AMPA-type ionotropic glutamate receptors mediate a majority of fast excitatory transmission in the mammalian central nervous system. AMPA receptors associate with an assortment of auxiliary proteins, which distinctly modulate gating kinetics and trafficking in different neuronal tissues. Chief among the auxiliary proteins are the transmembrane AMPA receptor regulatory proteins (TARPs) γ2–γ8, which can enhance agonist efficacy and potency, slow desensitization, and modify recovery from desensitization or attenuate polyamine pore block to differing extents. Electrophysiological studies have demonstrated the TARPs’ functionally critical extracellular β1-β2 loop is important for mediating some of these modulatory effects. However, the structural basis of β1-β2 loop action remains unclear, in large part because the loop remains unresolved in structural models. The relative position of this loop can be measured in complex cellular environments using fluorescent dye labeling and Förster energy transfer (FRET), but great care must be taken to ensure minimal background signal. To that end, we implement here a combination of genetic code expansion (GCE) and Click chemistry to selectively fluorescently label TARPs at compatible sites on the β1-β2 loop. This specificity is leveraged in combination with plasma membrane- and AMPA receptor-localized FRET acceptors to investigate β1-β2 loop ensemble position and motion. Together, our work highlights the utility of GCE-based Click chemistry labeling and FRET to reveal ensemble relative position between structurally unresolved regions in functioning membrane proteins and their putative interactors.
Stallone et al. (Sun,) studied this question.