2-guanidine-4-methylquinazoline and Pain

Sensory nerve fibers innervating the ocular anterior surface detect external stimuli producing innocuous and painful sensations. Protons are among the first mediators released by damaged cells during inflammation, tissue injury, or other chronic ophthalmic conditions. We studied whether acid-sensing ion channels (ASICs) are expressed in corneal sensory neurons and their roles in the response to moderate acidifications of the ocular surface and in pathologies producing ocular surface inflammation. Moderate acidic pH (6.6) activated ASIC-like currents in corneal sensory neurons, which were blocked by ASIC1- or ASIC3-specific toxins. Acidic pH depolarizes corneal sensory neurons to fire action potentials, an effect blocked by the ASIC3 inhibitor APETx2. 2-Guanidino-4-methylquinazoline, an ASIC3 agonist, activated a population of corneal polymodal sensory nerve fibers and significantly increased the blinking and tearing rate. The nocifensive behaviors produced by application of either a moderate acidic stimulus or ophthalmic drugs formulated in acidic solution were abolished by ASIC blockers. In a model of allergic keratoconjunctivitis, nocifensive behavior was greatly reduced by ASIC3 blockade, presumably by reducing nociceptor sensitization during the inflammatory process. Our results show that, in addition to the established role of TRPV1, ASICs play a significant role in the detection of acidic insults at the ocular surface. The identification of ASICs in corneal neurons and their alterations during different diseases is critical for the understanding of sensory ocular pathophysiology. They are likely to mediate some of the discomfort sensations accompanying several ophthalmic formulations and may represent novel targets for the development of new therapeutics for ocular pathologies.

Topics: Acid Sensing Ion Channel Blockers; Acid Sensing Ion Channels; Acids; Action Potentials; Amiloride; Animals; Blinking; Cnidarian Venoms; Cornea; Disease Models, Animal; Dry Eye Syndromes; Eye; Guanidines; Guinea Pigs; Hydrogen-Ion Concentration; Male; Nerve Fibers; Ocular Motility Disorders; Ovalbumin; Pain; Patch-Clamp Techniques; Quinazolines; Rats; Rats, Sprague-Dawley; RNA, Messenger; RNA, Small Interfering; Sensory Receptor Cells; Statistics, Nonparametric

Acid-sensing ion channels (ASICs) have long been considered as extracellular proton (H(+))-gated cation channels, and peripheral ASIC3 channels seem to be a natural sensor of acidic pain. Here, we report the identification of a nonproton sensor on ASIC3. We show first that 2-guanidine-4-methylquinazoline (GMQ) causes persistent ASIC3 channel activation at the normal pH. Using GMQ as a probe and combining mutagenesis and covalent modification analysis, we then uncovered a ligand sensor lined by residues around E423 and E79 of the extracellular "palm" domain of the ASIC3 channel that is crucial for activation by nonproton activators. Furthermore, we show that GMQ activates sensory neurons and causes pain-related behaviors in an ASIC3-dependent manner, indicating the functional significance of ASIC activation by nonproton ligands. Thus, natural ligands beyond protons may activate ASICs under physiological and pathological conditions through the nonproton ligand sensor, serving for channel activation independent of abrupt and marked acidosis.

Topics: Acid Sensing Ion Channels; Acids; Amiloride; Animals; Behavior, Animal; Biophysics; Calcium; Cells, Cultured; Cricetinae; Cricetulus; Disease Models, Animal; Dithionitrobenzoic Acid; Dose-Response Relationship, Drug; Electric Stimulation; Ganglia, Spinal; Glutamates; Guanidines; Hydrogen-Ion Concentration; Ion Channel Gating; Ligands; Membrane Potentials; Mice; Mice, Knockout; Mutagenesis, Site-Directed; Mutation; Nerve Tissue Proteins; Pain; Pain Measurement; Patch-Clamp Techniques; Protein Structure, Tertiary; Protons; Quinazolines; Rats; Sensory Receptor Cells; Sodium Channels; Sulfhydryl Reagents; Time Factors; Transfection