Pharmacological disruption of calcium channel trafficking by the alpha2delta ligand gabapentin

Abstract

The mechanism of action of the antiepileptic and antinociceptive drugs of the gabapentinoid family has remained poorly understood. Gabapentin (GBP) binds to an exofacial epitope of the alpha(2)delta-1 and alpha(2)delta-2 auxiliary subunits of voltage-gated calcium channels, but acute inhibition of calcium currents by GBP is either very minor or absent. We formulated the hypothesis that GBP impairs the ability of alpha(2)delta subunits to enhance voltage-gated Ca(2+)channel plasma membrane density by means of an effect on trafficking. Our results conclusively demonstrate that GBP inhibits calcium currents, mimicking a lack of alpha(2)delta only when applied chronically, but not acutely, both in heterologous expression systems and in dorsal root-ganglion neurons. GBP acts primarily at an intracellular location, requiring uptake, because the effect of chronically applied GBP is blocked by an inhibitor of the system-L neutral amino acid transporters and enhanced by coexpression of a transporter. However, it is mediated by alpha(2)delta subunits, being prevented by mutations in either alpha(2)delta-1 or alpha(2)delta-2 that abolish GBP binding, and is not observed for alpha(2)delta-3, which does not bind GBP. Furthermore, the trafficking of alpha(2)delta-2 and Ca(V)2 channels is disrupted both by GBP and by the mutation in alpha(2)delta-2, which prevents GBP binding, and we find that GBP reduces cell-surface expression of alpha(2)delta-2 and Ca(V)2.1 subunits. Our evidence indicates that GBP may act chronically by displacing an endogenous ligand that is normally a positive modulator of alpha(2)delta subunit function, thereby impairing the trafficking function of the alpha(2)delta subunits to which it binds.

Fig. 1.

GBP is effective to inhibit I_Ba after heterologous expression, when applied chronically but not acutely. (A) (Left) Current density–voltage (IV) relationships for Ca_V2.1/β4/α₂δ-2 currents in the absence or presence of chronic GBP (1 mM, red circles, n = 11) for ≈40 h (or H₂O as control, black squares, n = 13) from immediately after transfection of tsA-201 cells until recordings were performed. The reduction in peak I_Ba at +15 mV was statistically significant (P = 0.0013, Student's two-tailed t test). (Right) Examples of currents resulting from step potentials from −90 mV to between −15 and +15 mV in 5-mV increments, under control conditions and in the presence of GBP. Calibration bars refer to both sets of traces. (B) Steady-state inactivation data were obtained from the cells treated as described in A. Chronic GBP (1 mM, red circles, n = 12) and control (black circles, n = 13). The steady-state inactivation in the absence of α₂δ subunits is provided for comparison (dashed line, n = 11). Data are fit to a single Boltzmann equation. The V_50,inact was 37.6 ± 2.1 mV in the presence of α₂δ-2, −30.8 ± 4.4 mV in the absence of α₂δ (dashed line), and −28.3 ± 2.7 mV in the continued presence of GBP (P = 0.0103 compared with the absence of GBP, Student's two-tailed t test). (C) GBP (1 mM) applied acutely for 10 min had no effect on Ca_V2.1/β4/α₂δ-2 currents. Examples of currents resulting from step potentials from −90 mV to +10 mV, under control conditions and after application of GBP for 10 min, to a cell whose initial current amplitude had stabilized. Currents are representative of n = 5 cells, where the peak current (red trace) after application of GBP was 99.0 ± 7.1% of its initial value, before GBP application (black trace). (D) IV relationships for Ca_V2.1/β4/α₂δ-2 currents from cells cultured in the absence or presence of 100 μM GBP applied chronically as described in A. GBP (red circles, n = 10) and control (black squares, n = 14). The reduction in peak I_Ba at +15 mV was statistically significant (P = 0.0215, Student's two-tailed t test). (E) The percentage of inhibition of peak current density by GBP (100 μM, hatched red bar, n = 10; and 1 mM, solid red bar, n = 11) was determined for each experiment and compared with the reduction observed in the absence of α₂δ (gray bar, n = 14). The statistical significance of the reduction was determined relative to the data in the presence of α₂δ-2 and absence of GBP for each individual set of experiments; *, P = 0.0013; **, P = 0.021; ***, P = 0.00004, Student's two-tailed t test. (F) (Left) IV relationships for Ca_V2.2/β1b/α₂δ-1 currents in the absence or presence of chronic GBP (1 mM, red circles, n = 10) for ≈40 h or the equivalent amount of H₂O as control (black squares, n = 8). (Right) Examples of currents resulting from step potentials from −90 mV to between −15 and +15 mV in 5-mV increments, under control conditions (black traces) and in the presence of GBP (red traces). Calibration bars refer to both sets of traces.

Fig. 2.

The effect of GBP requires both binding to the α₂δ subunit and uptake by neutral amino acid transporter. (A) (Left) IV relationships for Ca_V2.1/β4/R282-α₂δ-2 currents in the absence or presence of chronic GBP (1 mM, red circles, n = 12) for ≈40 h or the equivalent amount of H₂O as control (black squares, n = 12), from immediately after transfection until recordings were performed. (Right) Examples of currents resulting from step potentials from −90 mV to between −15 and +15 mV in 5-mV increments under control conditions and in the presence of 1 mM GBP. Calibration bars refer to both sets of traces. (B) Steady-state inactivation data were obtained from the same cells as described in A. Chronic GBP (1 mM, red circles, n = 5) and control (black squares, n = 7). (C) (Upper) IV relationships for Ca_V2.2/β1b/R217A-α₂δ-1 currents in the absence or presence of chronic GBP. The cDNAs were transfected into tsA-201 cells that were then incubated with GBP (1 mM, red circles, n = 5) for ≈40 h or the equivalent amount of H₂O as control (black squares, n = 4), from immediately after transfection until recordings were performed. (Lower) Examples of currents resulting from step potentials from −90 mV to between −15 and +15 mV in 5-mV increments under control conditions and in the presence of 1 mM GBP. Calibration bars refer to both sets of traces. (D) Diagram illustrating some of the various sites at which GBP (red circles) or the putative endogenous ligand (green circles) may act on α₂δ subunits. The effect of GBP may be to displace an endogenous ligand and impair the ability of α₂δ-1 and α₂δ-2 to increase VGCC concentration at the plasma membrane. Thus, GBP may exert its effect on intracellular α₂δ subunits during maturation and trafficking of the VGCC complex to the plasma membrane and/or bind to cell surface α₂δ subunits and affect recycling from the plasma membrane. BCH is a competitive inhibitor of system-L transport. (E) IV relationships for Ca_V2.1/β4/α₂δ-2 currents either under control conditions (black squares, n = 8) or after chronic treatment with GBP (1 mM), in the absence (red filled circles, n = 7) or presence (red open circles, n = 10) of the inhibitor of system-L transport, BCH (10 mM) applied chronically from 1 h before GBP. The reduction in peak I_Ba by GBP alone at +10 mV was statistically significant compared with control (P = 0.0464, Student's two-tailed t test). (F) I_Ba was measured in Xenopus oocytes after injection of Ca_V2.2/β1b/α₂δ-2 cDNAs, together with either a control cDNA (nonconducting Kir2.1-AAA (30) (Left) or with mLAT4 (27) (Right). Oocytes were incubated without (black bars) or with 200 μM GBP (red bars) from 1 h after the time of injection until recording between 40 and 48 h later. To combine data from several experiments, the mean peak control I_Ba at +5 mV was normalized and the effect of GBP determined relative to control. The numbers of determinations are shown on the bars. Statistical significance: **, P = 0.0042 for the effect of GBP in the mLAT4 condition only (two-way ANOVA and Bonferroni's post hoc test). (G) As in E, with the combinations of transfected subunits indicated below the bars. Oocytes were incubated without (black bars) or with (white bars) 400 μM l-leucine. The statistical significance is P = 0.011 (*) for the effect of l-leucine in the mLAT4 condition and only in the presence of α₂δ-2.

Fig. 3.

Effect of chronic GBP on the plasma membrane localization of Ca_V2.1 and α₂δ-2 in COS-7 cells. (A) Ca_V2.1–2HA was cotransfected with β4 and α₂δ-2 and cultured for 48 h either in the absence or in the presence of GBP (100 μM or 1 mM). Cells were then fixed and permeabilized before immunocytochemical localization of Ca_V2.1 (HA Ab, Left) and α₂δ-2 [α₂-2 (102–117) Ab, Center], by using a magnification ×63 objective. Merged images are shown (Right) (Ca_V2.1 is shown in green and α₂δ-2 in red, with regions of colocalization in orange–yellow). Nuclear staining (blue, DAPI) is shown in the merged images. Images show 1-μm optical sections of data representative of three independent experiments. (Scale bar: 30 μm.) No signal was observed in nontransfected cells or in the absence of primary Abs (data not shown). (B) Images are obtained as in A, but cells were not permeabilized. The transfected cell(s) in each image are identified by an arrow. Similar results were obtained in three independent experiments. (Scale bar: 30 μm.) (C) (Left) Merged images of nonpermeabilized cells taken with a magnification ×20 objective to show a larger field of view for cells transfected as in A and cultured for 48 h either in the absence or in the presence of GBP (100 μM or 1 mM). The optical section for these images is 4.5 μm, and, as the cells are flattened, staining is seen over most of the cell surface. (Scale bar: 60 μm.) (Right) Quantification of cell-surface fluorescence intensity per cell area for Ca_V2.1 (white bars) and α₂δ-2 (gray bars) from magnification ×20 images under control conditions (n = 22 cells) and in the presence of 100 μM GBP (n = 19 cells) and 1 mM GBP (n = 24 cells). **, P < 0.01; ***, P < 0.001; one-way ANOVA, with Bonferroni's post hoc test. (D) The effect of GBP (100 μM and 1 mM) was determined on cell-surface expression of α₂δ-2 (Upper), with the intracellular protein Akt as control (Lower). (Left) Whole-cell lysate (WCL). (Right) Streptavidin pull-down of biotinylated proteins, immunoblotted with α₂-2 (102–117) or Akt Ab. The leftmost lane is from nonbiotinylated control cells (con). The proportion of α₂δ-2 at the cell surface was estimated from the ratio of the biotinylated α₂δ-2 to the amount of α₂δ-2 in the WCL. Data are from five experiments; 4.93 ± 1.92% of total α₂δ-2 was at the plasma membrane, and this was reduced compared with control by 19.8 ± 7.6% and 30.4 ± 14.4% in the chronic presence of 100 μM and 1 mM GBP, respectively (n = 5, P < 0.05, repeated-measures ANOVA and Bonferroni's post hoc test).

Fig. 4.

Effect of GBP on native calcium currents in DRGs. (A) (Upper Left) IV relationships for HVA I_Ba recorded from rat DRGs cultured for 3 days in the absence (black squares, n = 22) or presence of GBP (1 mM, red circles, n = 19). (Upper Right) Example HVA currents from a holding potential (V_H) of −40 mV to between −30 and +10 mV in 10-mV steps. Upper traces (black) control; lower traces (red) chronic GBP (1 mM). I_Ba was measured at 20 ms, as indicated by the dotted line. I_Ba at 0 mV was significantly inhibited by chronic GBP (P = 0.02, Student's two-tailed t test). (Lower) As above, except V_H was −80 mV, and I_Ba amplitude was measured at the end of the 220-ms step, as indicated by the dotted line. I_Ba was obtained in the absence (black squares, n = 12), or presence of GBP (1 mM, red circles, n = 13). (B) Acute application of GBP (1 mM) had no effect on native DRG HVA currents. (Left) Bar chart showing lack of effect of perfusion for 10 min of control medium (black bar) or GBP (1 mM, red bar) to cells whose initial current amplitude had stabilized (n = 6 for each). (Right) Examples of currents resulting from a step potential from V_H −40 mV to +10 mV under control conditions (black trace) and from the same cell after application of GBP (red trace) for 10 min.