Anne Baron

A new sea anemone peptide, APETx2, inhibits ASIC3, a major acid-sensitive channel in sensory neurons

From a systematic screening of animal venoms, we isolated a new toxin (APETx2) from the sea anemone Anthopleura elegantissima, which inhibits ASIC3 homomeric channels and ASIC3‐containing heteromeric channels both in heterologous expression systems and in primary cultures of rat sensory neurons. APETx2 is a 42 amino‐acid peptide crosslinked by three disulfide bridges, with a structural organization similar to that of other sea anemone toxins that inhibit voltage‐sensitive Na+ and K+ channels. APETx2 reversibly inhibits rat ASIC3 (IC50=63 nM), without any effect on ASIC1a, ASIC1b, and ASIC2a. APETx2 directly inhibits the ASIC3 channel by acting at its external side, and it does not modify the channel unitary conductance. APETx2 also inhibits heteromeric ASIC2b+3 current (IC50=117 nM), while it has less affinity for ASIC1b+3 (IC50=0.9 μM), ASIC1a+3 (IC50=2 μM), and no effect on the ASIC2a+3 current. The ASIC3‐like current in primary cultured sensory neurons is partly and reversibly inhibited by APETx2 with an IC50 of 216 nM, probably due to the mixed inhibitions of various co‐expressed ASIC3‐containing channels.

A tarantula peptide against pain via ASIC1a channels and opioid mechanisms

Psalmotoxin 1, a peptide extracted from the South American tarantula Psalmopoeus cambridgei, has very potent analgesic properties against thermal, mechanical, chemical, inflammatory and neuropathic pain in rodents. It exerts its action by blocking acid-sensing ion channel 1a, and this blockade results in an activation of the endogenous enkephalin pathway. The analgesic properties of the peptide are suppressed by antagonists of the μ and δ-opioid receptors and are lost in Penk1−/− mice.

ASIC‐like, proton‐activated currents in rat hippocampal neurons

The expression of mRNA for acid sensing ion channels (ASIC) subunits ASIC1a, ASIC2a and ASIC2b has been reported in hippocampal neurons, but the presence of functional hippocampal ASIC channels was never assessed. We report here the first characterization of ASIC‐like currents in rat hippocampal neurons in primary culture. An extracellular pH drop induces a transient Na+ current followed by a sustained non‐selective cation current. This current is highly sensitive to pH with an activation threshold around pH 6.9 and a pH0.5 of 6.2. About half of the total peak current is inhibited by the spider toxin PcTX1, which is specific for homomeric ASIC1a channels. The remaining PcTX1‐resistant ASIC‐like current is increased by 300 μm Zn2+ and, whereas not fully activated at pH 5, it shows a pH0.5 of 6.0 between pH 7.4 and 5. We have previously shown that Zn2+ is a co‐activator of ASIC2a‐containing channels. Thus, the hippocampal transient ASIC‐like current appears to be generated by a mixture of homomeric ASIC1a channels and ASIC2a‐containing channels, probably heteromeric ASIC1a+2a channels. The sustained non‐selective current suggests the involvement of ASIC2b‐containing heteromeric channels. Activation of the hippocampal ASIC‐like current by a pH drop to 6.9 or 6.6 induces a transient depolarization which itself triggers an initial action potential (AP) followed by a sustained depolarization and trains of APs. Zn2+ increases the acid sensitivity of ASIC channels, and consequently neuronal excitability. It is probably an important co‐activator of ASIC channels in the central nervous system.

Zn2+ and H+ Are Coactivators of Acid-sensing Ion Channels

Acid-sensing ion channels (ASICs) are cationic channels activated by extracellular protons. They are expressed in sensory neurons, where they are thought to be involved in pain perception associated with tissue acidosis. They are also expressed in brain. A number of brain regions, like the hippocampus, contain large amounts of chelatable vesicular Zn2+. This paper shows that Zn2+ potentiates the acid activation of homomeric and heteromeric ASIC2a-containing channels (i.e.ASIC2a, ASIC1a+2a, ASIC2a+3), but not of homomeric ASIC1a and ASIC3. The EC50 for Zn2+ potentiation is 120 and 111 μm for the ASIC2a and ASIC1a+2a current, respectively. Zn2+ shifts the pH dependence of activation of the ASIC1a+2a current from a pH0.5 of 5.5 to 6.0. Systematic mutagenesis of the 10 extracellular histidines of ASIC2a leads to the identification of two residues (His-162 and His-339) that are essential for the Zn2+ potentiating effect. Mutation of another histidine residue, His-72, abolishes the pH sensitivity of ASIC2a. This residue, which is located just after the first transmembrane domain, seems to be an essential component of the extracellular pH sensor of ASIC2a.

Knockout of the ASIC2 channel in mice does not impair cutaneous mechanosensation, visceral mechanonociception and hearing

Mechanosensitive cation channels are thought to be crucial for different aspects of mechanoperception, such as hearing and touch sensation. In the nematode C. elegans, the degenerins MEC‐4 and MEC‐10 are involved in mechanosensation and were proposed to form mechanosensitive cation channels. Mammalian degenerin homologues, the H+‐gated ASIC channels, are expressed in sensory neurones and are therefore interesting candidates for mammalian mechanosensors. We investigated the effect of an ASIC2 gene knockout in mice on hearing and on cutaneous mechanosensation and visceral mechanonociception. However, our data do not support a role of ASIC2 in those facets of mechanoperception.

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 (T0.5 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 (T0.5 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.

Effects of neuropeptide SF and related peptides on acid sensing ion channel 3 and sensory neuron excitability.

Acid sensing ion channel 3 (ASIC3) is a cation channel gated by extracellular protons. It is highly expressed in sensory neurons, including small nociceptive neurons and has been proposed to participate in pain perception associated with tissue acidosis and in mechanoperception. Neuropeptide FF (NPFF) and FMRFamide have been shown to potentiate proton-gated currents from cultured sensory neurons and acid sensing ion channel (ASIC) cDNA transfected cells. In this study, we report that another mammalian peptide neuropeptide SF (NPSF), derived from the same precursor, also considerably increases the amplitude of the sustained current of heterologously expressed ASIC3 (12-fold vs. 19- and nine-fold for FMRFamide and NPFF, respectively) with an EC(50) of approximately 50 microM. Similar effects were also observed on endogenous ASIC3-like sustained current recorded from DRG neurons although of smaller amplitudes (two-, three- and seven-fold increase for NPSF, NPFF and FMRFamide, respectively), and essentially related to a slowing down of the inactivation rate. Importantly, this modulation induced changes in neuronal excitability in response to an electrical stimulus applied during extracellular acidification. ASIC3-mediated sustained depolarisation, and its regulation by neuropeptides, could thus be important in regulating polymodal neuron excitability particularly under inflammatory conditions where the expression levels of both NPFF precursor and ASIC3 are increased.

The receptor site of the spider toxin PcTx1 on the proton-gated cation channel ASIC1a.

Acid‐sensing ion channels (ASICs) are excitatory neuronal cation channels, involved in physiopathological processes related to extracellular pH fluctuation such as nociception, ischaemia, perception of sour taste and synaptic transmission. The spider peptide toxin psalmotoxin 1 (PcTx1) has previously been shown to inhibit specifically the proton‐gated cation channel ASIC1a. To identify the binding site of PcTx1, we produced an iodinated form of the toxin (125I‐PcTx1YN) and developed a set of binding and electrophysiological experiments on several chimeras of ASIC1a and the PcTx1‐insensitive channels ASIC1b and ASIC2a. We show that 125I‐PcTx1YN binds specifically to ASIC1a at a single site, with an IC50 of 128 pm, distinct from the amiloride blocking site. Results obtained from chimeras indicate that PcTx1 does not bind to ASIC1a transmembrane domains (M1 and M2), involved in formation of the ion pore, but binds principally on both cysteine‐rich domains I and II (CRDI and CRDII) of the extracellular loop. The post‐M1 and pre‐M2 regions, although not involved in the binding site, are crucial for the ability of PcTx1 to inhibit ASIC1a current. The linker domain between CRDI and CRDII is important for their correct spatial positioning to form the PcTx1 binding site. These results will be useful for the future identification or design of new molecules acting on ASICs.

Venom toxins in the exploration of molecular, physiological and pathophysiological functions of acid-sensing ion channels

Acid-sensing ion channels (ASICs) are voltage-independent proton-gated cation channels that are largely expressed in the nervous system as well as in some non-neuronal tissues. In rodents, six different isoforms (ASIC1a, 1b, 2a, 2b, 3 and 4) can associate into homo- or hetero-trimers to form a functional channel. Specific polypeptide toxins targeting ASIC channels have been isolated from the venoms of spider (PcTx1), sea anemone (APETx2) and snakes (MitTx and mambalgins). They exhibit different and sometimes partially overlapping pharmacological profiles and are usually blockers of ASIC channels, except for MitTx, which is a potent activator. This review focuses on the use of these toxins to explore the structure-function relationships, the physiological and the pathophysiological roles of ASIC channels, illustrating at the same time the therapeutic potential of some of these natural compounds.

Current perspectives on acid‑sensing ion channels: new advances and therapeutic implications

Acid-sensing ion channels (ASICs) form a family of voltage-independent cation channels that predominantly conduct Na+ ions, and were identified at the molecular level a little more than a decade ago. ASICs are activated by extracellular acidification within the physiological range, and they form effective proton sensors in both central and peripheral sensory neurons. A combination of genetic and pharmacologic approaches has revealed their implication in an increasing number of physiological and pathophysiological processes – most of them associated with extracellular pH fluctuations, ranging from synaptic plasticity, learning, memory, fear, depression, seizure termination and neuronal degeneration to nociception and mechanosensation. ASICs, therefore, emerge as new potential therapeutic targets in the management of psychiatric disorders, stroke, neurodegenerative diseases and pain.