Mapping Neurosteroid Binding Sites on the Ionotropic Glutamate Receptor

Adam Lin
Western Washington University

Ionotropic glutamate receptors (iGluRs) are integral to the post-synaptic neural membrane and are major facilitators of neural cell plasticity, cognition, memory, and learning. They are classified according to their affinity for three natural products ?? (S)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazole) propionic acid (AMPA), N-methyl-D-aspartate (NMDA), and kainate. A functional iGluR ion channel is a homomeric or heteromeric rosette of four membrane spanning subunits; the NR1-1b NMDA subunit and the GluR2 (non-NMDA) subunit have been chosen as our representative models. Our focus is on the modulation of these iGluRs by two endogenous neurosteroids (NS) - pregnenolone sulfate (PS) and 3a/3ß-hydroxy-5ß-pregnan-20-one sulfate (Pregas), which differ structurally only by a double bond at the steroid A/B ring junction. While agonist activation by the neurotransmitter, glutamate, has been structurally characterized, the allosteric modulatory sites attributed to NS binding have not yet been located. It is known that NS bind extracellularly to both NMDA and non-NMDA receptors inducing some opposite effects. Pregas down-regulates all iGluRs; and while PS also down-regulates non-NMDA receptors, it interestingly up-regulates the NMDA receptor. Our goals are to identify the NS modulatory site(s), and to compare at the molecular level the structure/function relationship of the non-NMDA and NMDA receptors. Soluble iGluR constructs have previously been cloned and over-expressed showing near native ligand binding activity. Results will be presented here detailing both an improved purification protocol of these extracellular receptor domains, as well as biophysical studies suggesting which extracellular regions of these receptors are necessary for binding to PS and Pregas.