Structure /Function Of Neurotransmitter Receptor Channel

Child Health And Human Development

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Abstract

Ionotropic glutamate receptors (GluRs) are molecular pores which mediate signal transmission at the majority of excitatory synapses in the mammalian nervous system. Because of their essential role in normal brain function and development, and increasing evidence that dysfunction of GluR activity mediates multiple CNS diseases, as well as damage during stroke, a substantial effort in the Laboratory of Cellular and Molecular Neurophysiology is directed towards analysis of GluR function at the molecular level. The ultimate goal of this work is to obtain atomic resolution structural data which will provide a framework in which to design experiments to define the mechanisms underlying ligand recognition and gating. This will allow the development of subtype selective antagonists and allosteric modulators with novel therapeutic applications. The ionotropic glutamate receptors in humans are encoded by 7 gene families named after the ligands which were first used to identify the major subtypes on a functional basis: AMPA, kainate and NMDA. The recent crystallization of the ligand binding cores of an AMPA receptor subunit and a related bacterial receptor from the photosynthetic bacterium syncheocystis PCC 6803 has revealed for the first time the molecular mechanisms underlying the binding of agonists and antagonists as well as providing insight into the mechanisms of activation and desensitization. During the past year experimental efforts in structural biology have been directed towards similar studies on members of the kainate receptor gene family, as well as the delta receptor orphan subunits. Crystal structures of the GluR5 and GluR6 ligand binding cores (S1S2) have been solved in complexes with glutamate, 2S,4R-4-methylglutamate, kainate, and quisqualate. Agonists bind in a cavity between two globular domains. The volume of the ligand binding site cavities for GluR5 and GluR6 are 40% and 16% larger than for GluR2 and contain additional trapped water molecules not present in AMPA receptors. Modelling studies reveal that binding of the GluR5 selective agonists 5-iodowillardiine and ATPA to GluR6 is prevented by steric occlusion. The kainate receptor structures for glutamate and quisqualate are 4-6? more closed those of GluR2. Strikingly, the partial agonist kainate complex is 11? more closed for GluR6 than for GluR2 but more open than the GluR5 and GluR6 glutamate structures. This suggests that that kainate receptor efficacy is controlled by domain closure as previously determined for AMPA subtype iGluRs. In kainate receptors there is an extensive series of interdomain hydrogen bonds and salt links that are absent from AMPA receptors. These likely contribute to the higher stability of the closed cleft agonist bound complexes for kainate receptors which underlies their slow recovery from desensitization. Our functional studies over the same period had two goals. First, an analysis of the mechanism of partial agonist action, and second, identification of contacts in the dimer interface of the GluR2 AMPA receptor ligand binding core responsible for maintaining the active state of the receptor. Building on prior work we established that 5-substituted willardiines act as partial AMPA receptor agonists for which the extent of activation and desensitization are inversely related to the size of a 5 position substituent. Crystallographic studies revealed that due to steric hindrance large 5 substituents prevent domain closure of the agonist binding core to the same extent as produced by full agonists. Correlated with this, single channel studies revealed that full and partial agonists activate the same subconductance states, but with different relevant occupancy. Our studies on the GluR2 ligand binding core dimer interface used the crystal structure as a basis for designing mutants designed to test the role of ion pair interactions, hydrogen bonds, and van der Waals contactss of hydrophobic patches in maintaining the active state. The results obtained are in excellent agreement with those predicted by the crystal structure and reinforce our earlier proposal that this is a key structural element in the gating mechanism.