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Comparative Study
. 2014 Oct:85:57-66.
doi: 10.1016/j.neuropharm.2014.05.022. Epub 2014 May 27.

Functional insight into development of positive allosteric modulators of AMPA receptors

Autumn M Weeks  1 , Jonathan E Harms  2 , Kathryn M Partin  3 , Morris Benveniste  4
Affiliations
Comparative Study

Functional insight into development of positive allosteric modulators of AMPA receptors

Autumn M Weeks et al. Neuropharmacology. 2014 Oct.

Abstract

Positive allosteric modulators of α-amino-3-hydroxy-5-methyl-isoxazole-propionic acid (AMPA) ionotropic glutamate receptors facilitate synaptic plasticity and contribute essentially to learning and memory, properties which make AMPA receptors targets for drug discovery and development. One region at which several different classes of positive allosteric modulators bind lies at the dimer interface between the ligand-binding core of the second, membrane-proximal, extracellular domain of AMPA receptors. This solvent-accessible binding pocket has been the target of drug discovery efforts, leading to the recent delineation of five "subsites" which differentially allow access to modulator moieties, and for which distinct modulator affinities and apparent efficacies are attributed. Here we use the voltage-clamp technique in conjunction with rapid drug application to study the effects of mutants lining subsites "A" and "B" of the allosteric modulator pocket to assess affinity and efficacy of allosteric modulation by cyclothiazide, CX614, CMPDA and CMPDB. A novel analysis of the decay of current produced by the onset of desensitization has allowed us to estimate both affinity and efficacy from single concentrations of modulator. Such an approach may be useful for effective high throughput screening of new target compounds.

Keywords: AMPAkine; Allosteric modulation; Deactivation; Desensitization; Electrophysiology; Ion channel gating; Mutagenesis; Neuropharmacology.

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Figures

Figure 1
Figure 1. Chemical structures of four positive allosteric modulators of AMPA receptors
A solution-accessible binding site is formed between dimers of the ligand binding domain, allowing positive allosteric modulators to differentially bind and influence channel gating. A, B, Two views of CMPDA (magenta), overlaid with cyclothiazide (CTZ, green) and CX614 (blue). In each panel, the identity of subsites A, B, B', C, and C' are identified with circles and labels. Helices J and K, the flip/flop region, are identified. Residues hypothesized or known to be critical for modulator binding and/or function, and which are studied here, are shown. C, D, Two similar views of CMPDB (pink) in its binding site are shown, with similar labels as indicated above. The moieties of each modulator differentially occupy the five modulator subsites. Protein structures are visualized using PyMol with the PDB files 2AL4, 3H6T, 3RN8, 3RNN.
Figure 2
Figure 2. Mutations in the modulator binding site differentially perturb receptor desensitization
Outside-out membrane patches pulled from HEK293 cells transiently expressing WT GluA2o (flop) receptor isoforms and point mutations in the allosteric modulator binding site were screened for modulator efficacy, using 10 mM glutamate alone or in the presence of CTZ, CX614, CMPDA and CMPDB. Efficacious modulation of desensitization is observed as an absence of peak current decay during a prolonged (500 ms) pulse of 10 mM glutamate in the presence of 10 (CMPDA and B) or 100 (CTZ, CX614) μM. Traces shown are normalized to the largest peak amplitude, which was 500 pA, and ranged from10–500 pA. WT traces are re-published from Timm et al., 2011 with permission from the publisher.
Figure 3
Figure 3. Quantitative analysis of decay properties among modulators
Shown are three different types of decay patterns that were observed for WT GluA2o. The control trace has a rapid decay of the current from the peak, and almost no sustained current during the pulse; the black bar represents application of 10 mM glutamate for 500 ms. The CTZ current trace shows a slow decay throughout the 500 ms pulse of glutamate. The CMPDB trace shows an immediate, rapid decay to a sustained current that is larger than baseline. These three traces illustrate the three representative patterns that were observed for various combinations of modulators and mutant receptors shown in Figure 2. Traces have been normalized to their peak amplitudes to emphasize the different kinetics of decay. To quantitatively analyze these differences, measurements were made of the peak amplitude (IA), the amplitude measured at the time of 95% decay of control responses in the absence of modulator (IB), and the amplitude just prior to termination of the pulse of glutamate (IC). For the control trace, the current at which there is 95% decay from the peak amplitude is marked with an arrow. The inset shows an expanded view of the peak amplitudes. Values for each condition are presented in Table 2. Traces with modulators are single sweeps. Trace of 10 mM glutamate alone is an average of 25 sweeps.
Figure 4
Figure 4. Rapid decay to a sustained current likely represents a sub-saturating concentration of allosteric modulator
Shown are three averaged traces representing representing a 500 ms pulse of glutamate in the absence of modulator, or in the presence of 100 or 300 μM CX614. These traces are normalized to their peak responses to easily compare the kinetics of decay from peak. IA, IB and IC are shown to demarcate the peak amplitude, amplitude of 95% decay of control and the steady state current, respectively. IB Rapid decay to IB signifies desensitization of receptors not bound by CX614. Increasing the concentration of CX614 reduces the amplitude of this rapid decay without significantly affecting its kinetics. Traces for control, 100 and 300 μM CX614 are averages of 2, 3 and 8 sweeps, respectively.
Figure 5
Figure 5. Simulations of High and Low Efficacy Modulators at Different Receptor Occupancies
Upper Panel, 12-state model used to simulate effect of low and high efficacy modulators on AMPA receptor desensitization. Circled numbers indicate transitions whose rate constants can be found in Supplementary Table 1. R = receptor; M = modulator; Glu = glutamate; Rd = desensitized receptor. R*-Glu2 and R*M-Glu2 represent the open states for receptors without and with modulator bound. A, Simulations with a high efficiency modulator in which a saturating concentration of modulator yields full block of desensitization. Simulated AMPA receptor currents in the absence of modulator are depicted in the left panel. Middle and right panels represent AMPA receptor currents in the presence of modulator where approximately half the receptors or all the receptors are occupied by modulator. Inset. Expanded scale of the rapid component from the left (dotted line) and middle panels (solid line). B, Simulations utilizing a low-efficacy modulator. Left and right panels represent < 50% occupancy and full occupancy by modulator, respectively. Inset: Expanded scale of initial decay of currents. Dotted line represents a simulation in the absence of modulator (A, left panel). Arrows in A and B indicate the time to 95% decay where IB is measured and represents the fast component in the absence of modulator. C, Quantification of unoccupied receptors and the degree of desensitization. Unoccupied receptors can be quantified as (IA - IB) / IA while desensitization of receptors occupied by modulator (modulator efficacy) is characterized as IC / IB. D, The efficacy of the modulator measured as % desensitization of occupied receptors (IC / IB) does not change substantially with modulator concentration. Open circle indicates % Desensitization in the absence of modulator. Sat = saturating concentration of modulator.
Figure 6
Figure 6. Comparison of modulator affinity and efficacy for different AMPA receptor mutations
Efficacy and occupancy data from Table 2 for the effect of a given modulator on a given mutation in either flip (left) or flop (right) isoforms of GluA2 receptors has been normalized to the same modulator treatment for the respective wild type receptor isoform. Receptor occupancy is indicative of relative changes in modulator apparent affinity.
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