Acid-sensing Ion Channel Proteins Research Articles

Acid-Sensing Ion Channels' Immunoreactivity in Nerve Profiles and Glomus Cells of the Human Carotid Body.

The carotid body is a major peripheral chemoreceptor that senses changes in arterial blood oxygen, carbon dioxide, and pH, which is important for the regulation of breathing and cardiovascular function. The mechanisms by which the carotid body senses O2 and CO2 are well known; conversely, the mechanisms by which it senses pH variations are almost unknown. Here, we used immunohistochemistry to investigate how the human carotid body contributes to the detection of acidosis, analyzing whether it expresses acid-sensing ion channels (ASICs) and determining whether these channels are in the chemosensory glomic cells or in the afferent nerves. In ASIC1, ASIC2, and ASIC3, and to a much lesser extent ASIC4, immunoreactivity was detected in subpopulations of type I glomus cells, as well as in the nerves of the carotid body. In addition, immunoreactivity was found for all ASIC subunits in the neurons of the petrosal and superior cervical sympathetic ganglia, where afferent and efferent neurons are located, respectively, innervating the carotid body. This study reports for the first time the occurrence of ASIC proteins in the human carotid body, demonstrating that they are present in glomus chemosensory cells (ASIC1 < ASIC2 > ASIC3 > ASIC4) and nerves, presumably in both the afferent and efferent neurons supplying the organ. These results suggest that the detection of acidosis by the carotid body can be mediated via the ASIC ion channels present in the type I glomus cells or directly via sensory nerve fibers.

Cell-Type-Specific Expression Pattern of Proton-Sensing Receptors and Channels in Pituitary Gland

In mammalian cells, extracellular protons act as orthosteric and allosteric ligands for multiple receptors and channels. The aim of this study is to identify proton sensors in the rat pituitary gland. qRT-PCR analysis indicated the expression of G-protein-coupled receptor 68 gene (Gpr68) and acid-sensing ion channel (ASIC) genes Asic1, Asic2, and Asic4 in anterior pituitary cells and Asic1 and Asic2 in immortalized GH3 pituitary cells. Asic1a and Asic2b were the dominant splice isoforms. Single anterior pituitary cell RNA sequencing and immunocytochemical analysis showed that nonexcitable folliculostellate cells express GPR68 gene and protein, whereas excitable secretory cells express ASIC genes and proteins. Asic1 was detected in all secretory cell types, Asic2 in gonadotrophs, thyrotrophs, and somatotrophs, and Asic4 in lactotrophs. Extracellular acidification activated two types of currents in a concentration-dependent manner: a fast-developing, desensitizing current with an estimated EC50-value of pH 6.7 and a slow-developing, non-desensitizing current that required a higher proton concentration for activation. The desensitizing current was abolished by removal of bath sodium and application of amiloride, a blocker of ASIC channels, whereas the non-desensitizing current was amiloride insensitive and voltage dependent. Activation of both currents increased the excitability of secretory pituitary cells, consistent with their potential physiological relevance in control of voltage-gated calcium influx and calcium-dependent cellular functions.

High Expression of Acid-Sensing Ion Channel 2 (ASIC2) in Bone Cells in Osteoporotic Vertebral Fractures.

Little is known about the function of acid-sensing ion channels (ASICs) in bone cells or osteoporotic vertebral fractures (OVF). This study delineated ASICs expression in adult human bone marrow-mesenchymal stem cells- (BM-MSC-) derived osteoblasts and in OVF bone cells. Adult BM-MSC-derived osteoblasts were isolated and cultured in different pH values. Osteogenic markers as alkaline phosphatase (ALP), osteopontin (OPN), and osteocalcin (OC) mRNA were assessed. Western blots method was applied to analyze ASICs protein expression in different pH values. Amiloride was added into the osteogenic media to analyze the Na+/K+ ATPase change. We harvested the vertebral cancellous bone through a bone biopsy needle in 26 OVF patients when performing percutaneous vertebroplasty. Six vertebral bone specimens obtained from 4 patients with high-energy vertebral fractures were used as the control. The reverse transcription polymerase chain reaction was performed to analyze the quantitative mRNA expression of ASICs. Osteogenic markers as ALP, OPN, and OC mRNA were higher expressed in increasing pH values throughout osteoblastogenesis. ASIC proteins were higher expressed in lower pH media, especially ASIC3, and ASIC4. The highest protein expression at days 7, 14, and 21 was ASIC2, ASIC4, and ASIC3, respectively. Expression of Na+/K+ ATPase was significantly decreased in cultured osteoblasts by addition of amiloride into the pH 6.9 osteogenic media. ASIC2 mRNA was most highly expressed with a 65.93-fold increase in the biopsied vertebral bone cells in OVF compared with the control. In conclusion, we found osteoblastogenesis was reduced in an acidic environment, and ASIC2, ASIC3, and ASIC4 were most highly expressed in turn during osteoblastogenesis within acidic media. ASIC2 was the most abundantly expressed gene in human bone cells in OVF compared with the control. ASIC2 could be crucial in the pathogenesis of osteoporosis and could serve as a therapeutic target for antiosteoporotic therapies.

Dysfunction of Acid‐Sensing Ion Channel 5 (ASIC5) in Unipolar Brush Cells of the Vestibulocerebellum Causes Ataxia

Acid‐sensing ion channels (ASICs) are ligand‐gated ion channels in the Degenerin/Epithelial Na+ channel (Deg/ENaC) family. ASIC proteins are richly expressed in mammalian neurons. Mammals express five ASIC genes: ASIC1‐5. The gene product of ASIC5 is an orphan member of the family about which little is known. To assess the function of ASIC5, we created ASIC5 reporter and knockout mice. We find that ASIC5 is chiefly expressed in the brain in the cerebellum, specifically in the ventral uvula and nodulus of the vestibulocerebellum. Here, ASIC5 is restrictively expressed in a subset of interneurons in the granular layer. The locale, distinctive shape and immunohistochemical properties of these ASIC5‐expressing interneurons identify them as unipolar brush cells (UBC). ASIC5 is richly expressed in a subset of UBCs that also express the metabotropic glutamate receptor 1α (mGluR1α). Such observations are consistent with ASIC5 playing a key role in the physiology of UBCs and in the function of the vestibulocerebellum, which is essential for normal balance and movement. Consistent with this, knockout of ASIC5 results in ataxia and uncoordinated bodily movements.Support or Funding InformationThis work was funded by a CPRIT IIRG grant (RP150408) and a NIH/NCI R21 grant (CA194138)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Naked mole-rat acid-sensing ion channel 3 forms nonfunctional homomers, but functional heteromers

Acid-sensing ion channels (ASICs) form both homotrimeric and heterotrimeric ion channels that are activated by extracellular protons and are involved in a wide range of physiological and pathophysiological processes, including pain and anxiety. ASIC proteins can form both homotrimeric and heterotrimeric ion channels. The ASIC3 subunit has been shown to be of particular importance in the peripheral nervous system with pharmacological and genetic manipulations demonstrating a role in pain. Naked mole-rats, despite having functional ASICs, are insensitive to acid as a noxious stimulus and show diminished avoidance of acidic fumes, ammonia, and carbon dioxide. Here we cloned naked mole-rat ASIC3 (nmrASIC3) and used a cell-surface biotinylation assay to demonstrate that it traffics to the plasma membrane, but using whole-cell patch clamp electrophysiology we observed that nmrASIC3 is insensitive to both protons and the non-proton ASIC3 agonist 2-guanidine-4-methylquinazoline. However, in line with previous reports of ASIC3 mRNA expression in dorsal root ganglia neurons, we found that the ASIC3 antagonist APETx2 reversibly inhibits ASIC-like currents in naked mole-rat dorsal root ganglia neurons. We further show that like the proton-insensitive ASIC2b and ASIC4, nmrASIC3 forms functional, proton-sensitive heteromers with other ASIC subunits. An amino acid alignment of ASIC3s between 9 relevant rodent species and human identified unique sequence differences that might underlie the proton insensitivity of nmrASIC3. However, introducing nmrASIC3 differences into rat ASIC3 (rASIC3) produced only minor differences in channel function, and replacing the nmrASIC3 sequence with that of rASIC3 did not produce a proton-sensitive ion channel. Our observation that nmrASIC3 forms nonfunctional homomers may reflect a further adaptation of the naked mole-rat to living in an environment with high-carbon dioxide levels.

Acid-sensing ion channels in healthy and degenerated human intervertebral disc

Acid-sensing ion channels (ASICs) are a family of H+-gated voltage-insensitive ion channels that respond to extracellular acidification by regulating transmembrane Ca2+ flux. Moreover, ASICs can also be gated by mechanical forces and may function as mechanosensors. The cells of the intervertebral disc (IVD) have an unusual acidic and hyperosmotic microenvironment. Changes in the pH and osmolarity determine the viability of IVD cells and the composition of the extracellular matrix, and both are the basis of IVD degeneration. In this study, the expression of ASICs (ASIC1, ASIC2, ASIC3 and ASIC4) mRNAs and proteins in human healthy and degenerated IVD was evaluated by quantitative reverse transcription-quantitative polymerase chain reaction and Western blot. The distribution of ASIC proteins was determined by immunohistochemistry. The mRNAs for all ASICs were detected in normal human IVD, and significantly increased levels were found in degenerated IVD. Western blots demonstrated the presence of proteins with estimated molecular weights of approximately 68–72 kDa. In both the annulus fibrosus (AF) and nucleus pulposus (NP) of normal IVD, ASIC2 is the most frequently expressed ASIC followed by ASIC3, ASIC1 and ASIC4. In the AF of degenerated IVD, there was a significant increase in the number of ASIC1 and ASIC4 positive cells, whereas in the NP, we found significant increase of expression of ASIC1, ASIC2 and ASIC3. These results describe the occurrence and localization of different ASICs in human healthy IVD, and their increased expression in degenerated IVD, thus suggesting that ASICs may be involved in IVD degeneration.

Stomatin‐domain protein interactions with acid‐sensing ion channels modulate nociceptor mechanosensitivity

Acid-sensing ion channels (ASICs) and their interaction partners of the stomatin family have all been implicated in sensory transduction. Single gene deletion of asic3, asic2, stomatin, or stoml3 all result in deficits in the mechanosensitivity of distinct cutaneous afferents in the mouse. Here, we generated asic3−/−:stomatin−/−, asic3−/−:stoml3−/− and asic2−/−:stomatin−/− double mutant mice to characterize the functional consequences of stomatin–ASIC protein interactions on sensory afferent mechanosensitivity. The absence of ASIC3 led to a clear increase in mechanosensitivity in rapidly adapting mechanoreceptors (RAMs) and a decrease in the mechanosensitivity in both Aδ- and C-fibre nociceptors. The increased mechanosensitivity of RAMs could be accounted for by a loss of adaptation which could be mimicked by local application of APETx2 a toxin that specifically blocks ASIC3. There is a substantial loss of mechanosensitivity in stoml3−/− mice in which ∼35% of the myelinated fibres lack a mechanosensitive receptive field and this phenotype was found to be identical in asic3−/−:stoml3−/− mutant mice. However, Aδ-nociceptors showed much reduced mechanosensitivity in asic3−/−:stoml3−/− mutant mice compared to asic3−/− controls. Interestingly, in asic2−/−:stomatin−/− mutant mice many Aδ-nociceptors completely lost their mechanosensitivity which was not observed in asic2−/− or stomatin−/− mice. Examination of stomatin−/−:stoml3−/− mutant mice indicated that a stomatin/STOML3 interaction is unlikely to account for the greater Aδ-nociceptor deficits in double mutant mice. A key finding from these studies is that the loss of stomatin or STOML3 in asic3−/− or asic2−/− mutant mice markedly exacerbates deficits in the mechanosensitivity of nociceptors without affecting mechanoreceptor function.

Neurosensory mechanotransduction through acid‐sensing ion channels

Acid-sensing ion channels (ASICs) are voltage-insensitive cation channels responding to extracellular acidification. ASIC proteins have two transmembrane domains and a large extracellular domain. The molecular topology of ASICs is similar to that of the mechanosensory abnormality 4- or 10-proteins expressed in touch receptor neurons and involved in neurosensory mechanotransduction in nematodes. The ASIC proteins are involved in neurosensory mechanotransduction in mammals. The ASIC isoforms are expressed in Merkel cell–neurite complexes, periodontal Ruffini endings and specialized nerve terminals of skin and muscle spindles, so they might participate in mechanosensation. In knockout mouse models, lacking an ASIC isoform produces defects in neurosensory mechanotransduction of tissue such as skin, stomach, colon, aortic arch, venoatrial junction and cochlea. The ASICs are thus implicated in touch, pain, digestive function, baroreception, blood volume control and hearing. However, the role of ASICs in mechanotransduction is still controversial, because we lack evidence that the channels are mechanically sensitive when expressed in heterologous cells. Thus, ASIC channels alone are not sufficient to reconstruct the path of transducing molecules of mechanically activated channels. The mechanotransducers associated with ASICs need further elucidation. In this review, we discuss the expression of ASICs in sensory afferents of mechanoreceptors, findings of knockout studies, technical issues concerning studies of neurosensory mechanotransduction and possible missing links. Also we propose a molecular model and a new approach to disclose the molecular mechanism underlying the neurosensory mechanotransduction.

Human odontoblasts express transient receptor protein and acid‐sensing ion channel mechanosensor proteins

Diverse proteins of the denegerin/epithelial sodium channel (DEG/ENa(+) C) superfamily, in particular those belonging to the acid-sensing ion channel (ASIC) family, as well as some members of the transient receptor protein (TRP) channel, function as mechanosensors or may be required for mechanosensation in a diverse range of species and cell types. Therefore, we investigated the putative mechanosensitive function of human odontoblasts using immunohistochemistry to detect ENa(+) C subunits (α, β, and γ) and ASIC (1, 2, 3, and 4) proteins, as well as TRPV4, in these cells. Positive and specific immunoreactivity in the odontoblast soma and/or processes was detected for all proteins studied except α-ENa(+) C. The intensity of immunostaining was high for β-ENa(+) C and ASIC2, whereas it was low for ASIC1, ASIC3, γ-ENa(+) C, and TRPV4, being absent for α-ENa(+) C and ASIC4. These results suggest that human odontoblasts in situ express proteins related to mechanosensitive channels that probably participate in the mechanisms involved in teeth sensory transmission.

Amiloride Docking to Acid-sensing Ion Channel-1

Amiloride is a small molecule diuretic, which has been used to dissect sodium transport pathways in many different systems. This drug is known to interact with the epithelial sodium channel and acid-sensing ion channel proteins, as well as sodium/hydrogen antiporters and sodium/calcium exchangers. The exact structural basis for these interactions has not been elucidated as crystal structures of these proteins have been challenging to obtain, though some involved residues and domains have been mapped. This work examines the interaction of amiloride with acid-sensing ion channel-1, a protein whose structure is available using computational and experimental techniques. Using molecular docking software, amiloride and related molecules were docked to model structures of homomeric human ASIC-1 to generate potential interaction sites and predict which analogs would be more or less potent than amiloride. The predictions made were experimentally tested using whole-cell patch clamp. Drugs previously classified as NCX or NHE inhibitors are shown to also inhibit hASIC-1. Potential docking sites were re-examined against experimental data to remove spurious interaction sites. The voltage sensitivity of inhibitors was also examined. Using the aggregated data from these computational and experimental experiments, putative interaction sites for amiloride and hASIC-1 have been defined. Future work will experimentally verify these interaction sites, but at present this should allow for virtual screening of drug libraries at these putative interaction sites.

Yes, No, Maybe So

The myogenic response is an intrinsic vascular response characterized by vasoconstriction in response to increases and vasodilation to decreases in perfusion pressure. Recent studies suggest this response may play a significant role in the protection of the renal microcirculation from pressure dependent injury, especially with concomitant renal disease.1 Although the myogenic response was first described more than 100 years ago, the molecular mechanisms underlying the transduction of pressure into a cellular event (ie, vasoconstriction) in vascular smooth muscle cells (VSMCs) has remained elusive. Several potential molecules with a connection to mechanosensitive responses have been identified including integrins, transient receptor potential (TRP) channels, and members of the Epithelial Na+ Channel (ENaC) family.2 Members of the ENaC family have received recent attention because of evidence for and against a role for ENaC proteins in myogenic constriction in the renal circulation. The current study by Guan et al helps to address the controversial role of ENaC in afferent arteriolar myogenic constriction.3 ENaC and closely related Acid Sensing Ion Channel (ASIC) proteins are considered potential mechanotransducers in VSMCs because of their strong evolutionary connection to a group of proteins termed degenerins. The degenerins are a group of nematode Caenorhabditis elegans proteins that comprise the ion channel pore in a large multimeric mechanosensory complex.4 Genetic evidence suggests the pore forming degenerins are tethered to the cytoskeleton and extracellular matrix and aid in force transduction.4 The …

The (F)low Down on the Endothelial Epithelial Sodium Channel

The epithelial sodium channel (ENaC) contributes to blood pressure homeostasis through renal salt and water transport; increases in ENaC activity increase salt and water reabsorption along the distal nephron, increasing extracellular fluid volume and blood pressure. The importance of tubular ENaC in cardiovascular homeostasis is manifested in Liddle’s disease and type I pseudohypoaldosteronism, diseases associated with the gain and loss of ENaC function, respectively. However, several recent studies suggest that ENaC proteins may play a more ubiquitous role in blood pressure control as mechanosensors. Because ENaC proteins are closely related to a family of mechanosensitive proteins in the nematode termed “degenerins,” several laboratories have addressed the potential role of ENaC proteins as mechanosensors.1–3 In addition to their role in salt and water homeostasis, ENaC proteins and their relatives, acid-sensing ion channels (ASICs), may contribute to control of blood pressure through reflex regulation of the autonomic nervous system (baroreflex, chemoreflex, and metaboreflex) and local control of vascular tone.1 Recent literature reviews suggest that ENaC proteins may function as sensors of pressure-induced vascular stretch and laminar flow.1–3 The role of ENaC as a mediator of pressure-induced constriction in certain vessels is supported by several investigations; however, the role of ENaC as a flow sensor remains unclear.1 Because of their evolutionary relationship to mechanotransduction in the nematode, several laboratories have suggested that ENaC channels may act as flow …

Sensing tension: epithelial sodium channel/acid-sensing ion channel proteins in cardiovascular homeostasis.

The epithelial sodium (Na+) channel (ENaC) plays a critical role in blood pressure regulation by controlling renal salt and water reabsorption. Channel overactivity can lead to severe hypertension and underactivity to salt wasting and hypotension.1 In addition to their role in salt/water homeostasis, recent studies suggest that ENaC proteins, and their relatives, the acid-sensing ion channel (ASIC) proteins, may play more ubiquitous roles in cardiovascular regulation than considered previously. Recent evidence suggests that ENaC/ASIC proteins may act as mechanosensors and chemosensors in the cardiovascular system. ENaC/ASIC proteins are expressed in mechanosensing and chemosensing tissues, such as vascular smooth muscle cells (VSMCs), carotid body glomus cells, and sensory neurons innervating arterial baroreceptors, heart, and skeletal muscle. Disruption of the ENaC/ASIC channels alters myogenic constriction, arterial chemoreceptor and baroreceptor responses, and acid-induced responses in heart and skeletal muscle. This brief review summarizes the evidence supporting a role for ENaC and ASIC proteins in diverse systems of cardiovascular mechanosensing and chemosensing. Together, these studies suggest that ENaC/ASIC proteins contribute to cardiovascular homeostasis by mediating neural and local regulatory mechanisms. ENaC and ASIC proteins are members of a protein family termed the degenerin (DEG)/ENaC/ASIC family. Members of this family are expressed in a wide range of species (nematode Caenorhabditis elegans , Drosophila , and mammals) and participate in diverse biological functions, including neurodegeneration, acid sensation, taste, learning and memory, proprioception, Na+/water transport, and mechanosensation. All of the members of the DEG/ENaC/ASIC family share a highly conserved structure: intracellular N and C termini and 2 membrane-spanning domains separated by a large extracellular domain. Most DEG/ENaC/ASIC proteins form amiloride sensitive, nonvoltage, gated cation channels.1,2 ### C elegans DEGs Members were first identified in the nematode, where a chemically induced mutation caused a subset of neurons to swell and lyse. This phenotype led to the first …

ASIC proteins regulate smooth muscle cell migration

The purpose of the present study was to investigate Acid Sensing Ion Channel (ASIC) protein expression and importance in cellular migration. We recently demonstrated that Epithelial Na + Channel (ENaC) proteins are required for vascular smooth muscle cell (VSMC) migration; however, the role of the closely related ASIC proteins has not been addressed. We used RT-PCR and immunolabeling to determine expression of ASIC1, ASIC2, ASIC3 and ASIC4 in A10 cells. We used small interference RNA to silence individual ASIC expression and determine the importance of ASIC proteins in wound healing and chemotaxis (PDGF-bb)-initiated migration. We found ASIC1, ASIC2, and ASIC3, but not ASIC4, expression in A10 cells. ASIC1, ASIC2, and ASIC3 siRNA molecules significantly suppressed expression of their respective proteins compared to non-targeting siRNA (RISC) transfected controls by 63%, 44%, and 55%, respectively. Wound healing was inhibited by 10, 20, and 26% compared to RISC controls following suppression of ASIC1, ASIC2, and ASIC3, respectively. Chemotactic migration was inhibited by 30% and 45%, respectively, following suppression of ASIC1 and ASIC3. ASIC2 suppression produced a small, but significant, increase in chemotactic migration (4%). Our data indicate that ASIC expression is required for normal migration and may suggest a novel role for ASIC proteins in cellular migration.

ASIC 2 and 3 proteins are required for VSMC wound healing

Vascular smooth muscle cell (VSMC) migration plays a key role in tissue repair after arterial wall injury. Although mechanosensitive ion channels (MSIC) may play an important role in cell migration, the molecular identity of MSIC in VSMCs has not been determined. Degenerin/Epithelial Na+ Channels (DEG/ENaC) are good canditates because they are mechanosensors in a diverse range of species and cell types. We recently demonstrated ENaC proteins are required for migration associated with wound healing in VSMCs. However, it is unknown if Acid Sensing Ion Channels (ASIC), other DEG/ENaC members, also contribute to wound healing in VSMCs. Expression of ASIC 1, 2, and 3 molecules was determined by RT-PCR, immunostaining, and Western blotting in cultured VSMCs (A-10 cells). To determine if ASIC proteins are required for VSMC migration associated with wound healing, we suppressed ASIC expression using siRNA and then examined healing. Confluent VSMC monolayers were transiently transfected with non-targeting control siRNA (RISC) and ASIC 1, 2, or 3 siRNA molecules, then wounded by scratching with a pipette tip. Healing, quantified as reinvasion of initial wounded area, was reduced by suppression of ASIC 2 (55.4+/−2.0%, n=12) and ASIC 3 (50.4+/−2.6%, n=12), but not ASIC 1 (61.9+/−2.1%, n=12) expression when compared to RISC controls (68.5+/−4.4%, n=12). These data suggest a new role for ASIC 2 and 3 proteins in VSMCs, suggesting they may be required for vascular tissue repair. Whether ASIC 2 and 3 channels are proton- or mechanically-gated in VSMCs remains to be determined. Supported:NIH-NHLBI

Expression and Localization of Acid‐Sensing Ion Channels in Mouse Nodose Ganglia

Transduction of mechanosensation and chemosensation in sensory neurons may be mediated by various subunits of the ASIC (acid-sensing ion channel) family. We and others have experimental data to suggest that ASIC2 subunits may be more mechanosensitive whereas ASIC1 and ASIC3 may be more proton sensitive. The expression of various ASIC subunits has been reported in DRG neurons. Because of our interest in cardiovascular sensory signals we used quantitative PCR to evaluate the mRNA expression of ASIC subunits in nodose ganglia of adult mice (n = 5). These ganglia contain the soma of vagal sensory nerves including cardiopulmonary and aortic baroreceptor afferents. The results (Fig. A) show similar relative mRNA levels for four subunits (ASIC1a, 1b, 2a and 3) with higher expression of ASIC2b subunits. In ASIC2 deficient mice the mRNA expression of other subunits was not altered significantly (Fig. B). Specific antibodies were used in the immunohistochemical assay to determine the locations of ASIC proteins. The colocalization of ASIC1, 2 and 3 subunits was observed in the same nodose neurons, supporting the heteromultimeric model of ASIC channels. It is also possible that specific subunits may target selectively the mechanosensitive versus proton sensitive nerve endings from the same neuron. These results provide the basis for evaluating changes in expression of various subunits in pathologic states. (HL14388)

Proton Sensitivity Ca2+Permeability and Molecular Basis of Acid-Sensing Ion Channels Expressed in Glabrous and Hairy Skin Afferents

We contrasted the physiology and peripheral targets of subclassified nociceptive and nonnociceptive afferents that express acid-sensing ion channel (ASIC)-like currents. The threshold for current activation was similar in eight distinct cell subclasses regardless of functional modality (pH 6.8). When potency was determined from concentration-response curves, nonnociceptors exhibited currents with significantly greater potency than that of all but one class of nociceptors (pH50 = 6.54 and 6.75 vs. 6.20-6.34). In nonnociceptive cells, acid transduction was also confined to a very narrow range (0.1-0.3 vs. 0.8-1.4 pH units for nociceptors). Simultaneous whole cell recording and ratiometric imaging of three peptidergic nociceptive classes were consistent with the expression of Ca2+ -permeable ASICs. Sensitivity to psalmotoxin and flurbiprofen indicated the presence of Ca2+ -permeable ASIC1a. Immunocytochemistry on these subclassified populations revealed a differential distribution of five ASIC proteins consistent with Ca2+ permeability and differential kinetics of proton-gated currents (type 5: ASIC1a, 1b, 2a, 2b, 3; type 8a: ASIC1a, 1b, 3; type 8b: ASIC1a, 1b, 2a, 2b, 3). Using DiI tracing, we found that nociceptive classes had discrete peripheral targets. ASIC-expressing types 8a and 9 projected to hairy skin, but only types 8a and 13 projected to glabrous skin. Non-ASIC-expressing types 2 and 4 were present only in hairy skin. We conclude that ASIC-expressing nociceptors differ from ASIC-expressing nonnociceptors mainly by range of proton reactivity. ASIC- as well as non-ASIC-expressing nociceptors have highly distinct cutaneous targets, and only one class was consistent with the existence of a generic C polymodal nociceptor (type 8a).

Stomatin Modulates Gating of Acid-sensing Ion Channels

Acid-sensing ion channels (ASICs) are H(+)-gated members of the degenerin/epithelial Na(+) channel (DEG/ENaC) family in vertebrate neurons. Several ASICs are expressed in sensory neurons, where they play a role in responses to nociceptive, taste, and mechanical stimuli; others are expressed in central neurons, where they participate in synaptic plasticity and some forms of learning. Stomatin is an integral membrane protein found in lipid/protein-rich microdomains, and it is believed to regulate the function of ion channels and transporters. In Caenorhabditis elegans, stomatin homologs interact with DEG/ENaC channels, which together are necessary for normal mechanosensation in the worm. Therefore, we asked whether stomatin interacts with and modulates the function of ASICs. We found that stomatin co-immunoprecipitated and co-localized with ASIC proteins in heterologous cells. Moreover, stomatin altered the function of ASIC channels. Stomatin potently reduced acid-evoked currents generated by ASIC3 without changing steady state protein levels or the amount of ASIC3 expressed at the cell surface. In contrast, stomatin accelerated the desensitization rate of ASIC2 and heteromeric ASICs, whereas current amplitude was unaffected. These data suggest that stomatin binds to and alters the gating of ASICs. Our findings indicate that modulation of DEG/ENaC channels by stomatin-like proteins is evolutionarily conserved and may have important implications for mammalian nociception and mechanosensation.