Effects of Regulator Binding on the Structure and Conformational Flexibility of the Amino Terminal 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. While the ligand binding domain binds glutamate, a major fast excitatory neurotransmitter, which activates the receptor channels the amino terminal domains of all three bind endogenous neurosteroids via an unknown mechanism. 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 amino terminal domains play in response to the neurosteroids we have used Multiwavelength Collisional Quenching (MWCQ) which reports on the exposure and charge environment of tyrosines and tryptophans in the protein and gel filtration to assess the impacts of steroid binding on quaternary structure. Finally CD and thermal melt studies have been used in investigate the effects of steroids on overall secondary structure and stability.The results provide insight into potential mechanisms of steroid regulation of ionotropic glutamate receptors. This work was supported by NSF Grants MCB-104995 to EB