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A Ca V 2.1 N-terminal fragment relieves the dominant-negative inhibition by an Episodic ataxia 2 mutant

View Article: PubMed Central - PubMed

ABSTRACT

Episodic ataxia 2 (EA2) is an autosomal dominant disorder caused by mutations in the gene CACNA1A that encodes the pore-forming CaV2.1 calcium channel subunit. The majority of EA2 mutations reported so far are nonsense or deletion/insertion mutations predicted to form truncated proteins. Heterologous expression of wild-type CaV2.1, together with truncated constructs that mimic EA2 mutants, significantly suppressed wild-type calcium channel function, indicating that the truncated protein produces a dominant-negative effect (Jouvenceau et al., 2001; Page et al., 2004). A similar finding has been shown for CaV2.2 (Raghib et al., 2001). We show here that a highly conserved sequence in the cytoplasmic N-terminus is involved in this process, for both CaV2.1 and CaV2.2 channels. Additionally, we were able to interfere with the suppressive effect of an EA2 construct by mutating key N-terminal residues within it. We postulate that the N-terminus of the truncated channel plays an essential part in its interaction with the full-length CaV2.1, which prevents the correct folding of the wild-type channel. In agreement with this, we were able to disrupt the interaction between EA2 and the full length channel by co-expressing a free N-terminal peptide.

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Effect of the CaV2.2 N-terminus and truncated domains on CaV2.2 HA trafficking.(A) Representative confocal images of Neuro2A cells transfected with CaV2.2 HA/α2δ-1/β1b in the presence of CaV3.1 Dom I–II (top panel), CaV2.2-Dom I–II (middle panel) or CaV2.2-(43-95)-CAAX (bottom panel). Surface staining was obtained using HA antibody in non-permeabilized cells. (B) CaV2.2 cell surface expression of was quantified based on the HA signal. Mean cell surface expression ± SEM for CaV2.2 HA and CaV3.1 Dom I–II (black, control), CaV2.2 HA and CaV2.2-Dom I–II (red) or CaV2.2 HA and CaV2.2-(43-95)-CAAX (blue). The data are pooled from 3 independent experiments; more than 50 cells have been included in the analysis for each condition. Statistical analysis: **p < 0.01, ns = non-significant difference. (C) Representative confocal images of Neuro2A cells, showing cell surface expression of CaV2.2 HA in the presence of CaV3.1 Dom I–II and GFP-CAAX (top panel), CaV2.2 HA, CaV2.2-Dom I–II and GFP-CAAX (middle panel) or CaV2.2 HA, CaV2.2-Dom I–II and CaV2.2-(43-95)-CAAX (bottom panel). Surface staining was obtained by using HA antibody before cell permeabilization. (D) Cell surface expression of CaV2.2 was quantified based on the HA signal using ImageJ software. Bar chart of mean ± SEM cell surface expression of CaV2.2 HA and CaV3.1 Dom I–II and GFP-CAAX (black), CaV2.2 HA, CaV2.2 Dom I–II and GFP-CAAX (red) or CaV2.2 HA, CaV2.2 Dom I–II and CaV2.2-(43-95)-CAAX (blue). The data are pooled from 3 independent experiments and more than 50 cells have been included in the analysis for each condition. Statistical analysis: *p < 0.05, ns = non-significant difference.
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f0030: Effect of the CaV2.2 N-terminus and truncated domains on CaV2.2 HA trafficking.(A) Representative confocal images of Neuro2A cells transfected with CaV2.2 HA/α2δ-1/β1b in the presence of CaV3.1 Dom I–II (top panel), CaV2.2-Dom I–II (middle panel) or CaV2.2-(43-95)-CAAX (bottom panel). Surface staining was obtained using HA antibody in non-permeabilized cells. (B) CaV2.2 cell surface expression of was quantified based on the HA signal. Mean cell surface expression ± SEM for CaV2.2 HA and CaV3.1 Dom I–II (black, control), CaV2.2 HA and CaV2.2-Dom I–II (red) or CaV2.2 HA and CaV2.2-(43-95)-CAAX (blue). The data are pooled from 3 independent experiments; more than 50 cells have been included in the analysis for each condition. Statistical analysis: **p < 0.01, ns = non-significant difference. (C) Representative confocal images of Neuro2A cells, showing cell surface expression of CaV2.2 HA in the presence of CaV3.1 Dom I–II and GFP-CAAX (top panel), CaV2.2 HA, CaV2.2-Dom I–II and GFP-CAAX (middle panel) or CaV2.2 HA, CaV2.2-Dom I–II and CaV2.2-(43-95)-CAAX (bottom panel). Surface staining was obtained by using HA antibody before cell permeabilization. (D) Cell surface expression of CaV2.2 was quantified based on the HA signal using ImageJ software. Bar chart of mean ± SEM cell surface expression of CaV2.2 HA and CaV3.1 Dom I–II and GFP-CAAX (black), CaV2.2 HA, CaV2.2 Dom I–II and GFP-CAAX (red) or CaV2.2 HA, CaV2.2 Dom I–II and CaV2.2-(43-95)-CAAX (blue). The data are pooled from 3 independent experiments and more than 50 cells have been included in the analysis for each condition. Statistical analysis: *p < 0.05, ns = non-significant difference.

Mentions: We next wanted to examine whether the suppression of CaV2.2 trafficking by CaV2.2 Dom I–II is also disrupted by co-expression of the equivalent CaV2.2 N-terminal peptide. In parallel with the result found for CaV2.1 currents, we found firstly that CaV2.2-(43-95)-CAAX, unlike CaV2.2 Dom I–II, did not alter the cell-surface expression of CaV2.2 when they were co-expressed, as shown by the unchanged mean intensity of the HA signal at the cell surface (Fig. 6A, B). Furthermore, co-expression of CaV2.2-(43-95)-CAAX restored CaV2.2 cell surface expression by decreasing the deleterious effect of CaV2.2 Dom I–II (Fig. 6C, D). These results nicely recapitulate the CaV2.1 results shown in Fig. 5.


A Ca V 2.1 N-terminal fragment relieves the dominant-negative inhibition by an Episodic ataxia 2 mutant
Effect of the CaV2.2 N-terminus and truncated domains on CaV2.2 HA trafficking.(A) Representative confocal images of Neuro2A cells transfected with CaV2.2 HA/α2δ-1/β1b in the presence of CaV3.1 Dom I–II (top panel), CaV2.2-Dom I–II (middle panel) or CaV2.2-(43-95)-CAAX (bottom panel). Surface staining was obtained using HA antibody in non-permeabilized cells. (B) CaV2.2 cell surface expression of was quantified based on the HA signal. Mean cell surface expression ± SEM for CaV2.2 HA and CaV3.1 Dom I–II (black, control), CaV2.2 HA and CaV2.2-Dom I–II (red) or CaV2.2 HA and CaV2.2-(43-95)-CAAX (blue). The data are pooled from 3 independent experiments; more than 50 cells have been included in the analysis for each condition. Statistical analysis: **p < 0.01, ns = non-significant difference. (C) Representative confocal images of Neuro2A cells, showing cell surface expression of CaV2.2 HA in the presence of CaV3.1 Dom I–II and GFP-CAAX (top panel), CaV2.2 HA, CaV2.2-Dom I–II and GFP-CAAX (middle panel) or CaV2.2 HA, CaV2.2-Dom I–II and CaV2.2-(43-95)-CAAX (bottom panel). Surface staining was obtained by using HA antibody before cell permeabilization. (D) Cell surface expression of CaV2.2 was quantified based on the HA signal using ImageJ software. Bar chart of mean ± SEM cell surface expression of CaV2.2 HA and CaV3.1 Dom I–II and GFP-CAAX (black), CaV2.2 HA, CaV2.2 Dom I–II and GFP-CAAX (red) or CaV2.2 HA, CaV2.2 Dom I–II and CaV2.2-(43-95)-CAAX (blue). The data are pooled from 3 independent experiments and more than 50 cells have been included in the analysis for each condition. Statistical analysis: *p < 0.05, ns = non-significant difference.
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f0030: Effect of the CaV2.2 N-terminus and truncated domains on CaV2.2 HA trafficking.(A) Representative confocal images of Neuro2A cells transfected with CaV2.2 HA/α2δ-1/β1b in the presence of CaV3.1 Dom I–II (top panel), CaV2.2-Dom I–II (middle panel) or CaV2.2-(43-95)-CAAX (bottom panel). Surface staining was obtained using HA antibody in non-permeabilized cells. (B) CaV2.2 cell surface expression of was quantified based on the HA signal. Mean cell surface expression ± SEM for CaV2.2 HA and CaV3.1 Dom I–II (black, control), CaV2.2 HA and CaV2.2-Dom I–II (red) or CaV2.2 HA and CaV2.2-(43-95)-CAAX (blue). The data are pooled from 3 independent experiments; more than 50 cells have been included in the analysis for each condition. Statistical analysis: **p < 0.01, ns = non-significant difference. (C) Representative confocal images of Neuro2A cells, showing cell surface expression of CaV2.2 HA in the presence of CaV3.1 Dom I–II and GFP-CAAX (top panel), CaV2.2 HA, CaV2.2-Dom I–II and GFP-CAAX (middle panel) or CaV2.2 HA, CaV2.2-Dom I–II and CaV2.2-(43-95)-CAAX (bottom panel). Surface staining was obtained by using HA antibody before cell permeabilization. (D) Cell surface expression of CaV2.2 was quantified based on the HA signal using ImageJ software. Bar chart of mean ± SEM cell surface expression of CaV2.2 HA and CaV3.1 Dom I–II and GFP-CAAX (black), CaV2.2 HA, CaV2.2 Dom I–II and GFP-CAAX (red) or CaV2.2 HA, CaV2.2 Dom I–II and CaV2.2-(43-95)-CAAX (blue). The data are pooled from 3 independent experiments and more than 50 cells have been included in the analysis for each condition. Statistical analysis: *p < 0.05, ns = non-significant difference.
Mentions: We next wanted to examine whether the suppression of CaV2.2 trafficking by CaV2.2 Dom I–II is also disrupted by co-expression of the equivalent CaV2.2 N-terminal peptide. In parallel with the result found for CaV2.1 currents, we found firstly that CaV2.2-(43-95)-CAAX, unlike CaV2.2 Dom I–II, did not alter the cell-surface expression of CaV2.2 when they were co-expressed, as shown by the unchanged mean intensity of the HA signal at the cell surface (Fig. 6A, B). Furthermore, co-expression of CaV2.2-(43-95)-CAAX restored CaV2.2 cell surface expression by decreasing the deleterious effect of CaV2.2 Dom I–II (Fig. 6C, D). These results nicely recapitulate the CaV2.1 results shown in Fig. 5.

View Article: PubMed Central - PubMed

ABSTRACT

Episodic ataxia 2 (EA2) is an autosomal dominant disorder caused by mutations in the gene CACNA1A that encodes the pore-forming CaV2.1 calcium channel subunit. The majority of EA2 mutations reported so far are nonsense or deletion/insertion mutations predicted to form truncated proteins. Heterologous expression of wild-type CaV2.1, together with truncated constructs that mimic EA2 mutants, significantly suppressed wild-type calcium channel function, indicating that the truncated protein produces a dominant-negative effect (Jouvenceau et al., 2001; Page et al., 2004). A similar finding has been shown for CaV2.2 (Raghib et al., 2001). We show here that a highly conserved sequence in the cytoplasmic N-terminus is involved in this process, for both CaV2.1 and CaV2.2 channels. Additionally, we were able to interfere with the suppressive effect of an EA2 construct by mutating key N-terminal residues within it. We postulate that the N-terminus of the truncated channel plays an essential part in its interaction with the full-length CaV2.1, which prevents the correct folding of the wild-type channel. In agreement with this, we were able to disrupt the interaction between EA2 and the full length channel by co-expressing a free N-terminal peptide.

No MeSH data available.


Related in: MedlinePlus