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

Page KM, Heblich F, Margas W, Pratt WS, Nieto-Rostro M, Chaggar K, Sandhu K, Davies A, Dolphin AC - J. Biol. Chem. (2009)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, Physiology, and Pharmacology, University College London, Gower Street, London WC1E 6BT, United Kingdom.

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.

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Examination of the effect of the N terminus of CaV2.2 on functional expression of CaV2.2. A, example of current traces for voltage steps from −40 mV to +40 mV from a holding potential of −100 mV for GFP-CaV2.2/α2δ-1/β1b alone (left), and together with CaV2.2 N terminus (right). Recordings were made with 10 mm Ba2+ in Xenopus oocytes. B, peak IBa for CaV2.2/α2δ-1/β1b alone (black bar, n = 22) or together with CaV2.2 N terminus 1–95 (open bar, n = 36), GFP-CAAX (gray bar, n = 16), CaV2.2 N terminus 1–95-CAAX (hatched bar, n = 37), CaV2.2 N terminus Δ2–42-CAAX (horizontal striped bar, n = 25), and R52A/R54A CaV2.2 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 CaV2.2/α2δ-1/β1b with GFP-CAAX (left), with CaV2.2 N terminus (center), and with CaV2.2 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 CaV2.2/α2δ-1/β1b expressed in Xenopus oocytes, co-expressed with GFP-CAAX (■, n = 18), CaV2.2 N terminus-CAAX (○, n = 18), or R52A/R54A CaV2.2 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 V50, act was −8.6 mV for GFP-CAAX, −7.1 mV for CaV2.2 N terminus-CAAX, and −8.4 mV for R52A/R54A CaV2.2 N terminus-CAAX. F, peak IBa (at 0 mV) for Δ1–55 CaV2.2/α2δ-1/β1b alone (black bar, n = 34) or together with CaV2.2 N terminus (open bar, n = 36), GFP-CAAX (gray bar, n = 10), and CaV2.2 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 (τact): left, for CaV2.2/α2δ-1/β1b without (■, n = 12) or with (○, n = 7) the free CaV2.2 N terminus; and right, for CaV2.2/α2δ-1/β1b with GFP-CAAX (■, n = 10) or with the free CaV2.2 N terminus-CAAX (○, n = 13).
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Figure 4: Examination of the effect of the N terminus of CaV2.2 on functional expression of CaV2.2. A, example of current traces for voltage steps from −40 mV to +40 mV from a holding potential of −100 mV for GFP-CaV2.2/α2δ-1/β1b alone (left), and together with CaV2.2 N terminus (right). Recordings were made with 10 mm Ba2+ in Xenopus oocytes. B, peak IBa for CaV2.2/α2δ-1/β1b alone (black bar, n = 22) or together with CaV2.2 N terminus 1–95 (open bar, n = 36), GFP-CAAX (gray bar, n = 16), CaV2.2 N terminus 1–95-CAAX (hatched bar, n = 37), CaV2.2 N terminus Δ2–42-CAAX (horizontal striped bar, n = 25), and R52A/R54A CaV2.2 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 CaV2.2/α2δ-1/β1b with GFP-CAAX (left), with CaV2.2 N terminus (center), and with CaV2.2 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 CaV2.2/α2δ-1/β1b expressed in Xenopus oocytes, co-expressed with GFP-CAAX (■, n = 18), CaV2.2 N terminus-CAAX (○, n = 18), or R52A/R54A CaV2.2 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 V50, act was −8.6 mV for GFP-CAAX, −7.1 mV for CaV2.2 N terminus-CAAX, and −8.4 mV for R52A/R54A CaV2.2 N terminus-CAAX. F, peak IBa (at 0 mV) for Δ1–55 CaV2.2/α2δ-1/β1b alone (black bar, n = 34) or together with CaV2.2 N terminus (open bar, n = 36), GFP-CAAX (gray bar, n = 10), and CaV2.2 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 (τact): left, for CaV2.2/α2δ-1/β1b without (■, n = 12) or with (○, n = 7) the free CaV2.2 N terminus; and right, for CaV2.2/α2δ-1/β1b with GFP-CAAX (■, n = 10) or with the free CaV2.2 N terminus-CAAX (○, n = 13).

Mentions: We had previously shown that an N-terminal construct of CaV2.2 did not inhibit GFP-CaV2.2 currents (12). This result was confirmed in the present study, the peak IBa resulting from expression of GFP-CaV2.2 was nonsignificantly reduced in the presence of the N terminus of CaV2.2 (residues 1–95), by 8.9 ± 19.5% (n = 13; Fig. 4A).


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

Page KM, Heblich F, Margas W, Pratt WS, Nieto-Rostro M, Chaggar K, Sandhu K, Davies A, Dolphin AC - J. Biol. Chem. (2009)

Examination of the effect of the N terminus of CaV2.2 on functional expression of CaV2.2. A, example of current traces for voltage steps from −40 mV to +40 mV from a holding potential of −100 mV for GFP-CaV2.2/α2δ-1/β1b alone (left), and together with CaV2.2 N terminus (right). Recordings were made with 10 mm Ba2+ in Xenopus oocytes. B, peak IBa for CaV2.2/α2δ-1/β1b alone (black bar, n = 22) or together with CaV2.2 N terminus 1–95 (open bar, n = 36), GFP-CAAX (gray bar, n = 16), CaV2.2 N terminus 1–95-CAAX (hatched bar, n = 37), CaV2.2 N terminus Δ2–42-CAAX (horizontal striped bar, n = 25), and R52A/R54A CaV2.2 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 CaV2.2/α2δ-1/β1b with GFP-CAAX (left), with CaV2.2 N terminus (center), and with CaV2.2 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 CaV2.2/α2δ-1/β1b expressed in Xenopus oocytes, co-expressed with GFP-CAAX (■, n = 18), CaV2.2 N terminus-CAAX (○, n = 18), or R52A/R54A CaV2.2 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 V50, act was −8.6 mV for GFP-CAAX, −7.1 mV for CaV2.2 N terminus-CAAX, and −8.4 mV for R52A/R54A CaV2.2 N terminus-CAAX. F, peak IBa (at 0 mV) for Δ1–55 CaV2.2/α2δ-1/β1b alone (black bar, n = 34) or together with CaV2.2 N terminus (open bar, n = 36), GFP-CAAX (gray bar, n = 10), and CaV2.2 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 (τact): left, for CaV2.2/α2δ-1/β1b without (■, n = 12) or with (○, n = 7) the free CaV2.2 N terminus; and right, for CaV2.2/α2δ-1/β1b with GFP-CAAX (■, n = 10) or with the free CaV2.2 N terminus-CAAX (○, n = 13).
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Figure 4: Examination of the effect of the N terminus of CaV2.2 on functional expression of CaV2.2. A, example of current traces for voltage steps from −40 mV to +40 mV from a holding potential of −100 mV for GFP-CaV2.2/α2δ-1/β1b alone (left), and together with CaV2.2 N terminus (right). Recordings were made with 10 mm Ba2+ in Xenopus oocytes. B, peak IBa for CaV2.2/α2δ-1/β1b alone (black bar, n = 22) or together with CaV2.2 N terminus 1–95 (open bar, n = 36), GFP-CAAX (gray bar, n = 16), CaV2.2 N terminus 1–95-CAAX (hatched bar, n = 37), CaV2.2 N terminus Δ2–42-CAAX (horizontal striped bar, n = 25), and R52A/R54A CaV2.2 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 CaV2.2/α2δ-1/β1b with GFP-CAAX (left), with CaV2.2 N terminus (center), and with CaV2.2 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 CaV2.2/α2δ-1/β1b expressed in Xenopus oocytes, co-expressed with GFP-CAAX (■, n = 18), CaV2.2 N terminus-CAAX (○, n = 18), or R52A/R54A CaV2.2 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 V50, act was −8.6 mV for GFP-CAAX, −7.1 mV for CaV2.2 N terminus-CAAX, and −8.4 mV for R52A/R54A CaV2.2 N terminus-CAAX. F, peak IBa (at 0 mV) for Δ1–55 CaV2.2/α2δ-1/β1b alone (black bar, n = 34) or together with CaV2.2 N terminus (open bar, n = 36), GFP-CAAX (gray bar, n = 10), and CaV2.2 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 (τact): left, for CaV2.2/α2δ-1/β1b without (■, n = 12) or with (○, n = 7) the free CaV2.2 N terminus; and right, for CaV2.2/α2δ-1/β1b with GFP-CAAX (■, n = 10) or with the free CaV2.2 N terminus-CAAX (○, n = 13).
Mentions: We had previously shown that an N-terminal construct of CaV2.2 did not inhibit GFP-CaV2.2 currents (12). This result was confirmed in the present study, the peak IBa resulting from expression of GFP-CaV2.2 was nonsignificantly reduced in the presence of the N terminus of CaV2.2 (residues 1–95), by 8.9 ± 19.5% (n = 13; Fig. 4A).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, Physiology, and Pharmacology, University College London, Gower Street, London WC1E 6BT, United Kingdom.

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.

Show MeSH
Related in: MedlinePlus