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. Author manuscript; available in PMC: 2018 Mar 28.
Published in final edited form as: Cell Rep. 2018 Feb 27;22(9):2307–2321. doi: 10.1016/j.celrep.2018.02.021

Figure 5. Gabapentin Diminishes Membrane Surface Expression of α2δ-1-Bound NMDARs.

Figure 5

(A) Luminescence resonance energy transfer (LRET) between terbium-labeled GluN2A (GluN2A*), GluN1, and YFP-α2δ-1. Black curve: without gabapentin; red curve: with gabapentin. Left: LRET lifetime signals show that gabapentin resulted in loss of the LRET signal between terbium labeled-GluN2A* and YFP-α2δ-1 on themembrane. Middle: gabapentin had no effect on the LRET signal when YFP-α2δ-1 was replaced with its R217A mutant. Right: donor-only curves of terbium-labeled GluN2A* and unlabeled α2δ-1. Data were from 4 or 5 independent experiments (Table S1).

(B) LRET between terbium-labeled GluN1 (GluN1*), GluN2A, and YFP-α2δ-1. Black curve: without gabapentin; red curve: with gabapentin. Left: LRET lifetime signals show that gabapentin diminished the interaction between terbium labeled-GluN1* and YFP-α2δ-1 on the membrane. Middle: gabapentin had no effect on the LRET signal when YFP-α2δ-1 was replaced with its R217A mutant. Right: donor-only curves of terbium-labeled GluN1* and unlabeled α2δ-1. Data were from 4 or 5 independent experiments (Table S1).

(C) Coimmunoprecipitation and immunoblotting (IB) analysis shows that gabapentin (GBP; 100 µM for 30 min) diminished the expression of α2δ-1-bound NMDARs in the membrane extract of HEK293 cells. Left: the GluN1/GluN2A heterodimer was cotransfected with YFP-α2δ-1, a YFP-α2δ-1 mutant (R217A, also called R241A), or IRES-α2δ-1 (no tag) in HEK293 cells. Right: the GluN1/GluN2B heterodimer was cotransfected with YFP-α2δ-1, a YFP-α2δ-1 mutant (R217A, also called R241A), or IRES-α2δ-1 (no tag) in HEK293 cells. Data were from four independent experiments.

(D and E) Membrane surface protein analysis (D) and mean levels (E) show that gabapentin treatment reversed the α2δ-1 coexpression-induced increase in NMDAR surface expression. Immunoblotting was performed using antibodies against GluN1, GluN2A, GluN2B, and α2δ-1 for the membrane surface proteins isolated with biotinylation. HEK293 cells were cotransfected and treated with gabapentin or vehicle as indicated above the gel images. Na+/K+-ATPase, a known membrane protein marker, was used as an internal control. Data are expressed as means ± SEM (n = 5 independent experiments). *p < 0.05 (versus GluN1/GluN2A or GluN1/GluN2B alone), one-way ANOVA followed by Tukey’s post hoc test; #p < 0.05 (versus GluN1/GluN2A/α2δ-1 or GluN1/GluN2B/α2δ-1 without gabapentin), two-tailed Student’s t test. See also Table S1.