Ophthalmic acid (OA; ophthalmate) is a glutathione-related tripeptide whose biological function has remained largely unknown. We recently identified OA as an endogenous regulator of motor function acting through the calcium-sensing receptor (CaSR). Here, we define the molecular basis and functional properties of the OA–CaSR interaction.
Our initial discovery emerged from studies of L-DOPA responses in mouse models of Parkinson’s disease. Unexpectedly, L-DOPA produced a robust and prolonged motor response even when its conversion to dopamine was blocked by inhibition of aromatic L-amino acid decarboxylase. Untargeted metabolomics revealed that this response coincided with an approximately 20-fold increase in brain OA. Direct brain administration of OA rescued motor deficits in Parkinsonian mice in a dose-dependent manner, whereas the CaSR antagonist NPS-2143 blocked this effect, establishing CaSR as a mediator of OA-dependent motor regulation.
We investigated how OA interacts with CaSR using computational modeling, site-directed mutagenesis, radioligand binding, and functional signaling assays. OA directly interacted with CaSR and activated receptor signaling in a concentration-dependent manner. Computational modeling identified residues within the CaSR ligand-binding domain predicted to mediate OA recognition, and mutation of key residues altered OA binding and signaling. Importantly, OA retained the ability to activate CaSR in the absence of added extracellular Ca²⁺, demonstrating intrinsic agonist activity. In addition, OA potentiated Ca²⁺-evoked CaSR responses, demonstrating positive allosteric modulation.
Together, these findings identify OA as an endogenous CaSR ligand with both agonist and positive allosteric properties and reveal an unexpected OA–CaSR signaling pathway involved in the regulation of motor function. These findings provide new insight into the diversity of endogenous CaSR ligands and the molecular mechanisms through which CaSR activity can be modulated in the brain.