Activation of Ionotropic Glutamate Receptors using Tethered Photoswitchable Ligands

Abstract

Ionotropic glutamate receptors (iGluRs) are ligand-gated ion channels, which mediate and modulate excitatory neurotransmission in the central nervous system. iGluRs are tetramers that are formed by identical or homologous subunits, but little is known about how ligand binding at individual subunits contributes to gating. Here we address this question using photoswitchable ligands that are tethered to specifically introduced cysteine residues. The ligand, termed MAG, consists of a cysteine-reactive maleimide group, an azobenzene photoswitch, and a glutamate analogue as head group. After coupling to an attachment site close to the binding cleft, light of specific wavelengths can be used to switch MAG between its cis and trans form, which allows to control ligand binding and unbinding with high temporal precision. We combined photoswitching with voltage-clamp recordings to measure the effect of partial receptor occupation on the extent and time course of activation and desensitization in GluK2, a member of the kainate receptor family. Extending these experiments to heteromeric complexes and complexes of defined subunit stochiometry will provide further information on how individual subunits contribute to the activation, deactivation and desensitization of this important class of signaling molecules.