Non-acidic activation of pain-related Acid-Sensing Ion Channel 3 by lipids.

Extracellular pH variations are seen as the principal endogenous signal that triggers activation of Acid-Sensing Ion Channels (ASICs), which are basically considered as proton sensors, and are involved in various processes associated with tissue acidification. Here, we show that human painful inflammatory exudates, displaying non-acidic pH, induce a slow constitutive activation of human ASIC3 channels. This effect is largely driven by lipids, and we identify lysophosphatidylcholine (LPC) and arachidonic acid (AA) as endogenous activators of ASIC3 in the absence of any extracellular acidification. The combination of LPC and AA evokes robust depolarizing current in DRG neurons at physiological pH 7.4, increases nociceptive C-fiber firing, and induces pain behavior in rats, effects that are all prevented by ASIC3 blockers. Lipid-induced pain is also significantly reduced in ASIC3 knockout mice. These findings open new perspectives on the roles of ASIC3 in the absence of tissue pH variation, as well as on the contribution of those channels to lipid-mediated signaling.

Analgesic effects of mambalgin peptide inhibitors of acid-sensing ion channels in inflammatory and neuropathic pain.

Mambalgins are 57-amino acid peptides isolated from snake venom that evoke naloxone-resistant analgesia after local (intraplantar) and central (intrathecal) injections through inhibition of particular subtypes of acid-sensing ion channels (ASICs). We now show that mambalgins also have an opioid-independent effect on both thermal and mechanical inflammatory pain after systemic intravenous (i.v.) administration and are effective against neuropathic pain. By combining the use of knockdown and knockout animals, we show the critical involvement of peripheral ASIC1b-containing channels, along with a contribution of ASIC1a-containing channels, in the i.v. effects of these peptides against inflammatory pain. The potent analgesic effect on neuropathic pain involves 2 different mechanisms depending on the route of administration, a naloxone-insensitive and ASIC1a-independent effect associated with i.v. injection and an ASIC1a-dependent and partially naloxone-sensitive effect associated with intrathecal injection. These data further support the role of peripheral and central ASIC1-containing channels in pain, demonstrate their participation in neuropathic pain, and highlight differences in the repertoire of channels involved in different pain conditions. They also strengthen the therapeutic potential of mambalgin peptides that are active in a broader range of experimental pain models and through i.v. systemic delivery.

Black mamba venom peptides target acid-sensing ion channels to abolish pain.

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.

Human ASIC3 channel dynamically adapts its activity to sense the extracellular pH in both acidic and alkaline directions.

In rodent sensory neurons, acid-sensing ion channel 3 (ASIC3) has recently emerged as a particularly important sensor of nonadaptive pain associated with tissue acidosis. However, little is known about the human ASIC3 channel, which includes three splice variants differing in their C-terminal domain (hASIC3a, hASIC3b, and hASIC3c). hASIC3a transcripts represent the main mRNAs expressed in both peripheral and central neuronal tissues (dorsal root ganglia [DRG], spinal cord, and brain), where a small proportion of hASIC3c transcripts is also detected. We show that hASIC3 channels (hASIC3a, hASIC3b, or hASIC3c) are able to directly sense extracellular pH changes not only during acidification (up to pH 5.0), but also during alkalization (up to pH 8.0), an original and inducible property yet unknown. When the external pH decreases, hASIC3 display a transient acid mode with brief activation that is relevant to the classical ASIC currents, as previously described. On the other hand, an external pH increase activates a sustained alkaline mode leading to a constitutive activity at resting pH. Both modes are inhibited by the APETx2 toxin, an ASIC3-type channel inhibitor. The alkaline sensitivity of hASIC3 is an intrinsic property of the channel, which is supported by the extracellular loop and involves two arginines (R68 and R83) only present in the human clone. hASIC3 is thus able to sense the extracellular pH in both directions and therefore to dynamically adapt its activity between pH 5.0 and 8.0, a property likely to participate in the fine tuning of neuronal membrane potential and to neuron sensitization in various pH environments.

Acid-sensing ion channels in postoperative pain.

Iatrogenic pain consecutive to a large number of surgical procedures has become a growing health concern. The etiology and pathophysiology of postoperative pain are still poorly understood, but hydrogen ions appear to be important in this process. We have investigated the role of peripheral acid-sensing ion channels (ASICs), which form depolarizing channels activated by extracellular protons, in a rat model of postoperative pain (i.e., hindpaw skin/muscle incision). We report high levels of ASIC-type currents (∼ 77%) in sensory neurons innervating the hindpaw muscles, with a prevalence of ASIC3-like currents. The ASIC3 protein is largely expressed in lumbar DRG neurons innervating the plantar muscle, and its mRNA and protein levels are increased by plantar incision 24 h after surgery. Pharmacological inhibition of ASIC3 channels with the specific toxin APETx2 or in vivo knockdown of ASIC3 subunit by small interfering RNA led to a significant reduction of postoperative spontaneous, thermal, and postural pain behaviors (spontaneous flinching, heat hyperalgesia, and weight bearing). ASIC3 appears to have an important role in deep tissue but also affects prolonged pain evoked by skin incision alone. The specific homomeric ASIC1a blocker PcTx1 has no effect on spontaneous flinching, when applied peripherally. Together, these data demonstrate a significant role for peripheral ASIC3-containing channels in postoperative pain.

ASIC3, a sensor of acidic and primary inflammatory pain.

Acid-sensing ion channels (ASICs) are cationic channels activated by extracellular acidosis that are expressed in both central and peripheral nervous systems. Although peripheral ASICs seem to be natural sensors of acidic pain (e.g., in inflammation, ischaemia, lesions or tumours), a direct demonstration is still lacking. We show that approximately 60% of rat cutaneous sensory neurons express ASIC3-like currents. Native as well as recombinant ASIC3 respond synergistically to three different inflammatory signals that are slight acidifications (approximately pH 7.0), hypertonicity and arachidonic acid (AA). Moderate pH, alone or in combination with hypertonicity and AA, increases nociceptors excitability and produces pain suppressed by the toxin APETx2, a specific blocker of ASIC3. Both APETx2 and the in vivo knockdown of ASIC3 with a specific siRNA also have potent analgesic effects against primary inflammation-induced hyperalgesia in rat. Peripheral ASIC3 channels are thus essential sensors of acidic pain and integrators of molecular signals produced during inflammation where they contribute to primary hyperalgesia.

Regulation of sensory neuron-specific acid-sensing ion channel 3 by the adaptor protein Na+/H+ exchanger regulatory factor-1.

Acid-sensing ion channels (ASICs) are cationic channels activated by extracellular protons. The ASIC3 subunit is largely expressed in the peripheral nervous system, where it contributes to pain perception and to some aspects of mechanosensation. We report here a PDZ-dependent and protein kinase C-modulated association between ASIC3 and the Na+/H+ exchanger regulatory factor-1 (NHERF-1) adaptor protein. We show that NHERF-1 and ASIC3 are co-expressed in dorsal root ganglion neurons. NHERF-1 enhances the ASIC3 peak current in heterologous cells, including F-11 dorsal root ganglion cells, by increasing the amount of channel at the plasma membrane. Perhaps more importantly, we show that the plateau current of ASIC3 can be dramatically increased (10-30-fold) by association with NHERF-1, leading to a significant sustained current at pH 6.6. In the presence of NHERF-1, the ASIC3 subcellular localization is modified, and the channel co-localizes with ezrin, a member of the ezrin-radixin-moesin family of actin-binding proteins, providing the first direct link between ASIC3 and the cortical cytoskeleton. Given the importance of the ASIC3 sustained current in nociceptor excitability, it is likely that NHERF-1 participates in channel functions associated with nociception and mechanosensation.

The multivalent PDZ domain-containing protein CIPP is a partner of acid-sensing ion channel 3 in sensory neurons.

Acid-sensing ion channels (ASICs) are cationic channels activated by extracellular pH. They are present in the brain, where they are thought to participate in signal transduction associated with local pH variations, and in sensory neurons, where they have been involved in pain perception associated with tissue acidosis and in mechanoperception. The ASIC3 subunit is mainly expressed in dorsal root ganglion neurons. Its expression is associated with a rapidly inactivating current followed by a slowly activating sustained current thought to be required for the tonic sensation of pain caused by acids. We report here the interaction of this channel subunit with the multivalent PDZ (PSD-95 Drosophila discs-large protein, Zonula occludens protein 1) domain-containing protein CIPP. This interaction requires the C-terminal region of ASIC3 and the fourth PDZ domain of CIPP. Co-expression of CIPP and ASIC3 in COS cells increases the maximal ASIC3 peak current density by a factor of 5 and slightly shifts the pH(0.5) for activation from pH 6.2 to pH 6.4. CIPP mRNA is found at a significant level in the same dorsal root ganglion neuronal cell population that expresses the ASIC3 subunit, i.e. mainly in the small nociceptive neurons. CIPP is thus a scaffolding protein that could both enhance the surface expression of ASIC3 and bring together ASIC3 and functionally related proteins in the membrane of sensory neurons.