Abstract
Cell signaling is often mediated by the binding of multiple ligands to multisubunit receptors. The probabilistic nature and sometimes slow rate of binding encountered with diffusible ligands can impede attempts to determine how the ligand occupancy controls signaling in such protein complexes. We describe a solution to this problem that uses a photoswitched tethered ligand as a 'ligand clamp' to induce rapid and stable binding and unbinding at defined subsets of subunits. We applied the approach to study gating in ionotropic glutamate receptors (iGluRs), ligand-gated ion channels that mediate excitatory neurotransmission and plasticity at glutamatergic synapses in the brain. We probed gating in two kainate-type iGluRs, GluK2 homotetramers and GluK2–GluK5 heterotetramers. Ultrafast (submillisecond) photoswitching of an azobenzene-based ligand on specific subunits provided a real-time measure of gating and revealed that partially occupied receptors can activate without desensitizing. The findings have implications for signaling by locally released and spillover glutamate.
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Acknowledgements
We thank J. Levitz for discussion, and we are grateful to K.M. Partin (Colorado State University) and P.H. Seeburg (Max Planck Institute Heidelberg) for the gift of clones as well as to D. Trauner (Ludwig-Maximilians-Universität München) for the gift of L-MAG-0 and many stimulating discussions. This work was supported by grants to E.Y.I. from the US National Institutes of Health (2PN2EY018241 and U24NS057631) as well as a postdoctoral fellowship to A.R. from the Deutsche Forschungsgemeinschaft (DFG RE 3101/1-1).
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A.R. and E.Y.I. designed experiments, A.R. performed and analyzed experiments, and A.R. and E.Y.I. wrote the manuscript.
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Reiner, A., Isacoff, E. Tethered ligands reveal glutamate receptor desensitization depends on subunit occupancy. Nat Chem Biol 10, 273–280 (2014). https://doi.org/10.1038/nchembio.1458
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DOI: https://doi.org/10.1038/nchembio.1458
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