Abstract
Kainate receptors (KARs), a distinct subfamily of ionotropic glutamate receptors, are critical modulators of synaptic transmission and network excitability. Their function is intricately regulated by auxiliary subunits and endogenous ions. The GluK3 subunit, in particular, exhibits unique gating and modulatory properties; however, the interplay between its known regulators, the Neto auxiliary proteins, and synaptic zinc remains poorly understood. We reveal a multi-layered regulatory system governing the function of GluK3. Using whole-cell electrophysiology, we demonstrate that the auxiliary subunits Neto1 and Neto2 differentially regulate the gating kinetics of GluK3. While both proteins markedly slow receptor desensitization and relieve the intrinsic polyamine block, they exert opposing effects on the rate of recovery from desensitization, with Neto1 accelerating and Neto2 decelerating recovery, suggesting distinct mechanisms for tuning synaptic fidelity. Crucially, we show that Neto proteins uniquely reshape the potentiation of GluK3 currents by zinc. Neto2, in particular, acts synergistically with zinc to produce a profound facilitation of peak currents. To dissect these regulatory pathways, we utilized a GluK3 (D759G) mutant, which ablates the LBD dimer interface zinc-binding site. This mutation unmasked a secondary, inhibitory zinc-binding site, revealing a previously unknown layer of modulation. While the (D759G) mutant preserved the fundamental modulatory actions of Neto proteins, the Neto isoforms differentially regulated this previously unidentified revealed inhibitory zinc effect. Cryo-electron microscopy confirms that the (D759G) mutation promotes a more compact arrangement of the ligand-binding domain (LBD), consistent with its stabilizing effect on gating. Together, these findings establish a distinct framework for understanding KAR function, where auxiliary subunits and ionic modulators converge to create a highly tunable signaling complex essential for synaptic plasticity.
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Data availability
The cryo-EM density reconstruction and final model for the GluK3 D759G mutant receptor complex with UBP310 and spermine are deposited in the Electron Microscopy Data Bank (accession codes EMD-32032) and the Protein Data Bank (accession codes 7VM2). All other data supporting the key findings of this study are included in the article and its Supplementary Information files or are available from the corresponding author upon reasonable request. Source data for all graphs are provided in the Supplementary Data.
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Acknowledgements
This work was supported by the DBT–Wellcome Trust India Alliance (IA/S/21/2/505937). J.K. is a Senior Fellow of the DBT–Wellcome Trust India Alliance. B.K.D. acknowledges support from the University Grants Commission (UGC) in the form of a Senior Research Fellowship. S.B. acknowledges the National Centre for Cell Science (NCCS), Pune, for a Senior Research Fellowship. We thank M. L. Mayer for providing GluK3 wild-type constructs used for construct optimization and mutational studies, and E. Gouaux for the pEGBacMam vector. We acknowledge the European Synchrotron Radiation Facility for access to beamline CM01 and the cryo-electron microscopy facility at CSIR–CCMB for data collection. We thank the facility staff for their assistance.
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R.V. performed electrophysiology experiments. B.K.D. and A.A. generated GluK3 mutant constructs, expressed and purified proteins, conducted molecular biology and biochemical experiments, and processed cryo-EM data. S.B. performed pull-down assays. J.K. conceived and supervised the project. R.V., B.K.D., S.B., A.A., and J.K. analyzed the data and wrote the manuscript. All authors approved the final manuscript.
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J.K. is an Editorial Board Member for Communications Biology, but was not involved in the editorial review of, nor the decision to publish this article. Other authors declare that they have no competing interests.
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Vinnakota, R., Dawath, B.K., Assaiya, A. et al. Multilayered regulation of GluK3 kainate receptors is mediated by Neto subunits and zinc. Commun Biol (2026). https://doi.org/10.1038/s42003-026-09707-7
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DOI: https://doi.org/10.1038/s42003-026-09707-7
