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. The structure of a GluR6 ligand binding core complex with glutamate was solved by SeMET MAD crystallography, and two additional glutamate complexes in different crystal forms were solved by molecular replacement. In addition the structures of complexes with the high affinity agonists quisqualate and 4-methylglutamate were solved by Fourier difference techniques. 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 contacts 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.
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