Single-Molecule Patch-Clamp FRET Anisotropy Microscopy Studies of NMDA Receptor Ion Channel Activation and Deactivation under Agonist Ligand Binding in Living Cells.
N-methyl-d-aspartate (NMDA) receptor ion channel is activated by the binding of two pairs of glycine and glutamate along with the application of action potential. Binding and unbinding of ligands changes its conformation that plays a critical role in the open-close activities of NMDA receptor. Conformation states and their dynamics due to ligand binding are extremely difficult to characterize either by conventional ensemble experiments or single-channel electrophysiology method. Here we report the development of a new correlated technical approach, single-molecule patch-clamp FRET anisotropy imaging and demonstrate by probing the dynamics of NMDA receptor ion channel and kinetics of glycine binding with its ligand binding domain. Experimentally determined kinetics of ligand binding with receptor is further verified by computational modeling. Single-channel patch-clamp and four-channel fluorescence measurement are recorded simultaneously to get correlation among electrical on and off states, optically determined conformational open and closed states by FRET, and binding-unbinding states of the glycine ligand by anisotropy measurement at the ligand binding domain of GluN1 subunit. This method has the ability to detect the intermediate states in addition to electrical on and off states. Based on our experimental results, we have proposed that NMDA receptor gating goes through at least one electrically intermediate off state, a desensitized state, when ligands remain bound at the ligand binding domain with the conformation similar to the fully open state.
Center for Photochemical Sciences, Department of Chemistry, Bowling Green State University , Bowling Green, Ohio 43403, United States.
Dibyendu Kumar
DK
Sasmal
Center for Photochemical Sciences, Department of Chemistry, Bowling Green State University , Bowling Green, Ohio 43403, United States.
Rajeev
R
Yadav
Center for Photochemical Sciences, Department of Chemistry, Bowling Green State University , Bowling Green, Ohio 43403, United States.
H Peter
HP
Lu
D016880:Anisotropy; D002470:Cell Survival; D031541:Fluorescence Resonance Energy Transfer; D005998:Glycine / Q000378:metabolism* / Q000494:pharmacology*; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008853:Microscopy; D008958:Models, Molecular; D011485:Protein Binding; D000072417:Protein Domains; D016194:Receptors, N-Methyl-D-Aspartate / Q000819:agonists* / Q000737:chemistry / Q000378:metabolism*
D008024:Ligands; D016194:Receptors, N-Methyl-D-Aspartate; D005998:Glycine