Modulation of muscle spindle stretch-evoked firing has an atypical glutamate receptor pharmacology; L-cysteinesulfinic acid, quisqualate and kainate are potent agonists of afferent firing. However, afferent firing is not modulated by Group I, II, or III mGluR ligands, kynurenate (broad-spectrum iGluR antagonist) or NBQX (kainate receptor antagonist). This glutamate pharmacological profile most closely matches that of the PLD-GluR described in the hippocampus by a number of groups1-3. To allow further investigation of this atypical receptor, a functionalised kainate moiety amenable to ‘click’ chemistry (ZCZ-90) has been synthesised4. Here we describe how ZCZ-90 allowed us to make progress towards the isolation of the atypical spindle glutamate receptor by using biotin (ZCZ-180) and fluorescein (ZCZ-172) conjugated forms to visualise ligand binding in spindle homogenates and whole mount tissues, and the subsequent identification of putative glutamate receptor-like proteins associated with spindles. To obtain sufficient spindle protein for screening, a method to extract a high yield of spindle material was first developed. The column of ~100 spindles in rat deep masseter muscle5 was disrupted by collagenase digestion, visualised with methylene blue, then isolated by microdissection and the protein extracted. Far Western blotting with ZCZ-180 confirmed that spindle homogenate contained a protein capable of binding this biotinylated kainate analogue, with bands at ~120 kDa and ~70 kDa. Bands of similar mass were detected in the hippocampus, our positive control where the PLD-GluR was first described. ZCZ-172 labelling in masseter whole mounts revealed fluorescent ligand binding on annulospiral nerve terminals. This suggests the protein to which the ZCZ ligands, and thus kainate, binds is indeed expressed in annulospiral nerve endings, and is well-placed to be involved in the glutamate-modulation of the stretch response. To further identify the atypical receptor, we screened for all known mGluRs and kainate receptors (KARs) using Western blotting of spindle homogenates and immunofluorescence of lumbrical spindle whole mounts. We identified a rapidly migrating isoform of mGluR5 but, consistent with previous studies6, mGluR5 immunofluorescence was only present on nociceptors running parallel to spindles. However, Western blotting did show spindle homogenates contained bands for GluK2, and immunofluorescence showed labelling for GluK2 on annulospiral nerve terminals, consistent in both molecular weight and labelling pattern to ZCZ-172 labelling, respectively. This suggests the GluK2 receptor subunit is indeed involved in modulating stretch-induced afferent firing. We are now pursuing the isolation of the atypical receptor protein. A band of a similar molecular weight to that probed by ZCZ-180 in homogenate enriched for lipophilic (membrane-associated) proteins could not be identified by mass spectrometry. However, the ‘unassigned’ peptides in this band showed strong sequence homology to glutamate receptors by NCBI-based sequence alignment. Furthermore, functional clustering analysis of the proteins associated with the unassigned peptides identified PLD as a potential regulatory hub, suggesting the protein(s) in the band likely signal through PLD. These data are consistent, therefore, with the receptor being an atypical glutamate receptor coupled to PLD. We have thus developed a functionalised kainate analogue and used it to demonstrate the expression of a kainate-binding protein in muscle spindles. This seems most likely to be GluK2, despite the spindle pharmacology resembling that of mGluRs. Metabotropic actions of kainate receptors have been described elsewhere7, yet the exact functions of these remain somewhat elusive. Our current working hypothesis is that we have uncovered a functional system which uses only the metabotropic function of these iGluR subunits. Future work will focus on isolating sufficient protein for sequencing to validate whether it is the iGluR subunit which is responsible for glutamatergic modulation of this class of mechanoreceptors. Ultimately, the receptor will be fully characterised by expression in a cell line to determine protein-protein interactions and detailed pharmacological profile. Preliminary evidence presented previously to the Society suggests this receptor is expressed on baroreceptors. If confirmed, this atypical receptor may provide a novel drug target for treating hypertension.