Allosteric mechanism in AMPA receptors: a FRET-based investigation of conformational changes.
Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are the primary mediators of fast excitatory synaptic transmission in the mammalian CNS. Structures of the extracellular ligand-binding domain suggest that the extent of cleft closure in the ligand-binding domain controls the extent of activation of the receptor. Here we have developed a fluorescence resonance energy transfer-based probe that allows us to study the extent of cleft closure in the isolated ligand-binding domain in solution. These investigations show that the wild-type protein exhibits a graded cleft closure that correlates to the extent of activation, which is in qualitative agreement with the crystal structures. However, the changes in extent of cleft closure between the apo and agonist-bound states are smaller than that observed in the crystal structures. We have also used this method to study the L650T mutant and show that in solution the alpha-amino-5-methyl-3-hydroxy-4-isoxazole propionate-bound form of this mutant exists primarily in a conformation that is more closed than predicted based on the activity, indicating that the degree of cleft closure alone cannot be used as a measure of extent of activation of the receptor, and there are possibly other mechanisms in addition to cleft closure that mediate the subtleties in extent of activation by a given agonist.
Ramanoudjame
Gomathi
G
Department of Integrative Biology and Pharmacology, 6431 Fannin, University of Texas Health Science Center, Houston, TX 77030.
Du
Mei
M
Department of Integrative Biology and Pharmacology, 6431 Fannin, University of Texas Health Science Center, Houston, TX 77030.
Mankiewicz
Kimberly A
KA
Department of Integrative Biology and Pharmacology, 6431 Fannin, University of Texas Health Science Center, Houston, TX 77030.
Jayaraman
Vasanthi
V
Department of Integrative Biology and Pharmacology, 6431 Fannin, University of Texas Health Science Center, Houston, TX 77030 vasanthi.jayaraman@uth.tmc.edu.
D000494:Allosteric Regulation; D002460:Cell Line; D004594:Electrophysiology; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D008958:Models, Molecular; D018408:Patch-Clamp Techniques; D017434:Protein Structure, Tertiary; D018091:Receptors, AMPA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D018091:Receptors, AMPA