetic Manipulation	Outcome	Reference(s)
ASIC1a
Carrageenan injection in the gastrocnemius muscle	KO	Did not develop primary muscle hyperalgesia	138
Subcutaneous injection of formalin in the plantar surface of the paw	KO	↑Pain behaviors	129
Complete Freund’s adjuvant injection in the paw	KO	No change in mechanical allodynia or thermal hyperalgesia	129
ASIC2
Subcutaneous injection of formalin in the plantar surface of the paw	KO	↑Pain behaviors	129
Complete Freund’s adjuvant injection in the paw	KO	No change in mechanical allodynia or thermal hyperalgesia	129
Mechanical hyperalgesia induced by repeated intramuscular injections of acidic solutions	KO	Prevents the development of mechanical hyperalgesia	125
ASIC3
Carrageenan injection in the gastrocnemius muscle	KO	Did not develop secondary paw hyperalgesia	138
Intraperitoneal administration of 0.6% acetic acid solutions	KO	↓Latency to the onset of pain responses ↑Pain behaviors	20
Tail-pressure assay	KO	↓Pain threshold	20
Activity-induced mechanical hyperalgesia	KO	↑Sensitivity to activity-induced muscle fatigue in male animals (three 1-h runs)	13
Postoperative pain (plantar incisional model)	Intrathecal injections of siRNA targeting ASIC3	↓Postoperative spontaneous, thermal, and postural pain behaviors	29
Carrageenan injection in the gastrocnemius muscle	miRNA-mediated knockdown of ASIC3 in muscle primary afferent neurons	Prevents the development of primary muscle and secondary paw hyperalgesia	138
Muscular ischemia-reperfusion injury	Nerve-specific siRNA knockdown	Attenuates ischemia-reperfusion-induced alterations in voluntary activity and pain behaviors	115
Visceral pain	KO	Partially impaired visceromotor response to colon distension	62

ASIC, acid-sensing ion channel; KO, knockout; ↑, increase; ↓, decrease.

GRANTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants R01-DK-084060 and DK-119183 and by the Cellular Physiology and Kidney Imaging Cores of the Pittsburgh Center for Kidney Research (P30-DK-079307).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

M.D.C. prepared figures; M.D.C. and N.M. drafted manuscript; M.D.C. and N.M. edited and revised manuscript; M.D.C. and N.M. approved final version of manuscript.