Effects of Agonist and Regulator Binding on the Structure and Conformational Flexibility of the Ligand Binding Domains of Ionotropic Glutamate Receptors

Abstract

Ionotropic Glutamate receptors (iGluRs) are tetrameric proteins with 4 domains, an amino terminal domain, the ligand binding domain, the transmembrane ion channel and a largely disordered intracellular C terminal region. The ligand binding domain binds glutamate, a major fast excitatory neurotransmitter, which activates the receptor channels. There are three main families of iGluRs, AMPA, NMDA, and kainite which among other things differ in their responses to two endogenous neurosteroids, pregnenalone sulfate (PS) and pregnanalone sulfate (PREGAS). To investigate the role that conformational flexibility of the ligand binding domain plays in response to either glutamate or the neurosteroids (in the presence or absence of glutamate) we have used Multiwavelength Collisional Quenching (MWCQ) which reports on the exposure and charge environment of multiple tyrosines and tryptophans in the protein. While Glutamate has different effects on flexibility depending upon the family of receptor, the use of differently charged quenching molecules shows glutamate induced changes in the charge environment that can be correlated with steroid binding ability and that the presence of glutamate augments the effects of the steroid leading to enhanced global rigidity of the domains. NSF Grant MCB-104995 to EB