ADAM17 Mediates Proteolytic Maturation of Voltage-Gated Calcium Channel Auxiliary α2δ Subunits, and Enables Calcium Current Enhancement

Abstract

The auxiliary α₂δ subunits of voltage-gated calcium (Ca_V) channels are key to augmenting expression and function of Ca_V1 and Ca_V2 channels, and are also important drug targets in several therapeutic areas, including neuropathic pain. The α₂δ proteins are translated as preproteins encoding both α₂ and δ, and post-translationally proteolyzed into α₂ and δ subunits, which remain associated as a complex. In this study, we have identified ADAM17 as a key protease involved in proteolytic processing of pro-α₂δ-1 and α₂δ-3 subunits. We provide three lines of evidence: First, proteolytic cleavage is inhibited by chemical inhibitors of particular metalloproteases, including ADAM17. Second, proteolytic cleavage of both α₂δ-1 and α₂δ-3 is markedly reduced in cell lines by knockout of ADAM17 but not ADAM10. Third, proteolytic cleavage is reduced by the N-terminal active domain of TIMP-3 (N-TIMP-3), which selectively inhibits ADAM17. We have found previously that proteolytic cleavage into mature α₂δ is essential for the enhancement of Ca_V function, and in agreement, knockout of ADAM17 inhibited the ability of α₂δ-1 to enhance both Ca_V2.2 and Ca_V1.2 calcium currents. Finally, our data also indicate that the main site of proteolytic cleavage of α₂δ-1 is the Golgi apparatus, although cleavage may also occur at the plasma membrane. Thus, our study identifies ADAM17 as a key protease required for proteolytic maturation of α₂δ-1 and α₂δ-3, and thus a potential drug target in neuropathic pain.

Keywords: ADAM17; calcium channel; calcium currents; matrix metalloprotease; trafficking; α2δ subunit.

Graphical Abstract

Figure 1.

Effect of chemical inhibitors of ADAMs and MMPs on α₂δ-1 proteolytic cleavage. (A) Diagram of post-translational processing of α₂δ proteins, including glycosylation (V), GPI anchoring, and proteolytic cleavage. It also shows the approximate position of inserted HA tag (red) and disulfide bonding between α₂ (black) and δ (white). (B) Effect of GM-6001 (0, 10, and 25 µM; lanes 1–3, respectively) on cleavage in whole cell membranes of HA-α₂δ-1 expressed in tsA-201 cells (upper panel: HA immunoblot), deglycosylated with PNGase-F to allow resolution between pro-α₂δ-1 (upper band) and the cleaved form, α₂–1 (lower band). The absolute % cleavage was 12.8 ± 2.0% in control conditions. Lower panel, loading control: Endogenous GAPDH. (C) Quantification of the effect of 10 µM (squares) and 25 µM (triangles) GM-6001 on relative cleavage of α₂δ-1 (normalized to that under control conditions (circles)). Data are mean ± SEM and individual data in three separate experiments, including that in (B), denoted by red, green, and blue symbols. Statistical differences determined using 1-way ANOVA and Tukey post hoc test; ** P = .0084; ***P = .0090. (D) Effect of SB-3CT (0, 1, and 100 µM; lanes 1–3, respectively) and CL-82198 (60 µM; lane 4) on cleavage in whole cell membranes of HA-α₂δ-1 expressed in tsA-201 cells. Top panel: α₂δ-1 immunoblot and middle panel: HA immunoblot, both deglycosylated to allow resolution between pro-α₂δ-1 (upper band) and the cleaved form, α₂–1 (lower band). Bottom panel: Loading control endogenous GAPDH. The absolute % cleavage was 11.2 ± 1.0% in control conditions. (E) Quantification of the effect of 1 and 100 µM SB-3CT on relative cleavage of α₂δ-1, measured from HA immunoblots (normalized to that under control conditions). Data are mean ± SEM and individual data in three separate experiments, including that in (D), denoted by red, green, and blue symbols). Statistical differences determined using 1-way ANOVA and Tukey post hoc test; ***P < .0001.

Figure 2.

Cleavage of α₂δ-1 and α₂δ-3 is reduced in CRISPR ADAM17^–/–/ADAM10^–/– and ADAM17^–/– compared to CRISPR WT cells. (A) Effect of expression of α₂δ-1 in CRISPR WT (lanes 1–3) compared to CRISPR ADAM17^–/–/ADAM10^–/– (A17/10-KO) HEK293 cells (lanes 4–6) on cleavage of HA-α₂δ-1 (α₂–1 immunoblot) in WCL, deglycosylated to allow resolution between pro-α₂δ-1 (upper band) and the cleaved form, α₂–1 (lower band). The absolute % cleavage was 21.2 ± 1.4% in CRISPR WT cells and 11.05 ± 1.2% in ADAM17^–/–/ADAM10^–/– cells (n = 4 experiments). (B) Effect of expression of α₂δ-1 in CRISPR WT (lanes 1–3) compared to ADAM17^–/–/ADAM10^–/– (A17/10-KO) HEK293 cells (lanes 4–6) on cleavage of HA-α₂δ-1 (α₂–1 immunoblot) in deglycosylated cell surface biotinylated fractions. The absolute % cleavage was 37.4 ± 2.4% in CRISPR WT cells (n = 6 experiments). (C) Quantification of the effect of expression in ADAM17^–/–/ADAM10^–/– cells on relative cleavage of α₂δ-1 in cell surface biotinylated fraction, normalized to that in CRISPR WT cells. Data are mean ± SEM and individual data from six separate experiments, performed on two different transfections (red and green symbols). Statistical difference determined using Student's t-test; P = .0004. (D) Effect of expression of α₂δ-3 in CRISPR WT (lanes 1 and 2) compared to ADAM17^–/–/ADAM10^–/– HEK293 cells (lanes 3 and 4) on cleavage of HA-α₂δ-3 in cell surface biotinylated fraction (α₂–3 immunoblot), deglycosylated to allow resolution between pro-α₂δ-3 (upper band) and the cleaved form, α₂–3 (lower band). The absolute % cleavage was 23.3 ± 2.5% in CRISPR WT cells (n = 4). (E) Quantification of the effect of ADAM17^–/–/ADAM10^–/– on relative cleavage of α₂δ-3 in cell surface biotinylated fraction (normalized to that in CRISPR WT cells). Data are mean ± SEM and individual data in 4–5 separate experiments, performed on two separate transfections (red and green symbols). Statistical difference determined using Student's t-test; ***P = .0002. (F) Effect of expression of α₂δ-1 in CRISPR WT (lanes 1 and 2) compared to ADAM17^–/– (lanes 3 and 4) and ADAM10^–/– (lanes 5 and 6) HEK293 cells on cleavage of HA-α₂δ-1 (α₂–1 immunoblot) in cell surface biotinylated fractions, deglycosylated to allow resolution between pro-α₂δ-1 (upper band) and the cleaved form, α₂–1 (lower band). The absolute % cleavage was 44.5 ± 2.7% in CRISPR WT cells (n = 9). (G) Quantification of the effect of expression in ADAM10^–/– and ADAM17^–/– cells on relative cleavage of α₂δ-1 in cell surface biotinylated fraction, normalized to that in CRISPR WT cells. Data are mean ± SEM and individual data for nine separate experiments from four different transfections (all including WT and ADAM17^–/– cells, and four also including ADAM10^–/–cells; colored symbols refer to different experiments). Statistical differences determined using one-way ANOVA and Tukey's multiple comparison test; ^****P < .0001.

Figure 3.

Knockout of ADAM17, but not ADAM10, decreases Ca_V2.2 and Ca_V1.2 currents. (A) Examples of I_Ba currents for Ca_V2.2 expressed in CRISPR WT HEK293 cells (black), ADAM10^–/– cells (red) and ADAM17^–/– cells (blue). Ca_V2.2 is co-expressed with β1b and α₂δ-1. Holding potential −80 mV, steps between −50 mV and +60 mV for 50 ms. (B) Mean (± SEM) current–voltage (I–V) relationships for the conditions shown in (A). Control (black; n = 23), ADAM10^–/– (red, n = 29), and ADAM17^–/– (blue; n = 21). The individual and mean I–V data were fit with a modified Boltzmann equation (see Methods). V_{50, act} values are −5.9 ± 1.4 mV (Control), −5.8 ± 0.8 mV (ADAM10^–/–), and −2.5 ± 1.36 mV (ADAM17^–/–). Two-Way ANOVA with Sidak's post hoc test correction for multiple comparisons was performed for the I–V data: ^∗P < .05, ^∗∗P < .01, ^∗∗∗P < .001, and ^∗∗∗∗P < .0001. (C) Examples of I_Ba currents for Ca_V1.2 expressed in CRISPR WT HEK293 cells (black) and ADAM17^–/– cells (blue). Ca_V1.2 is co-expressed with β3 and α₂δ-1. Holding potential −80 mV, steps between −60 and +90 mV for 50 ms. (D) Mean (± SEM) I–V relationships for the conditions shown in (C). Control (black; n = 26) and ADAM17^–/– (blue; n = 19). The individual and mean I–V data were fit with a modified Boltzmann equation as in (B). V_{50, act} values are 5.8 ± 1.0 mV (Control) and 6.5 ± 1.9 mV (ADAM17^–/–). Statistical differences between the two sets of I–V data were examined at each potential and corrected for multiple t-tests with Holm Sidak's post hoc correction: ^∗∗P < .01, ^∗∗∗∗P < .0001.

Figure 4.

ADAM17 knockout does not alter the cell surface expression of Ca_V2.2 at the plasma membrane. (A) Confocal images of GFP_Ca_V2.2-HA expressed in CRISPR WT (top), ADAM17^–/– (middle) or ADAM10^–/– (bottom) HEK293 cells. All conditions contained β1b and α₂δ-1. Cell surface staining of GFP_Ca_V2.2-HA was obtained by incubating the cells with HA Ab in nonpermeabilized cells (left, white). The total expression of GFP_Ca_V2.2-HA is determined by the cytoplasmic GFP signal (middle), the merged images are shown on the right (HA in red). Scale bars, 10 μm. (B) Scatter plot (mean ± SEM with individual data points for six independent transfections, with all conditions in parallel), showing the ratio of cell surface to total Ca_V2.2 (HA/GFP) in CRISPR WT, ADAM17^–/–or ADAM10^–/– HEK293 cells. Each individual data point represents a mean of ratio HA/GFP of ∼100 cells/experiment. ns = nonsignificant for WT vs. ADAM17^–/–; *P = .0379 for WT vs. ADAM10^–/–, one-way ANOVA, and Tukey's post hoc test, correcting for multiple comparisons.

Figure 5.

Effect of block of ER–Golgi trafficking and stimulation of alternative route to cell surface by Arf1(Q71L) on cleavage and N-glycosylation pattern of α₂δ-1 in CRISPR WT and ADAM17^–/– cells. (A) Schematic representation of post-translational modifications of α₂δ-1. Immature glycosylation, occurring in the ER, is sensitive to Endo-H, whereas mature glycosylation, occurring in the Golgi, is resistant to Endo-H. There are differential effects of Endo-H on uncleaved (top) and cleaved (bottom) α₂δ-1. (B) Effect of expression of α₂δ-1 in CRISPR WT (lanes 1, 2, and 5) and ADAM17^–/– (A17-KO) cells (lanes 3 and 4) in the absence (lanes 1, 3, and 5) and presence (lanes 2 and 4) of the ER-to-Golgi blocker, Arf1(Q71L), in cell surface biotinylated samples, to show fraction at the plasma membrane, either treated with Endo-H (lanes 1–4) or left untreated (lane 5) for comparison. The sizes of the Endo-H-resistant bands (after mature glycosylation in the Golgi) and Endo-H-sensitive bands (after blocking ER-to-Golgi transport using Arf1(Q71L)) are indicated with arrows. Representative of n = 3 separate experiments. (C) Effect of expression of α₂δ-1 in CRISPR WT (lanes 1 and 2) and ADAM17^–/– (A17-KO) cells (lanes 3 and 4) in the absence (lanes 1 and 3) and presence (lanes 2 and 4) of Arf1(Q71L). Samples are biotinylated and fully deglycosylated with PNGase-F to show uncleaved-α₂δ-1 (upper band) and cleaved α₂–1 (lower band). The absolute % cleavage of α₂δ-1 to cleaved α₂–1 was 27.3 ± 1.3% in control CRISPR WT cells (n = 3). *indicates an intermediate species, which may represent cleavage of α₂δ-1 at an alternative site, or an intermediate product. (D) Quantification of the effect of of Arf1(Q71L) (shaded compared to open bars) in WT (black bars) and ADAM17^–/– cells (blue bars) on cleavage of cell surface biotinylated α₂δ-1, normalized to that in control CRISPR WT cells. Data are mean ± SEM and individual data for three separate experiments, including that in (C). Statistical differences determined using one-way ANOVA and Sidak's multiple comparison test; ^****P < .0001; **P = .0017; ## P = .0037.

Figure 6.

N-TIMP-3 inhibits cleavage of α₂δ-1 on cell surface of tsA-201 cells. (A) Effect of extracellular application of N-TIMP-3 (lanes 2 and 5) compared to control (lanes 1 and 4), on cleavage in WCL (lanes 1 and 2) and cell surface biotinylated fractions (lanes 4 and 5) of HA-α₂δ-1 expressed in tsA-201 cells (upper panel: α₂δ-1 immunoblot), deglycosylated to allow resolution between pro-α₂δ-1 (upper band) and the cleaved form, α₂–1 (lower band). Lower panel, loading and biotinylation control: Endogenous GAPDH. (B) and (C) Quantification of the effect of N-TIMP-3 application on relative cleavage of α₂δ-1 in WCL (B) and at plasma membrane (C) (mean ± SEM and individual data shown for three separate experiments, each normalized to that under control conditions). *P = .0231 (Student's t-test).