Subunit-dependent high-affinity zinc inhibition of acid-sensing ion channels.
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Acid-sensing ion channels (ASICs), a novel class of ligand-gated cation channels activated by protons, are highly expressed in peripheral sensory and central neurons. Activation of ASICs may play an important role in physiological processes such as nociception, mechanosensation, and learning-memory, and in the pathology of neurological conditions such as brain ischemia. Modulation of the activities of ASICs is expected to have a significant influence on the roles that these channels can play in both physiological and/or pathological processes. Here we show that the divalent cation Zn2+, an endogenous trace element, dose-dependently inhibits ASIC currents in cultured mouse cortical neurons at nanomolar concentrations. With ASICs expressed in Chinese hamster ovary cells, Zn2+ inhibits currents mediated by homomeric ASIC1a and heteromeric ASIC1a-ASIC2a channels, without affecting currents mediated by homomeric ASIC1beta, ASIC2a, or ASIC3. Consistent with ASIC1a-specific modulation, high-affinity Zn2+ inhibition is absent in neurons from ASIC1a knock-out mice. Current-clamp recordings and Ca2+-imaging experiments demonstrated that Zn2+ inhibits acid-induced membrane depolarization and the increase of intracellular Ca2+. Mutation of lysine-133 in the extracellular domain of the ASIC1a subunit abolishes the high-affinity Zn2+ inhibition. Our studies suggest that Zn2+ may play an important role in a negative feedback system for preventing overexcitation of neurons during normal synaptic transmission and ASIC1a-mediated excitotoxicity in pathological conditions. (+info)
Stomatin modulates gating of acid-sensing ion channels.
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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. (+info)
Analysis of the membrane topology of the acid-sensing ion channel 2a.
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Acid-sensing ion channels, or ASICs, are members of the amiloride-sensitive cationic channel superfamily that are predicted to have intracellular amino and carboxyl termini and two transmembrane domains connected by a large extracellular loop. This prediction comes from biochemical studies of the mammalian epithelial sodium channels where glycosylation mutants identified the extracellular regions of the channel and a combination of antibody sensitivity and protease action substantiated the intracellular nature of the amino and carboxyl termini. However, although there are highly conserved regions within the different cation channel family members, membrane topology prediction programs provide several alternative structures for the ASICs. Thus, we used glycosylation studies to define the actual membrane topology of the ASIC2a subtype. We deleted the five predicted endogenous asparagine-linked glycosylation sites (Asn-Xaa-(Ser/Thr)) at Asn-22, Asn-365, Asn-392, Asn-478, and Asn-487 to map the extracellular topology. We then introduced exogenous asparagine-linked glycosylation sites at Lys-4, Pro-37, Arg-63, Tyr-67, His-72, Ala-81, Tyr-414, Tyr-423, and Tyr-453 to define the transmembrane domain borders. Finally, we used cell permeabilization studies to confirm the intracellular amino termini of ASIC2a. The data show that Asn-365 and Asn-392 are extracellular and that the introduction of asparagine-linked glycosylation sites at His-72, Ala-81, Tyr-414, and Tyr-423 leads to an increase in molecular mass consistent with an extracellular apposition. In addition, heterologous expression of ASIC2a requires membrane permeabilization for antibody staining. These data confirm the membrane topology prediction that the ASIC2a subtype consists of intracellular amino and carboxyl termini and two transmembrane domains connected by a large extracellular loop. (+info)
Acid-sensing ion channel 2 contributes a major component to acid-evoked excitatory responses in spiral ganglion neurons and plays a role in noise susceptibility of mice.
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Ion channels in the degenerin-epithelial sodium channel (DEG-ENaC) family perform diverse functions, including mechanosensation. Here we explored the role of the vertebrate DEG-ENaC protein, acid-sensing ion channel 2 (ASIC2), in auditory transduction. Contributions of ASIC2 to hearing were examined by comparing hearing threshold and noise sensitivity of wild-type and ASIC2 null mice. ASIC2 null mice showed no significant hearing loss, indicating that the ASIC2 was not directly involved in the mechanotransduction of the mammalian cochlea. However, we found that (1) ASIC2 was present in the spiral ganglion (SG) neurons in the adult cochlea and that externally applied protons induced amiloride-sensitive sodium currents and action potentials in SG neurons in vitro, (2) proton-induced responses were greatly reduced in SG neurons obtained from ASIC2 null mice, indicating that activations of ASIC2 contributed a major portion of the proton-induced excitatory response in SG neurons, and (3) ASIC2 null mice were considerably more resistant to noise-induced temporary, but not permanent, threshold shifts. Together, these data suggest that ASIC2 contributes to suprathreshold functions of the cochlea. The presence of ASIC2 in SG neurons could provide sensors to directly convert local acidosis to excitatory responses, therefore offering a cellular mechanism linking hearing losses caused by many enigmatic causes (e.g., ischemia or inflammation of the inner ear) to excitotoxicity. (+info)
Acid-induced pain and its modulation in humans.
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Despite the discovery of ion channels that are activated by protons, we still know relatively little about the signaling of acid pain. We used a novel technique, iontophoresis of protons, to investigate acid-induced pain in human volunteers. We found that transdermal iontophoresis of protons consistently caused moderate pain that was dose-dependent. A marked desensitization occurred with persistent stimulation, with a time constant of approximately 3 min. Recovery from desensitization occurred slowly, over many hours. Acid-induced pain was significantly augmented in skin sensitized by acute topical application of capsaicin. However, skin desensitized by repeated capsaicin application showed no significant reduction in acid-induced pain, suggesting that both capsaicin-sensitive and insensitive sensory neurons contribute to acid pain. Furthermore, topical application of non-steroidal anti-inflammatory drugs (NSAIDs) significantly attenuated acid-evoked pain but did not affect the heat pain threshold, suggesting a specific interaction between NSAIDs and peripheral acid sensors. Subcutaneous injection of amiloride (1 mm) also significantly inhibited the pain induced by iontophoresis of acid, suggesting an involvement of acid-sensing ion channel (ASIC) receptors. Conversely, iontophoresis of acid over a wide range of skin temperatures from 4 to 40 degrees C produced only minor changes in the induced pain. Together these data suggest a prominent role for ASIC channels and only a minor role for transient receptor potential vanilloid receptor-1 as mediators of cutaneous acid-induced pain. (+info)
Functional properties and pharmacological inhibition of ASIC channels in the human SJ-RH30 skeletal muscle cell line.
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The rhabdomyosarcoma cell line (SJ-RH30) exhibits both ultrastructural and electrophysiological hallmarks of mammalian skeletal muscle. We have used patch-clamp electrophysiology to study acid-gated currents in these cells. At a holding potential of -60 mV, rapid application of extracellular solutions of pH 6.5 produced inward current responses in approximately 85% of cells. The amplitude of these responses exhibited a marked pH dependence. In addition, the kinetics of both activation and desensitization were faster at more acidic pH, whereas the deactivation rate was pH independent. Repeated applications of a pH 6.0 solution produced a tachyphalaxis that could be substantially attenuated by reducing the duration of the acid challenge and increasing the interstimulus interval. The current-voltage relationship of the acid-induced currents was linear at positive potentials but an area of negative slope conductance was observed in the negative potential range. This was not eliminated by removal of extracellular Ca(2+), a manoeuvre which did, however, substantially increase current amplitude. Changing the transmembrane Na(+) gradient altered the current-voltage relationship in a fashion commensurate with an underlying conductance predominantly permeable to Na(+). Pharmacologically, acid-induced currents were blocked 84.4 +/- 1.2% by 30 microm amiloride and 91.8 +/- 3.0% by a 1 : 1000 dilution of Psalmopoeus cambridgei venom. Inhibition by both agents could be reversed by a short period of compound washout. By contrast, APETx2 had no significant effect on acid-evoked currents. These observations strongly suggest the acid-induced current is mediated by ASIC1 channels. In agreement with this, current responses of SJ-RH30 cells to a pH 6.0 challenge were greatly enhanced by extracellular lactate. These data demonstrate the presence of ASIC1 channels in a cell line with skeletal muscle characteristics. In addition, significant levels of ASIC1 and ASIC3 mRNA were found in skeletal muscle tissue samples. These findings are discussed with regard to the role such a conductance might play if present in skeletal muscle in vivo. (+info)
Role of different proton-sensitive channels in releasing calcitonin gene-related peptide from isolated hearts of mutant mice.
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OBJECTIVE: Calcitonin gene-related peptide (CGRP), a potent vasodilator released from a subset of sensory Adelta- and C-fiber afferents, has been suggested to play a beneficial role in myocardial ischemia. The aim of the present study was to investigate some receptors possibly involved in the proton-mediated CGRP release from the heart. METHODS: CGRP release from freshly isolated hearts of mice lacking the capsaicin receptor (TRPV1-/-), the bradykinin receptor type 2 (B2-/-), or the acid-sensing ion channel type 3 (ASIC3-/-) and their wild-type littermates (TRPV1+/+, B2+/+, ASIC3+/+) were compared. Hearts were passed through a series of solutions based on oxygenated synthetic interstitial fluid (SIF). SIF buffered to pH 5.7 or 5.2 was used as an acidic test stimulus, and capsaicin (5x10(-7) M) was finally applied as a positive control. All eluates were processed using an enzyme immunoassay (EIA) for measurement of CGRP concentrations. RESULTS: SIF at pH 5.7 and 5.2 caused significant increases in CGRP release in TRPV1+/+ but not in mice lacking the TRPV1 receptor. The same acid stimuli caused no significant differences in CGRP release between ASIC3+/+ and ASIC3-/- or between B2+/+ and B2-/-, respectively. Capsaicin caused massive CGRP release in all mouse genotypes with the exception of TRPV1-/-. CONCLUSION: We conclude that cardiac acidosis is a strong stimulus to release CGRP from the mouse heart. This effect seems to be primarily mediated through activation of TRPV1 receptors that are known to be expressed by slowly conducting nociceptive primary afferent nerve fibers. (+info)
Acid-sensing properties in rat gastric sensory neurons from normal and ulcerated stomach.
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Gastric acid contributes to dyspeptic symptoms, including abdominal pain, in patients with disorders of the proximal gastrointestinal tract. To examine the molecular sensor(s) of gastric acid chemonociception, we characterized acid-elicited currents in dorsal root ganglion (DRG) and nodose ganglion (NG) neurons that innervate the stomach and examined their modulation after induction of gastric ulcers. A fluorescent dye (DiI) was injected into the stomach wall to retrogradely label gastric sensory neurons. After 1-2 weeks, gastric ulcers were induced by 45 s of luminal exposure of the stomach to 60% acetic acid injected into a clamped area of the distal stomach; control animals received saline. In whole-cell voltage-clamp recordings, all gastric DRG neurons and 55% of NG neurons exhibited transient, amiloride-sensitive, acid-sensing ion-channel (ASIC) currents. In the remaining 45% of NG neurons, protons activated a slow, sustained current that was attenuated by the transient receptor potential vanilloid subtype 1 antagonist, capsazepine. The kinetics and proton sensitivity of amiloride-sensitive ASIC currents differed between NG and DRG neurons. NG neurons had a lower proton sensitivity and faster kinetics, suggesting expression of specific subtypes of ASICs in the vagal and splanchnic innervation of the stomach. Effects of Zn2+ and N,N,N',N'-tetrakis-(2-pyridylmethyl)-ethylenediamine on acid-elicited currents suggest contributions of ASIC1a and ASIC2a subunits. Gastric ulcers altered the properties of acid-elicited currents by increasing pH sensitivity and current density and changing current kinetics in gastric DRG neurons. The distinct properties of NG and DRG neurons and their modulation after injury suggest differential contributions of vagal and spinal afferent neurons to chemosensation and chemonociception. (+info)