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In my lab we are attempting to understand how ligand gated ion channels function at the molecular level. To do this we use x-ray crystallography to solve receptor structures, and high resolution biochemical and electrophysiological techniques to study the assembly and activity of native and mutant ion channels. Binding of agonists and antagonists independent of gating is measured using purified water soluble ligand binding cores. We are also interested in the thermodynamics of these proteins. Our goal is to understand in detail the mechanisms underlying subtype selectivity, gating, desensitization, and ion selectivity. Our major focus is the large family of glutamate receptors which mediate synaptic transmission at ≈ 60% of synapses in the brain, and which have diverse functional properties which arise from structural diferences in the 18 iGluR gene products.
My lab studies the ligand gated ion channels which mediate synaptic transmission in the CNS. The growing success of x-ray diffraction analysis for membrane proteins, provides a structural framework for interpretation of ion channel function. By using crystallography combined with biochemistry and physiology we can for the 1st time infer molecular mechanisms from structures, and then test the resulting hypotheses about receptor and ion channel function via site directed mutagenesis and studies on channels expressed in native membranes.
Our main focus for many years has been iGluRs from the AMPA, kainate and NMDA receptor gene familes which are used at about 60% of synapses in the brain. In a collaboration with the Gouaux lab at HHMI we used this approach to define for the 1st time the mechanism underlying AMPA receptor desensitization. Prior to this, work in my lab using biophysical approaches established many of the key functional parameters of iGluRs including Mg block (1984) and the high Ca permeability of NMDA receptors (1987); the development of 5-substituted willardiines as tools which differentiate AMPA and kainate receptors (1994); the allosteric activity and channel blocking action of polyamines (1995); the mechanism of action of AMPA receptor allosteric modulators (1995). Much of this earlier work lay the foundation for questions now being addressed using structural approaches.
In our current experiments we use a combined experimental approach involving mostly crystallographic, biochemical, and some electrophysiological techniques. Our best success in protein expression and crystallization has been with isolated ligand binding domains, and more recently the ATDs which control assembly. Recent projects identified the structure of the binding sites and mechanism of action of allosteric anions and cations which form integral components of kainate receptor ligand binding domains (Cl site PDB 2OJT and Na site 3C32) and created non-desensitizing kainate receptors using protein engineering and disulfide crosslinking (PDB 2I0C). Structures of the NMDA receptor NR3A (PDB 2RC7) and NR3B (PDB 2RCA) ligand binding domains in complex with glycine and D-serine were solved and analyzed by MD simulations in collaboration with the group of Klaus Schulten. Biophysical studies using sedimentation velocity and equilibrium anaysls, light scattering, and other approaches are being used to explore the thermodynamics of intersubuniit communication, with a focus on the role of ATDs in hetero-oligomer assembly (GluR6/KA2 assembly PDB 3QLV).
In earlier work we solved structures for GluR6 and GluR5 kainate receptors in complex with the agonists glutamate at 1.65 and 1.8 A (PDB 1S7Y and 1S50); 2(S),4(R),4-methylglutamate at 1.8 A (PDB 1SD3); kainate at 1.93 A (PDB 1TT1); and quisqualate at 1.8 A (PDB 1S9T). GluR5 dimer assemblies with glutamate (PDB 2F36) and the novel competitive antagonists UBP302 (PDB 2F35) and UBP310 (PDB 2F34) have been solved at resolutions of 2.1, 1.8 and 1.74 A. We are continuing to work on additional complexes with kainate receptor selective ligands and on the role of receptor dimer assemblies in desensitization and gating. Additional iGluR subtypes are at less advanced stages of analysis. Functional and computational studies on AMPA and kainate receptors continue to probe the dimer interface to give a unique insight into ion channel function and allosteric regulation at the molecular level.
The lab tour shows some of the equipment needed to pursue this diverse research program which requires protein purification, X-ray crystallography, and patch clamp recording. The lab is well equipped and offers an exceptional training environment for highly motivated postdoctoral fellows with strong backgrounds in protein chemistry or ion channel biophysics who wish to work on the cutting edge of ion channel research.