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 þ and K þ channels. APETx2 reversibly inhibits rat ASIC3(IC 50 ¼ 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 þ 3 current (IC 50 ¼117 nM), while it has less affinity for ASIC1b þ 3 (IC 50 ¼ 0.9 lM), ASIC1a þ 3 (IC 50 ¼ 2 lM), and no effect on the ASIC2a þ 3 current. The ASIC3-like current in primary cultured sensory neurons is partly and reversibly inhibited by APETx2 with an IC 50 of 216 nM, probably due to the mixed inhibitions of various co-expressed ASIC3-containing channels.

Tissue acidosis is an important feature of inflammation. It is a direct cause of pain and hyperalgesia. Protons activate sensory neurons mainly through acid-sensing ion channels (ASICs) and the subsequent membrane depolarization that leads to action potential generation. We had previously shown that ASIC transcript levels were increased in inflammatory conditions in vivo. We have now found that this increase is caused by the proinflammatory mediators NGF, serotonin, interleukin-1, and bradykinin. A mixture of these mediators increases ASIC-like current amplitude on sensory neurons as well as the number of ASIC-expressing neurons and leads to a higher sensory neuron excitability. An analysis of the promoter region of the ASIC3 encoding gene, an ASIC specifically expressed in sensory neurons and associated with chest pain that accompanies cardiac ischemia, reveals that gene transcription is controlled by NGF and serotonin.

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Polypeptide toxins have played a central part in understanding physiological and physiopathological functions of ion channels. In the field of pain, they led to important advances in basic research and even to clinical applications. Acid-sensing ion channels (ASICs) are generally considered principal players in the pain pathway, including in humans. A snake toxin activating peripheral ASICs in nociceptive neurons has been recently shown to evoke pain. Here we show that a new class of three-finger peptides from another snake, the black mamba, is able to abolish pain through inhibition of ASICs expressed either in central or peripheral neurons. These peptides, which we call mambalgins, are not toxic in mice but show a potent analgesic effect upon central and peripheral injection that can be as strong as morphine. This effect is, however, resistant to naloxone, and mambalgins cause much less tolerance than morphine and no respiratory distress. Pharmacological inhibition by mambalgins combined with the use of knockdown and knockout animals indicates that blockade of heteromeric channels made of ASIC1a and ASIC2a subunits in central neurons and of ASIC1b-containing channels in nociceptors is involved in the analgesic effect of mambalgins. These findings identify new potential therapeutic targets for pain and introduce natural peptides that block them to produce a potent analgesia.

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 mu and delta-opioid receptors and are lost in Penk1-/- mice.

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 Zn 2؉ . This paper shows that Zn 2؉ 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 EC 50 for Zn 2؉ potentiation is 120 and 111 M for the ASIC2a and ASIC1a؉2a current, respectively. Zn 2؉ shifts the pH dependence of activation of the ASIC1a؉2a current from a pH 0.5 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 Zn 2؉ 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.

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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.