N terminus is key to the dominant negative suppression of Ca(V)2 calcium channels: implications for episodic ataxia type 2

Abstract

Expression of the calcium channels Ca(V)2.1 and Ca(V)2.2 is markedly suppressed by co-expression with truncated constructs containing Domain I. This is the basis for the phenomenon of dominant negative suppression observed for many of the episodic ataxia type 2 mutations in Ca(V)2.1 that predict truncated channels. The process of dominant negative suppression has been shown previously to stem from interaction between the full-length and truncated channels and to result in downstream consequences of the unfolded protein response and endoplasmic reticulum-associated protein degradation. We have now identified the specific domain that triggers this effect. For both Ca(V)2.1 and Ca(V)2.2, the minimum construct producing suppression was the cytoplasmic N terminus. Suppression was enhanced by tethering the N terminus to the membrane with a CAAX motif. The 11-amino acid motif (including Arg(52) and Arg(54)) within the N terminus, which we have previously shown to be required for G protein modulation, is also essential for dominant negative suppression. Suppression is prevented by addition of an N-terminal tag (XFP) to the full-length and truncated constructs. We further show that suppression of Ca(V)2.2 currents by the N terminus-CAAX construct is accompanied by a reduction in Ca(V)2.2 protein level, and this is also prevented by mutation of Arg(52) and Arg(54) to Ala in the truncated construct. Taken together, our evidence indicates that both the extreme N terminus and the Arg(52), Arg(54) motif are involved in the processes underlying dominant negative suppression.

FIGURE 1.

Effect of Ca_V2.2-truncated domains on Ca_V2.2 I_Ba when expressed in Xenopus oocytes. A, diagram of the main Ca_V2.2 constructs used in this study, as described under “Experimental Procedures.” B, peak I_Ba for Ca_V2.2/α₂δ-2/β1b expressed in Xenopus oocytes without any truncated domains (black bar, 100%) or with Ca_V2.2-Dom I (white bar, n = 12; ***, p < 0.001), Ca_V2.2-Dom I-4TMs (hatched bar, n = 17; ***, p < 0.001), Ca_V2.2-Dom I-4TMs no charges (cross-hatched bar, n = 14; **, p < 0.01), Ca_V2.2-Dom I-4TMs no charges C110S (light gray bar, n = 23; **, p < 0.01). Data are pooled from several experiments all recorded in 5 mm Ba²⁺ and normalized to the respective control in each experiment, and the statistical differences were determined compared with their respective control data, using one-way ANOVA and Bonferroni's post hoc test. Error bars indicate S.E. The symbols above the bars refer to the I-V relationship for the representative data in C. C, mean I-V relationship from two pooled experiments for Ca_V2.2/α₂δ-2/β1b expressed in Xenopus oocytes without any truncated domains (■, n = 7) or with Ca_V2.2-Dom I (○, n = 8), Ca_V2.2-Dom I 4TMs (▵, n = 4), Ca_V2.2-Dom I-4TMs no charges (□, n = 11), Ca_V2.2-Dom I-4TMs no charges C110S (●, n = 7). The symbols are identified above the bars in B. All recordings are in 5 mm Ba²⁺.

FIGURE 2.

Effect of N-terminally truncated Ca_V2.2 domains on Ca_V2.2 I_Ba when expressed in tsA-201 cells. A, peak I_Ba was determined from I-V relationships in 1 mm Ba²⁺ following expression in tsA-201 cells. The currents in the presence of the stated truncated domain are expressed as a percentage of control currents in its absence for Ca_V2.2/α₂δ-2/β1b (filled bars) or Δ1–55 Ca_V2.2/α₂δ-2/β1b (open bars). Data were pooled from several experiments, each examining the effect of one truncated construct, and normalized to the respective control in each experiment. The statistical significances of the differences compared with control were determined by Student's t test; *, p < 0.05. The numbers of determinations are given above each bar. Error bars indicate S.E. B, representative current traces (from −30 to +15 mV at Δ5 mV, from a holding potential of −90 mV), for Ca_V2.2/α₂δ-2/β1b (upper panel) and Δ1–55 Ca_V2.2/α₂δ-2/β1b (lower panel) in the absence or presence of Ca_V2.2-Dom I, Δ1–55 Ca_V2.2-Dom I, or Δ2–91 Ca_V2.2-Dom I. The scale bars refer to all traces.

FIGURE 3.

Effect of Δ2–91 Ca_V2.2 and YFP-tagged Ca_V2.2 on Ca_V2.2 I_Ba. A, mean I-V relationship for Ca_V2.2 (■, n = 12) or Δ2–91 Ca_V2.2 (▵, n = 3) co-expressed with α₂δ-2/β1b in Xenopus oocytes, either alone or together (○, n = 13). All recordings are in 10 mm Ba²⁺. The I-V curves are fit with a modified Boltzmann relationship, as described under “Experimental Procedures.” Inset, bar chart of peak I_Ba determined from these I-V relationships. The currents in the presence of Δ2–91 Ca_V2.2 (open bar, n = 13) are expressed as a percentage of control I_Ba in its absence (black bar), for Ca_V2.2/α₂δ-2/β1b. B, lack of effect of co-expression of Ca_V2.2 with Δ2–91 Ca_V2.2 (○, n = 6) on the voltage-dependence of steady-state inactivation of Ca_V2.2/α₂δ-2/β1b I_Ba (■, n = 8) from the same experiments as in A. Data are fit with a Boltzmann function, as described under “Experimental Procedures.” C, peak I_Ba was determined from I-V relationships in 1 mm Ba²⁺ following expression of constructs in tsA-201 cells. The currents in the presence of Δ2–91 Ca_V2.2 are expressed as a percentage of control I_Ba in its absence (black bar), for Ca_V2.2/α₂δ-2/β1b (open bar, n = 20), or Δ1–55 Ca_V2.2/α₂δ-2/β1b (gray bar, n = 16). Data were pooled from several experiments and normalized to the respective control in each experiment. The statistical significances of the differences compared with control were determined by Student's t test, p < 0.05. Error bars indicate S.E. D, lack of effect of YFP-Ca_V2.2-Dom I on YFP-Ca_V2.2 I_Ba in Xenopus oocytes. Peak currents at +5 mV are shown for YFP-Ca_V2.2/α₂δ-1/β1b alone (black bar, n = 24) or plus YFP-Ca_V2.2-Dom I (open bar, n = 20). Data were obtained in three different experiments, all with similar results. No significant differences were observed between the conditions, p > 0.05, Student's t test.

FIGURE 4.

Examination of the effect of the N terminus of Ca_V2.2 on functional expression of Ca_V2.2. A, example of current traces for voltage steps from −40 mV to +40 mV from a holding potential of −100 mV for GFP-Ca_V2.2/α₂δ-1/β1b alone (left), and together with Ca_V2.2 N terminus (right). Recordings were made with 10 mm Ba²⁺ in Xenopus oocytes. B, peak I_Ba for Ca_V2.2/α₂δ-1/β1b alone (black bar, n = 22) or together with Ca_V2.2 N terminus 1–95 (open bar, n = 36), GFP-CAAX (gray bar, n = 16), Ca_V2.2 N terminus 1–95-CAAX (hatched bar, n = 37), Ca_V2.2 N terminus Δ2–42-CAAX (horizontal striped bar, n = 25), and R52A/R54A Ca_V2.2 N terminus-CAAX (cross-hatched bar, n = 19). The statistical significances of the differences indicated were determined by one-way ANOVA and Bonferroni's post hoc test. **, p = 0.0016; ***, p < 0.001. Error bars indicate S.E. C, example of current traces for voltage steps from −40 mV to +40 mV for Ca_V2.2/α₂δ-1/β1b with GFP-CAAX (left), with Ca_V2.2 N terminus (center), and with Ca_V2.2 N terminus-CAAX (right). Recordings were made with 10 mm Ba²⁺. D, representative images showing the distribution of GFP-CAAX (upper panel) and free GFP (lower panel) expression in tsA-201 cells. Scale bars, 20 μm. E, mean I-V relationship for Ca_V2.2/α₂δ-1/β1b expressed in Xenopus oocytes, co-expressed with GFP-CAAX (■, n = 18), Ca_V2.2 N terminus-CAAX (○, n = 18), or R52A/R54A Ca_V2.2 N terminus-CAAX (▵, n = 19). All recordings were performed in parallel using 10 mm Ba²⁺. The I-V curves are fit with a modified Boltzmann relationship, as described under “Experimental Procedures.” The V_{50, act} was −8.6 mV for GFP-CAAX, −7.1 mV for Ca_V2.2 N terminus-CAAX, and −8.4 mV for R52A/R54A Ca_V2.2 N terminus-CAAX. F, peak I_Ba (at 0 mV) for Δ1–55 Ca_V2.2/α₂δ-1/β1b alone (black bar, n = 34) or together with Ca_V2.2 N terminus (open bar, n = 36), GFP-CAAX (gray bar, n = 10), and Ca_V2.2 N terminus-CAAX (hatched bar, n = 9). The statistical significances of the differences indicated were determined by Student's two-tailed t test. **, p = 0.002; ***, p < 0.0001. Recordings were made with 10 mm Ba²⁺. G, voltage dependence of time constant of activation (τ_act): left, for Ca_V2.2/α₂δ-1/β1b without (■, n = 12) or with (○, n = 7) the free Ca_V2.2 N terminus; and right, for Ca_V2.2/α₂δ-1/β1b with GFP-CAAX (■, n = 10) or with the free Ca_V2.2 N terminus-CAAX (○, n = 13).

FIGURE 5.

Examination of the role of the N terminus of Ca_V2.1 on functional expression of Ca_V2.1. A, mean I-V relationship for Ca_V2.1/α₂δ-2/β4 in Xenopus oocytes, either alone (■, n = 15) or co-expressed with Ca_V2.1 N terminus (○, n = 15). All recordings were performed in parallel, using 10 mm Ba²⁺. The I-V curves were fit with a modified Boltzmann relationship, up to +35 mV. The V_50,act was −11.9 mV for control and −9.3 mV in the presence of the Ca_V2.1 N terminus. B, left panel, peak I_Ba (at 0 mV) for Ca_V2.1/α₂δ-2/β4 alone (black bar, n = 28) or together with Ca_V2.1 N terminus (open bar, n = 26), from two independent experiments, including that depicted in A. Right panel, peak I_Ba for Ca_V2.1/α₂δ-2/β4 alone (hatched bar, n = 9) or together with Ca_V2.1 N terminus-CAAX (cross-hatched bar, n = 20) or Ca_V2.1 N terminus R57A/R59A-CAAX (gray bar, n = 20). The statistical significances of the differences indicated were determined by Student's two-tailed t test. **, p = 0.0046; ***, p < 0.001. Error bars indicate S.E. C, peak I_Ba (at 0 mV) for Ca_V2.1/α₂δ-2/β4 alone (black bar, n = 23) or together with Ca_V2.2 N terminus-CAAX (open bar, n = 22) or R52A/R54A Ca_V2.2 N terminus-CAAX (gray bar, n = 22). The statistical significances of the differences indicated were determined by ANOVA and Bonferroni's post hoc test. *, p < 0.01; **, p < 0.001.

FIGURE 6.

Examination of the effect of the N terminus of Ca_V2.2 on Ca_V2.2 protein expression. A, expression of Ca_V2.2 (upper panel) and α₂δ-1 (lower panel) protein in untransfected tsA-201 cells (first lane), when Ca_V2.2/α₂δ-1/β1b were expressed, alone (second lane) and together with Ca_V2.2 N terminus-CAAX (third lane), or R52A/R54A Ca_V2.2 N terminus-CAAX (fourth lane). The same amount of total protein was loaded for all samples on a gel, for accurate comparison among lanes. B, bar chart from quantification of results, including those in A, showing the effect of Ca_V2.2 N terminus-CAAX (open bars, n = 6) or R52A/R54A Ca_V2.2 N terminus-CAAX (gray bars, n = 4) relative to control levels (black bars), for Ca_V2.2 (left) and α₂δ-1 (right) protein levels. The statistical significance of the differences indicated were determined by Student's t test. *, p = 0.0162; **, p = 0.0041. Error bars indicate S.E.

FIGURE 7.

Effect of truncated constructs containing Ca_V2.2 on expression of endogenous calcium channel currents in DRG neurons. A, I-V relationship recorded in the presence of 10 μm nifedipine for DRG neurons expressing YFP (control, ■, n = 14), Ca_V2.2 Dom I (○, n = 12), and Ca_V2.2 N terminus-CAAX) (▴, n = 8). All recordings were performed 4 days after transfection. The mean ± S.E. cell capacitances were 26.4 ± 3.8, 28.4 ± 6.0, and 27.3 ± 5.5 picofarads, respectively, for the three different conditions. B, peak I_Ba (recorded in the presence of 10 μm nifedipine, at +10 mV) for DRG neurons expressing Ca_V2.2 Dom I (open bar, n = 12) and Ca_V2.2 N terminus-CAAX) (hatched bar, n = 8), normalized as a percentage of control (black bar, n = 14). The statistical significances of the differences were determined by one-way ANOVA followed by post-hoc Dunnett's test. *, p < 0.05. Error bars indicate S.E. C, example of current traces for voltage steps between −40 mV and +65 mV for neurons expressing YFP only (control) or with Ca_V2.2 Dom I or Ca_V2.2 N terminus-CAAX (left to right). Recordings were made with 10 mm Ba²⁺ in the presence of 10 μm nifedipine. D, I_Ba (recorded in the presence of 10 μm nifedipine at +10 mV) for DRG neurons expressing R52A/R54A Ca_V2.2 N terminus-CAAX (white bar, n = 10), normalized as a percentage of control (black bar, n = 9).