Chopra DA et al. (2017), A single-channel mechanism for pharmacological ...

XB-ART-55792

Sci Rep 2017 Jul 31;71:6933. doi: 10.1038/s41598-017-07292-8.

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A single-channel mechanism for pharmacological potentiation of GluN1/GluN2A NMDA receptors.

Chopra DA , Sapkota K , Irvine MW , Fang G , Jane DE , Monaghan DT , Dravid SM .

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NMDA receptors (NMDARs) contribute to several neuropathological processes. Novel positive allosteric modulators (PAMs) of NMDARs have recently been identified but their effects on NMDAR gating remain largely unknown. To this end, we tested the effect of a newly developed molecule UBP684 on GluN1/GluN2A receptors. We found that UBP684 potentiated the whole-cell currents observed under perforated-patch conditions and slowed receptor deactivation. At the single channel level, UBP684 produced a dramatic reduction in long shut times and a robust increase in mean open time. These changes were similar to those produced by NMDAR mutants in which the ligand-binding domains (LBDs) are locked in the closed clamshell conformation by incorporating a disulfide bridge. Since the locked glutamate-binding clefts primarily contributes to receptor efficacy these results suggests that UBP684 binding may induce switch in conformation similar to glutamate LBD locked state. Consistent with this prediction UBP684 displayed greater potentiation of NMDARs with only the GluN1 LBD locked compared to NMDARs with only the GluN2 LBD locked. Docking studies suggest that UBP684 binds to the GluN1 and GluN2 LBD interface supporting its potential ability in stabilizing the LBD closed conformation. Together these studies identify a novel pharmacological mechanism of facilitating the function of NMDARs.

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Genes referenced: pam

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	Figure 1. UBP684 potentiates GluN1/GluN2A receptor responses and slows their deactivation kinetics. (A) UBP684 structure and PAM activity on GluN1/GluN2A receptors expressed in Xenopus oocytes. After obtaining a stable steady-state response to 10 µM L-glutamate and 10 µM glycine, application of 50 µM UBP684 potentiates the agonist response (upper panel). Dose-response of UBP684 potentiation of GluN1/GluN2A receptor responses under saturating agonist conditions (300µM L-glutamate/300 µM glycine) is shown in the lower panel. (B) Whole-cell recordings (left panel) were obtained from HEK293 cells either in dialyzed (top row) or perforated mode (bottom row). Individual cell peak current and deactivation time in response to UBP684 application (right two panels). Using the fast concentration-jump technique, cells were exposed to a solution containing no drug and switched to a solution containing glutamate and glycine (black traces) or glutamate, glycine and UBP694 (red traces). UBP684 (100 µM) potentiated glutamate (100 µM) and glycine (100 µM) induced peak currents in the perforated condition, but not in the dialyzed condition. Slowing of the deactivation rate by UBP684 was observed under both modes. N = 6, *P < 0.05, paired t-test.
	Figure 2. UBP684 induced changes in channel gating resemble effect of locking ligand-binding domain in closed conformation. Cell-attached patches containing one active channel were obtained with agonists alone (glutamate and glycine) (N = 7) or agonists plus UBP684 (N = 7) or from mutant GluN1c/GluN2Ac (N = 4) where the LBD was locked closed by engineered cysteine bridges. (A) Representative single-channel traces are shown. Single-channel recordings were filtered at 5 kHz (2 kHz for presentation) and digitized at 20 kHz. (B) Shut time histograms were fitted with 5 exponential components. Fitting of composite shut time histograms is shown. Individual shut time histograms were fitted and the changes in time constants and their areas were compared. The fold change in the normalized time constants and area of time constants is shown. Data is compared using one-way ANOVA followed by Tukey’s post-hoc analysis. P < 0.05, P < 0.01 and **P < 0.001 compared to control. #P < 0.05, ###P < 0.001 compared to UBP684. SQRT stands for square root.
	Figure 3. UBP684 robustly affects the mean open time in addition to its effects on shut states. (A) Open time histograms were fitted with 2 exponential components. Fitting of composite open time histograms is shown. Individual open time histograms were fitted and the time constants and areas are presented in Table 1. UBP684 (N = 7) increased (B) the mean open time and reduced (C) the mean shut time and lead to an overall increase in (D) open probability compared to control (N = 7). The GluN1c/GluN2Ac mutant (N = 4) shared the reduction in mean shut time and overall increase in open probability but did not lead to a significant increase in mean open time. P < 0.05 and **P < 0.001. SQRT stands for square root.
	Figure 4. Kinetic mechanism describing the effects of UBP684 on GluN1/GluN2A receptor activation. (A) MIL fit of single-channel data to assess the gating mechanism underlying potentiation of UBP684 is shown. All rates are in sec−1. Bold numbers with asterisks denote the rates which were significantly different from glutamate/glycine control patches. Rates ± SEM are presented in Table 2. Data was analyzed using unpaired t-test. (B) Free-energy trajectories for the kinetic states in the different models are presented. The free energies of the active states for the mutant GluN1c/GluN2Ac is lower than control but is still higher than UBP684. Scale bar represents 1 kBT. (C) Macroscopic current profiles were obtained by whole-cell recordings (black traces). Cells were equilibrated to glycine ± UBP684 before exposing to glycine + glutamate ± UBP684 as detailed in Methods. Current profiles were fitted to the kinetic Markov models for control (green) and UBP684 (red) that included glutamate binding and unbinding steps. Except the desensitization rates and the agonist binding and unbinding rates all other rates were fixed to those depicted in the kinetic models. The models were able to accurately predict the activation and deactivation kinetics. Rates obtained from least squares fitting are presented in Table 3.
	Figure 5. UBP684 potentiation is dependent on GluN2 LBD flexibility. (A) Current traces showing the effect of UBP684 on wildtype, GluN1 LBD-locked (GluN1C/GluN2WT) or GluN2 LBD-locked (GluN1WT/GluN2C) receptors. (B) UBP684 potentiated wildtype and GluN1 LBD-locked receptors but not GluN2 LBD-locked receptors (one-way ANOVA followed by Tukey’s test; P = 0.0012 and 0.0093 for GluN1WT/GluN2Ac compared to GluN1WT/GluN2AWT and GluN1C/GluN2AWT respectively; N = 19–32). (C) Molecular modeling of UBP684 binding to the GluN1/GluN2A receptor LBD dimer. Top: UBP684 (space filled) is shown docked into the GluN1/GluN2A LBD intersubunit interface. Modeling suggests that the carboxylic acid group of UBP684 interacts with positively charged residues on the top of the LBD away from the transmembrane domains and near the N-terminal domains. The alkyl side chain terminates near the LBD hinge region near GluN1 Y535 shown in green. Bottom: The same docking of UBP684 is shown rotated 90° in the horizontal plane with the GluN1 LBD removed and GluN1’s Y535 shown for reference.

References [+] :

Banke, Activation of NR1/NR2B NMDA receptors. 2003, Pubmed