Fast Photoswitching of Tethered Ligands to Study Ionotropic Glutamate Receptor Activation and Desensitization

Abstract

Binding of glutamate triggers both the activation and subsequent desensitization of ionotropic glutamate receptors (iGluRs). Matching their role in excitatory neurotransmission, iGluR activation and desensitization are fast processes occurring on the submillisecond and millisecond timescale, respectively. However, little is known about how ligand binding to the four subunits of the tetrameric channel assemblies mediates these processes. Here we address this question using photo-switchable ligands (MAGs) that can be tethered to individual subunits via a cysteine-reactive maleimide group. An azobenzene group serves as photo-switch that allows binding of the glutamate head group in its cis, but not its trans state [1]. This allows us to control ligand binding and unbinding with short (<100 μs) pulses of light and to follow channel activation and desensitization in real-time. The experiments give insight into the gating mechanism of GluK2 homotetramers and will be extended to study subunit specific activation in heteromeric GluK2/GluK5 complexes, which assemble with defined 2:2 subunit stoichiometry [2].1. Gorostiza P. et al., Proc. Natl. Acad. Sci. USA (2007) 104: 10865.2. Reiner A. et al., Cell. Rep. (2012) 1: 234.