Ligand-Directed Chemistry of AMPA Receptors Confers Live-Cell Fluorescent Biosensors

Abstract

AMPA-type glutamate receptors (AMPARs) mediate fast excitatory synaptic transmission in the central nervous system. Dysregulation of AMPAR function is associated with many kinds of neurological, neurodegenerative, and psychiatric disorders. As a result, molecules capable of controlling AMPAR functions are potential therapeutic agents. Fluorescent semisynthetic biosensors have attracted considerable interest for the discovery of ligands selectively acting on target proteins. Given the large protein complex formation of AMPARs in live cells, biosensors using full-length AMPARs retaining original functionality are ideal for drug screening. Here, we demonstrate that fluorophore-labeled AMPARs prepared by ligand-directed acyl imidazole chemistry can act as turn-on fluorescent biosensors for AMPAR ligands in living cells. These biosensors selectively detect orthosteric ligands of AMPARs among the glutamate receptor family. Notably, the dissociation constants of agonists and antagonists for AMPARs were determined in live cells, which revealed that the ligand-binding properties of AMPARs to agonists are largely different in living cells, compared with noncellular conditions. We also show that these sensors can be applied to detecting allosteric modulators or subunit-selective ligands of AMPARs. Thus, our protein-based biosensors can be useful for discovering pharmaceutical agents to treat AMPAR-related neurological disorders.

Recent Progress in Chemical Modification of Proteins

“…It has been demonstrated that LD and AG chemistries are applicable for visualization of endogenous proteins, 211,215,217,218 biosensor construction, 11,12,[219][220][221] protein-protein interaction analyses, [222][223][224] design of a new type of covalent inhibitor 199 and drug screening in live cell environments. 221 For example, Hamachi, Kiyonaka and coworkers recently demonstrated that fluorophore-labeled AMPA-type glutamate receptors (AMPARs) prepared by LDAI chemistry act as turn-on-type fluorescent biosensors for AMPAR ligands under live cell conditions.…”

“…221 For example, Hamachi, Kiyonaka and coworkers recently demonstrated that fluorophore-labeled AMPA-type glutamate receptors (AMPARs) prepared by LDAI chemistry act as turn-on-type fluorescent biosensors for AMPAR ligands under live cell conditions. 220 These biosensors selectively detect orthosteric ligands for AMPARs. Notably, the dissociation constants of competitive ligands to AMPAR (both agonists and antagonists) were successfully determined in live cells, by which they clearly demonstrated that the agonists-binding properties of AMPARs are largely different between live cell environments and noncellular conditions.…”

Considerations on Probe Design for Affinity‐Guided Protein Conjugation

“…The imidazole carbamate reactive moiety has been extensively used to study the biology of proteins (Figure C), especially membrane bound receptors . During the conjugation reaction, the imidazole acts as a leaving group transferring the label containing alkyl formate to the protein.…”

“…It showed superior labeling of the folate receptor on live cells compared to a benzenesulfonate probe. While the kinetics of imidazole carbamate probes have not been further investigated in vitro, imidazole carbamate probes have been successfully employed to investigate the biology of both GABA A and AMPA receptors by live cell labeling. Through the highly selective complexation of the guiding moiety and the delicate reactivity of the imidazole carbamate, it was possible to label the AMPA receptors in cultured neurons, and more impressively, in brain tissue of acute hippocampal slices.…”

“…By pairing with a suitable reactive moiety, it has been possible to covalently modify proteins of interest on or in live cells, in brain tissue slices, and even in mice . In most cases, the probes have been used for labeling of receptors or other membrane bound proteins, thereby, avoiding the issue of traversing the cell membrane. However, there has also been reports on selective conjugation to target proteins inside cells using ligand‐guided affinity probes, but the mechanism of crossing the membrane has not been addressed.…”