Citric Acid in Drug Formulations Causes Pain by Potentiating Acid-Sensing Ion Channel 1

Pain at the injection site is a common complaint of patients receiving therapeutic formulations containing citric acid. Despite the widely acknowledged role of acid-sensing ion channels (ASICs) in acid-related perception, the specific ASIC subtype mediating pain caused by subcutaneous acid injection and the mechanism by which citrate affects this process are less clear. Here, male mice subjected to intraplantar acid injection responded by executing a withdrawal reflex, and this response was abolished by ASIC1 but not ASIC2 knockout. Although intraplantar injection of neutral citrate solution did not produce this response, intraplantar injection of acidic citrate solution produced a withdrawal reflex greater than that produced by acidity alone. Consistent with the behavioral data, neutral citrate failed to produce an electrophysiological response in HEK293 cells, which express ASIC1, but acidic citrate produced a whole-cell inward current greater than that produced by acidity alone. Saturating the intracellular solution with citrate had no effect on the potentiating effect of extracellular citrate, suggesting that citrate acted extracellularly to potentiate ASIC1. Moreover, exposure to citrate immediately before acid stimulation failed to potentiate ASIC1 currents, which ruled out the involvement of a metabotropic receptor gated by a citrate metabolite. Finally, removal of calcium ions from the extracellular solution mimicked the potentiating effect of citrate and prevented citrate from further potentiating ASIC1. Our data demonstrate that ASIC1 is necessary for the nociceptive response caused by subcutaneous acid infusion and that neutral citrate, despite not inducing ASIC1 currents or nociceptive behavior on its own, potentiates acid nociception by removing the inhibitory effect of extracellular calcium ions on ASIC1.