Probing the Channel Gating of a Glutamate Receptor with Photoactive Unnatural Amino Acids

Abstract

Ionotropic glutamate receptors (iGluRs) are responsible for fast synaptic transmission throughout the nervous system. Despite considerable study, the conformational change of the transmembrane domain (TMD) underlying ion channel activation remains unclear. Here, we aim to explore the function and dynamics of the transmembrane region of AMPA-type glutamate receptors using unnatural amino acid (UAA) photo-cross-linkers, p-benzoyl-L-phenylalanine (BzF) and p-azido-L-phenylalanine (AzF). Using mammalian cells as expression system, AzF and BzF were individually introduced throughout the TMD of the AMPA receptor GluA2 by genetically-encoded UAA mutagenesis. Outside-out patch clamp recording of receptors activated by a fast-perfusion system was combined with synchronized exposures to UV light via epi-illumination, to characterize the functionality of the AzF and BzF containing iGluR constructs, as well as their individual photo-controllable profiles. AzF and BzF were individually inserted in 23 sites throughout the TMD. Glutamate induced currents could be measured from 18 constructs containing AzF at different sites, while only 10 constructs containing BzF resulted in a glutamate-activated current. The glutamate activated currents of mutant receptors had similar characteristics to wild type channels. Exposing channels harboring AzF to UV light had a range of effects, from inhibition to potentiation, dependent on the insertion site. Exposure of channels incorporating BzF in the TMD to UV light did not have any effect on currents. In contrast, photoactivation of BzF in the Pre-M1 linker was found to increase peak current by up to 50%. Our results demonstrate that UAAs can be incorporated site specifically in the TMD and can trap key moving parts of the ion channel.