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Structural basis for partial agonist action at ionotropic glutamate receptors

Nature Neuroscience volume 6, pages 803–810 (2003)Cite this article

Abstract

An unresolved problem in understanding neurotransmitter receptor function concerns the mechanism(s) by which full and partial agonists elicit different amplitude responses at equal receptor occupancy. The widely held view of 'partial agonism' posits that resting and active states of the receptor are in equilibrium, and partial agonists simply do not shift the equilibrium toward the active state as efficaciously as full agonists. Here we report findings from crystallographic and electrophysiological studies of the mechanism of activation of an AMPA-subtype glutamate receptor ion channel. In these experiments, we used 5-substituted willardiines, a series of partial agonists that differ by only a single atom. Our results show that the GluR2 ligand-binding core can adopt a range of ligand-dependent conformational states, which in turn control the open probability of discrete subconductance states of the intact ion channel. Our findings thus provide a structure-based model of partial agonism.

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Figure 1: Ionotropic glutamate receptor domain organization and agonist structure.
Figure 2: The 5-substituted willardiines are highly potent agonists and act as partial agonists on the GluR2 receptor.
Figure 3: Electron density |Fo| − |Fc| 'omit' maps for willardiines and selected interacting residues.
Figure 4: The 5-substituted willardiines produce greater domain closure and separation of residue Pro632 as the size of the 5-substituent decreases.
Figure 5: Partial agonists differentially activate subconductance levels.
Figure 6: Subunit-linked channel opening is correlated with degree of domain closure.

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Acknowledgements

We thank J. Lidestri for maintenance of the x-ray laboratory at Columbia University, C. Ogata for assistance at X4A, C. Glasser for technical assistance, and W.N. Zagotta for helpful discussions. Synchrotron diffraction data were collected at beamlines X26C and X4A at the National Synchrotron Light Source. This work was supported by the Klingenstein Foundation (E.G.), the National Alliance for Research on Schizophrenia and Depression (E.G.) and the National Institutes of Health (E.G., M.L.M., S.T.), the Benzon Society (T.B.), and the Danish MRC (T.B.). E.G. is also an assistant investigator of the Howard Hughes Medical Institute. We thank D. Colquhoun for supplying software for single-channel analysis.

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biophysics, Columbia University, 650 West 168 Street, New York, 10032, New York, USA

    Rongsheng Jin & Eric Gouaux

  2. Howard Hughes Medical Institute, Columbia University, 650 West 168 Street, New York, 10032, New York, USA

    Eric Gouaux

  3. Department of Pharmacology, Emory University, School of Medicine, Rollins Research Center, Atlanta, 30322, Georgia, USA

    Tue G Banke & Stephen F Traynelis

  4. Laboratory of Cellular and Molecular Neurophysiology, National Institute of Child Health and Development, National Institutes of Health, Bethesda, 20892, Maryland, USA

    Mark L Mayer

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Correspondence to Eric Gouaux.

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Supplementary information

Supplementary Fig. 1.

The willardiines bind similarly to glutamate but induce a more open cleft conformation in comparison to the glutamate complex. (a) Stereo view of the binding pocket in superimposed HW and glutamate structures. The superposition was carried out using the Ca atoms of all residues in domain 1. The HW and glutamate complexes are shown in pink and green while HW and glutamate are yellow and purple, respectively. Ion pair and hydrogen bond interactions between HW and the protein are indicated as dashes: direct interactions to the protein are in cyan and water mediated interactions are in black. Selected water molecules (W1-4) in the HW and glutamate structures are drawn as cyan and pink spheres, respectively. Shown in panels (b-d) are stereo views of the binding pocket in superimposed FW/glutamate, BrW/glutamate and IW/glutamate structures, respectively. The glutamate complex is colored as in a, and the FW, BrW and IW structures are colored the same as HW is colored in a. Note that water W5 is observed in the BrW and IW complexes, and not in the HW and FW complexes. Panels b and d were taken from Jin, R. & Gouaux, E. Biochemistry 42, 5201-5213 (2003). (JPG 138 kb)

Supplementary Fig. 2.

The willardiines bind similarly to glutamate but induce a more open cleft conformation in comparison to the glutamate complex. (a) Stereo view of the binding pocket in superimposed HW and glutamate structures. The superposition was carried out using the Ca atoms of all residues in domain 1. The HW and glutamate complexes are shown in pink and green while HW and glutamate are yellow and purple, respectively. Ion pair and hydrogen bond interactions between HW and the protein are indicated as dashes: direct interactions to the protein are in cyan and water mediated interactions are in black. Selected water molecules (W1-4) in the HW and glutamate structures are drawn as cyan and pink spheres, respectively. Shown in panels (b-d) are stereo views of the binding pocket in superimposed FW/glutamate, BrW/glutamate and IW/glutamate structures, respectively. The glutamate complex is colored as in a, and the FW, BrW and IW structures are colored the same as HW is colored in a. Note that water W5 is observed in the BrW and IW complexes, and not in the HW and FW complexes. Panels b and d were taken from Jin, R. & Gouaux, E. Biochemistry 42, 5201-5213 (2003). (JPG 31 kb)

Supplementary Fig. 3.

Properties of GluR2 macroscopic currents. (a) Current response of an outside-out patch from a GluR2-L483Y transfected HEK-293 cell (Vhold -60 mV) to slow application 1.2 mM HW (bar). Data were filtered at 5 kHz (-3dB) and sampled at 10 kHz. The x-axis is broken between 10 and 30 s. The slowly changing current response during washout of agonist at the end of the trace (dotted box) was used for variance analysis, and is shown below after high-pass filtering (1 Hz, -3dB). (b) Current traces before and after high pass filtering were divided up into 50 fractions and the current variance was plotted against the mean current for each section. Data were fitted with Variance = i I - I 2 / N + base where N is the number of channels, i is the weighted mean unitary current, I is the mean current, and base is the baseline variance. Po was calculated as the ratio of the measured maximal current to the predicted current ( N i ). Po for this response was 0.83 (13.6 pS). (c-d) Peak response dose response curves were determined for glutamate (c) and IW (d) applied to outside-out patches excised from HEK cells transiently transfected with wiild type GluR2. Data are expressed + SEM. (JPG 29 kb)

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Jin, R., Banke, T., Mayer, M. et al. Structural basis for partial agonist action at ionotropic glutamate receptors. Nat Neurosci 6, 803–810 (2003). https://doi.org/10.1038/nn1091

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