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Tuning Activation of the AMPA-Sensitive Glur2 Ion Channel by Genetic Adjustment of Agonist-Induced Conformational Changes

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Tuning Activation of the AMPA-Sensitive Glur2 Ion Channel by Genetic Adjustment of Agonist-Induced Conformational Changes Tuning activation of the AMPA-sensitive GluR2 ion channel by genetic adjustment of agonist-induced conformational changes Neali Armstrong*, Mark Mayer†, and Eric Gouaux*‡§ *Department of Biochemistry and Molecular Biophysics and ‡Howard Hughes Medical Institute, Columbia University, New York, NY 10032; and †Laboratory of Cellular and Molecular Neurophysiology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892 Edited by Douglas C. Rees, California Institute of Technology, Pasadena, CA, and approved March 17, 2003 (received for review December 5, 2002) The (S)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazole) propionic acid (AMPA) receptor discriminates between agonists in terms of bind- ing and channel gating; AMPA is a high-affinity full agonist, whereas kainate is a low-affinity partial agonist. Although there is extensive literature on the functional characterization of partial agonist activity in ion channels, structure-based mechanisms are scarce. Here we investigate the role of Leu-650, a binding cleft residue conserved among AMPA receptors, in maintaining agonist specificity and regulating agonist binding and channel gating by using physiological, x-ray crystallographic, and biochemical tech- niques. Changing Leu-650 to Thr yields a receptor that responds more potently and efficaciously to kainate and less potently and efficaciously to AMPA relative to the WT receptor. Crystal struc- tures of the Leu-650 to Thr mutant reveal an increase in domain closure in the kainate-bound state and a partially closed and a fully Fig. 1. Mechanisms to describe the conformational behavior of ligand-gated closed conformation in the AMPA-bound form. Our results indicate ion channels. (A) The two-state model where the receptor is in equilibrium that agonists can induce a range of conformations in the GluR2 between two conformations, closed and open. Agonist binding stabilizes the ligand-binding core and that domain closure is directly correlated receptor in the open state. Full agonists stabilize the open state more effec- to channel activation. The partially closed, AMPA-bound confor- tively than partial agonists, and both types of agonists stabilize the same conformational states. (B) Multistate model where the agonist-binding region mation of the L650T mutant likely captures the structure of an of the receptor adopts a range of agonist-dependent conformations. Partial agonist-bound, inactive state of the receptor. Together with pre- agonists promote a submaximal conformational change and therefore are not viously solved structures, we have determined a mechanism of as effective in shifting the closed to open equilibrium of the ion channel to the agonist binding and subsequent conformational rearrangements. open state. igand-gated ion channels are allosteric proteins composed of Lagonist binding and ion channel domains (1). Agonists do agonist-binding domain and activation of the ion channel by work on the ion channel by coupling the energy derived from ‘‘tuning’’ ion channel gating via a specific combination of a agonist binding to the opening or gating of the ion channel. By partial agonist and a site-directed mutant in the agonist binding site. definition, full agonists produce maximal activation of the ion AMPA receptors (GluR1–4) are a subtype of the ionotropic channel, whereas partial agonists result in submaximal activa- glutamate receptor family of ligand-gated ion channels (7, 8) and tion, even when applied at saturating concentrations. There are have a high affinity for the full agonist AMPA and a low affinity two distinct models to describe the behavior of allosteric proteins for the partial agonist kainate (9–11). AMPA receptors also such as ligand-gated ion channels: a two-state or concerted bind and activate in response to the nonselective, full agonists model (2) and an induced-fit or multistate model (3, 4) (Fig. 1). L-glutamate and quisqualate (9–12). Crystallographic studies In the two-state model, the ligand-gated ion channel exists in reveal that full and partial agonists bind to the cleft of the two conformations, an inactive or ‘‘apo’’ conformation and an ‘‘clamshell-shaped’’ GluR2 S1S2J ligand-binding core (12–15): agonist-bound, activated conformation. Full and partial agonists The full agonists AMPA, glutamate, and quisqualate bring the can bind to both states, with full agonists more effectively domains of the ligand-binding core Ϸ21° closer together, relative stabilizing the activated state in comparison with partial agonists to the apo state, whereas the partial agonist kainate induces only (5). Thus, full agonists produce greater activation of the ion 12° of domain closure (14). Kainate induces only partial domain channel than partial agonists. Cyclic-nucleotide gated ion chan- closure because its isopropenyl group acts like a ‘‘foot in the nels from bovine rod photoreceptors respond maximally to door,’’ colliding with Tyr-450 and Leu-650. On the basis of these cGMP but only weakly to cAMP and are paradigms of the structural studies, we suggest that the ligand-binding core can two-state model (5, 6). adopt multiple, agonist-dependent conformations and that dif- According to the multistate hypothesis, the ligand-gated ion ferences in agonist efficacy at AMPA receptors can arise from channel can adopt a range of conformations that depends on the different conformations of the ligand-binding core. particular agonist (Fig. 1B). Full agonists stabilize a conforma- tion that maximally activates the ion channel and partial agonists stabilize different conformations that are less efficacious in This paper was submitted directly (Track II) to the PNAS office. channel activation; i.e., for partial agonists less binding energy is Abbreviations: AMPA, (S)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazole) propionic acid; AS, available for doing work necessary to open the ion channel gate. ammonium sulfate; Imax, maximal current measured at saturating agonist concentration. Using the GluR2 (S)-2-amino-3-(3-hydroxy-5-methyl-4- Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, isoxazole) propionic acid (AMPA)-sensitive ion channel, we www.rcsb.org (PDB ID codes 1P1N, 1P1O, 1P1Q, 1P1U, and 1P1W). have studied the relationships between the conformation of the §To whom correspondence should be addressed. E-mail: [email protected]. 5736–5741 ͉ PNAS ͉ May 13, 2003 ͉ vol. 100 ͉ no. 10 www.pnas.org͞cgi͞doi͞10.1073͞pnas.1037393100 Downloaded by guest on September 29, 2021 To test this hypothesis, we reasoned that if the steric clash a reservoir solution containing 24–28% polyethylene glycol between the isopropenyl group of kainate and neighboring (PEG) 4000 and 0.2–0.35 M ammonium sulfate (AS). The two residues in the ligand-binding core were reduced, then kainate crystal forms for the S1S2J L650T͞AMPA and L650T͞ binding should induce greater domain closure and therefore be quisqualate complexes were obtained with 12–16% PEG 8000, able to do more work on the ion channel; i.e., kainate should 0.1–0.3 M zinc acetate, and 0.1 M cacodylate, pH 6.5 (Zn form), become an agonist with greater efficacy. Because mutation of or with 14–18% PEG 4000 and 0.2–0.4 M AS (AS form). S1S2J the conserved Tyr-450 residue to smaller residues resulted in L483Y͞L650T was cocrystallized with 20 mM AMPA by using nonfunctional ligand-binding core protein, we focused our stud- 14–18% PEG 4000 and 0.2–0.4 M AS as precipitant. Crystals ies on residue 650. In fact, mutation of the equivalent residue in were cryoprotected with mother liquors supplemented with GluR1, Leu-646 to Thr (L646T), produces a decrease in the 14–18% glycerol before flash cooling in liquid nitrogen. kainate EC50 and an increase in the extent of kainate current potentiation by cyclothiazide, suggesting that kainate is both a Crystallography. All data sets were collected at the National more potent and strongly desensitizing agonist when acting on the L646T mutant, in comparison with the WT receptor (16). In Synchrotron Light Source beamline X4A (Upton, NY) by using a Quantum 4 charge-coupled device detector except for the addition, leucine is conserved at position 650 in AMPA recep- ͞ ϫ tors, whereas in kainate receptors, which respond maximally to L650T AMPA (Zn form) data set, which was collected at 25 kainate but weakly to AMPA, the residue is either a valine or an on a Brandeis B4 charge-coupled device detector. Data sets were isoleucine. Here we report complementary functional and struc- indexed and merged by using the HKL suite (21). The S1S2J ͞ tural studies of the Leu-650 to Thr (L650T) mutant of the GluR2 L650T kainate, AMPA (AS form), quisqualate (AS form), and receptor, illuminating relationships between agonist binding, the S1S2J L483Y͞L650T͞AMPA structures were solved by domain closure, and ion channel activation. molecular replacement using AMORE (22) with the S1S2J kainate protomer, S1S2J AMPA dimer, S1S2J quisqualate monomer, Materials and Methods and S1S2J AMPA dimer structures as the search probes, re- Molecular Biology. The S1S2J constructs (14) were derived from spectively (12, 14). The S1S2J L650T͞quisqualate(Zn) and the GluR2 (flop) gene (17), whereas the unedited GluR2 or L650T͞AMPA(Zn) crystal forms were refined beginning from GluRB (flip) gene (10), in the pGEM-HE expression vector (32), the WT structures by using X-PLOR (23) and CNS (24). The was used in the physiology. The L650T mutation was incorpo- protocols included rigid-body refinement, simulated annealing, rated into the GluR2 S1S2J, GluR2 S1S2J L483Y, and full- Powell minimization, individual B-value refinement, and bulk- length constructs (14, 18) by using the QuikChange protocol solvent modeling. After every round of refinement, the model (Stratagene) and the primers 5Ј-ATGGAACAACCGACTCT- Ј Ј was manually compared with the electron density by using omit GGATCCACTAAAG-3 and 5 -GTGGATCCAGAGTCGGT- maps, and the model was manually fit to the electron density. TGTTCCATAAGCA-3Ј. The correct clones were confirmed by Refinement continued until the crystallographic R factors con- sequencing both strands of the DNA.
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