Properties Of Acid-sensing Ion Channels Research Articles

Acid Sensing Ion Channels (ASICs) in NS20Y cells - potential role in neuronal differentiation.

Cultured neuronal cell lines can express properties of mature neurons if properly differentiated. Although the precise mechanisms underlying neuronal differentiation are not fully understood, the expression and activation of ion channels, particularly those of Ca2+-permeable channels, have been suggested to play a role. In this study, we explored the presence and characterized the properties of acid-sensing ion channels (ASICs) in NS20Y cells, a neuronal cell line previously used for the study of neuronal differentiation. In addition, the potential role of ASICs in cell differentiation was explored. Reverse Transcription Polymerase Chain Reaction and Western blot revealed the presence of ASIC1 subunits in these cells. Fast drops of extracellular pH activated transient inward currents which were blocked, in a dose dependent manner, by amiloride, a non-selective ASIC blocker, and by Psalmotoxin-1 (PcTX1), a specific inhibitor for homomeric ASIC1a and heteromeric ASIC1a/2b channels. Incubation of cells with PcTX1 significantly reduced the differentiation of NS20Y cells induced by cpt-cAMP, as evidenced by decreased neurite length, dendritic complexity, decreased expression of functional voltage gated Na+ channels. Consistent with ASIC1a inhibition, ASIC1a knockdown with small interference RNA significantly attenuates cpt-cAMP-induced increase of neurite outgrowth. In summary, we described the presence of functional ASICs in NS20Y cells and demonstrate that ASIC1a plays a role in the differentiation of these cells.

The Effect of Hydrophobic Monoamines on Acid-Sensing Ion Channels ASIC1B

Acid-sensing ion channels (ASICs) are widely distributed in both the central and peripheral nervous systems of vertebrates. The pharmacology of these receptors remains poorly investigated, while the search for new ASIC modulators is very important. Recently, we found that some monoamines, which are blockers of NMDA receptors, inhibit and/or potentiate acid-sensing ion channels, depending on the subunit composition of the channels. The effect of 9-aminoacridine, IEM-1921, IEM-2117, and memantine both on native receptors and on recombinant ASIC1a, ASIC2a, and ASIC3 homomers was studied. In the present study, we have investigated the effect of these four compounds on homomeric ASIC1b channels. Experiments were performed on recombinant receptors expressed in CHO cells using the whole-cell patch clamp technique. Only two compounds, 9-aminoacridine and memantine, inhibited ASIC1b channels. IEM-1921 and IEM-2117 were inactive even at a 1000 μM concentration. In most aspects, the effect of the compounds on ASIC1b was similar to their effect on ASIC1a. The distinguishing feature of homomeric ASIC1b channels is a steep activation-dependence, indicating cooperative activation by protons. In our experiments, the curve of the concentration dependence of ASIC1b inhibition by 9-aminoacridine also had a slope (Hill coefficient) of 3.8, unlike ASIC1a homomers, for which the Hill coefficient was close to 1. This finding indicates that the inhibitory effect of 9-aminoacridine is associated with changes in the activation properties of acid-sensing ion channels.

The Molecular Basis for Species-specific Activation of Human TRPA1 Protein by Protons Involves Poorly Conserved Residues within Transmembrane Domains 5 and 6

The surveillance of acid-base homeostasis is concerted by diverse mechanisms, including an activation of sensory afferents. Proton-evoked activation of rodent sensory neurons is mainly mediated by the capsaicin receptor TRPV1 and acid-sensing ion channels. In this study, we demonstrate that extracellular acidosis activates and sensitizes the human irritant receptor TRPA1 (hTRPA1). Proton-evoked membrane currents and calcium influx through hTRPA1 occurred at physiological acidic pH values, were concentration-dependent, and were blocked by the selective TRPA1 antagonist HC030031. Both rodent and rhesus monkey TRPA1 failed to respond to extracellular acidosis, and protons even inhibited rodent TRPA1. Accordingly, mouse dorsal root ganglion neurons lacking TRPV1 only responded to protons when hTRPA1 was expressed heterologously. This species-specific activation of hTRPA1 by protons was reversed in both mouse and rhesus monkey TRPA1 by exchange of distinct residues within transmembrane domains 5 and 6. Furthermore, protons seem to interact with an extracellular interaction site to gate TRPA1 and not via a modification of intracellular N-terminal cysteines known as important interaction sites for electrophilic TRPA1 agonists. Our data suggest that hTRPA1 acts as a sensor for extracellular acidosis in human sensory neurons and should thus be taken into account as a yet unrecognized transduction molecule for proton-evoked pain and inflammation. The species specificity of this property is unique among known endogenous TRPA1 agonists, possibly indicating that evolutionary pressure enforced TRPA1 to inherit the role as an acid sensor in human sensory neurons.

Primary Afferent Nociceptor as a Target for the Relief of Pain

Primary Afferent Nociceptor as a Target for the Relief of Pain

Developmental change in the electrophysiological and pharmacological properties of acid-sensing ion channels in CNS neurons

Acid-sensing ion channels (ASICs) are proton-gated cation channels that play important roles in the CNS including synaptic plasticity and acidosis-mediated neuronal injury. ASIC1a and ASIC2a subunits are predominant in CNS neurons, where homomultimeric and heteromultimeric channel configurations co-exist. Since ASIC1a and ASIC2a have dramatic differences in pH sensitivity, Ca(2+) permeability and channel kinetics, any change in the level of individual subunits may have significant effects on the properties and functions of ASICs. Using patch-clamp recording, fluorescent Ca(2+) imaging and molecular biological techniques, we show dramatic developmental changes in the properties of ASICs in mouse cortical neurons. For example, the amplitude of ASIC currents increases whereas desensitization decreases with neuronal maturation. Decreased H(+) affinity and acid-evoked [Ca(2+)](i) but increased Zn(2+) potentiation were also recorded in mature neurons. RT-PCR revealed significant increases in the ratio of ASIC2/ASIC1 mRNA with neuronal maturation. Thus, contributions of ASIC1a and ASIC2a to overall ASIC-mediated responses undergo distinct developmental changes. These findings may help in understanding the precise role of ASICs in physiological and pathological conditions at different developmental stages.

Characterization of acid-sensing ion channels in medium spiny neurons of mouse striatum

Acid-sensing ion channels (ASICs) regulate synaptic activities and play important roles in neurodegenerative diseases. They are highly expressed in the striatum, where medium spiny neurons (MSNs) are a major population. Given that the properties of ASICs in MSNs are unknown, in this study, we characterized ASICs in MSNs of the mouse striatum. A rapid drop in extracellular pH induced transient inward currents in all MSNs. The pH value for half-maximal activation was 6.25, close to that obtained in homomeric ASIC1a channels. Based on psalmotoxin 1 and zinc sensitivity, ASIC1a (70.5% of neurons) and heteromeric ASIC1a-2 channels (29.5% of neurons) appeared responsible for the acid-induced currents in MSNs. ASIC currents were diminished in MSNs from ASIC1, but not ASIC2, null mice. Furthermore, a drop in pH induced calcium influx by activating homomeric ASIC1a channels. Activation of ASICs increased the membrane excitability of MSNs and lowering extracellular Ca 2+ potentiated ASIC currents. Our data suggest that the homomeric ASIC1a channel represents a majority of the ASIC isoform in MSNs. The potential function of ASICs in the striatum requires further investigation.

Acid Sensing Ion Channels in Dorsal Spinal Cord Neurons

Acid-sensing ion channels (ASICs) are broadly expressed in the CNS, including the spinal cord. However, very little is known about the properties of ASICs in spinal cord neurons compared with brain. We show here that ASIC1a and ASIC2a are the most abundant ASICs in mouse adult spinal cord and are coexpressed by most neurons throughout all the laminas. ASIC currents in cultured embryonic day 14 mouse dorsal spinal neurons mainly flow through homomeric ASIC1a (34% of neurons) and heteromeric ASIC1a plus 2a channels at a ratio of 2:1 (83% of neurons). ASIC2b only has a minor contribution to these currents. The two channel subtypes show different active pH ranges and different inactivation and reactivation kinetics supporting complementary functional properties. One striking property of native dorsal spinal neuron currents and recombinant currents is the pH dependence of the reactivation process. A light sustained acidosis induces a threefold slow-down of the homomeric ASIC1a (from pH 7.4 to pH 7.3) and heteromeric ASIC1a plus 2a (from pH 7.4 to pH 7.2) current reactivation (T(0.5) increasing from 5.77 to 16.84 s and from 0.98 to 3.2 s, respectively), whereas a larger acidosis to pH 6.6 induces a 32-fold slow-down of the ASIC1a plus 2a current reactivation (T(0.5) values increasing to 31.30 s). The pH dependence of ASIC channel reactivation is likely to modulate neuronal excitability associated with repetitive firing in response to extracellular pH oscillations, which can be induced, for example, by intense synaptic activity of central neurons.

Modulation of acid-sensing ion channels by Cu 2+ in cultured hypothalamic neurons of the rat

Acid-sensing ion channels (ASICs) are known to distribute throughout the nervous system and serve important roles in various physiological and pathological processes. However, the properties of ASICs in the hypothalamus, an important region of diencephalon, are little known. We herein used whole-cell patch-clamp recordings to characterize proton-induced cation currents in cultured hypothalamic neurons of the rat, and attributed these transient inward currents to ASICs based on their electrophysiological and pharmacological properties. We further examined the effects of Cu 2+, the third most abundant trace element, on ASICs in hypothalamic neurons. Our results showed that this divalent cation reversibly and concentration-dependently inhibited the amplitude of ASIC currents, and slowed down the desensitization of ASIC channels. Our results also displayed that Cu 2+ modulated ASICs independent of change in membrane potential and extracellular protons, suggesting a noncompetitive mechanism. Furthermore, micromolar concentration of Cu 2+ attenuated the acid-induced membrane depolarization. Taken together, our data demonstrate a modulatory effect of Cu 2+ on ASICs in native hypothalamic neurons and suggest a role of this endogenous metal ion in negatively modulating the increased neuronal membrane excitability caused by activation of ASICs.

Physiology and pharmacology of copper

Acid-sensing ion channels (ASICs) are known to distribute throughout the nervous system and serve important roles in various physiological and pathological processes. However, the properties of ASICs in the hypothalamus, an important region of diencephalon, are little known. We herein used whole-cell patch-clamp recordings to characterize proton-induced cation currents in cultured hypothalamic neurons of the rat, and attributed these transient inward currents to ASICs based on their electrophysiological and pharmacological properties. We further examined the effects of Cu2+, the third most abundant trace element, on ASICs in hypothalamic neurons. Our results showed that this divalent cation reversibly and concentration-dependently inhibited the amplitude of ASIC currents, and slowed down the desensitization of ASIC channels. Our results also displayed that Cu2+ modulated ASICs independent of change in membrane potential and extracellular protons, suggesting a noncompetitive mechanism. Furthermore, micromolar concentration of Cu2+ attenuated the acid-induced membrane depolarization. Taken together, our data demonstrate a modulatory effect of Cu2+ on ASICs in native hypothalamic neurons and suggest a role of this endogenous metal ion in negatively modulating the increased neuronal membrane excitability caused by activation of ASICs.