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

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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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Co-immunoprecipitation of full-length and truncated CaV2.2 is disrupted by the N-terminal construct.A: Representative Western blot showing proteins immunoprecipitated with rat anti-HA antibody and immunoblotted with anti-CaV2.2. tsA-201 cells expressed CaV2.2 HA, α2δ-1 and β1b, together with CaV3.1 Dom I–II (lane 1), CaV2.2 Dom I–II (lane 2), CaV2.2 Dom I–II plus GFP-CAAX (lane 3) or CaV2.2 Dom I–II plus CaV2.2-(43-95)-CAAX (lane 4). This result is representative of 3 independent experiments. B: Western-blot showing 30 μg WCL of the same samples shown in A. C: Quantification of Western blots. Band intensities for CaV2.2 Dom I–II were measured using ImageJ and normalised for expression of full-length CaV2.2 HA for each condition and relative to the condition of CaV2.2 HA + CaV2.2 Dom I–II (lane 2 in A and B). Data are mean band intensities (+ SEM) from three co-immunoprecipitation experiments from three different transfections. Statistical analysis: ns = non-significant difference, **p < 0.01.
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f0020: Co-immunoprecipitation of full-length and truncated CaV2.2 is disrupted by the N-terminal construct.A: Representative Western blot showing proteins immunoprecipitated with rat anti-HA antibody and immunoblotted with anti-CaV2.2. tsA-201 cells expressed CaV2.2 HA, α2δ-1 and β1b, together with CaV3.1 Dom I–II (lane 1), CaV2.2 Dom I–II (lane 2), CaV2.2 Dom I–II plus GFP-CAAX (lane 3) or CaV2.2 Dom I–II plus CaV2.2-(43-95)-CAAX (lane 4). This result is representative of 3 independent experiments. B: Western-blot showing 30 μg WCL of the same samples shown in A. C: Quantification of Western blots. Band intensities for CaV2.2 Dom I–II were measured using ImageJ and normalised for expression of full-length CaV2.2 HA for each condition and relative to the condition of CaV2.2 HA + CaV2.2 Dom I–II (lane 2 in A and B). Data are mean band intensities (+ SEM) from three co-immunoprecipitation experiments from three different transfections. Statistical analysis: ns = non-significant difference, **p < 0.01.

Mentions: Statistical analysis between groups was performed using one-way ANOVA with Bonferroni post-hoc test, using Graphpad Prism software. Statistical analysis between two groups (Fig. 4) was carried out using Student's t test.


A Ca V 2.1 N-terminal fragment relieves the dominant-negative inhibition by an Episodic ataxia 2 mutant
Co-immunoprecipitation of full-length and truncated CaV2.2 is disrupted by the N-terminal construct.A: Representative Western blot showing proteins immunoprecipitated with rat anti-HA antibody and immunoblotted with anti-CaV2.2. tsA-201 cells expressed CaV2.2 HA, α2δ-1 and β1b, together with CaV3.1 Dom I–II (lane 1), CaV2.2 Dom I–II (lane 2), CaV2.2 Dom I–II plus GFP-CAAX (lane 3) or CaV2.2 Dom I–II plus CaV2.2-(43-95)-CAAX (lane 4). This result is representative of 3 independent experiments. B: Western-blot showing 30 μg WCL of the same samples shown in A. C: Quantification of Western blots. Band intensities for CaV2.2 Dom I–II were measured using ImageJ and normalised for expression of full-length CaV2.2 HA for each condition and relative to the condition of CaV2.2 HA + CaV2.2 Dom I–II (lane 2 in A and B). Data are mean band intensities (+ SEM) from three co-immunoprecipitation experiments from three different transfections. Statistical analysis: ns = non-significant difference, **p < 0.01.
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Related In: Results  -  Collection

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f0020: Co-immunoprecipitation of full-length and truncated CaV2.2 is disrupted by the N-terminal construct.A: Representative Western blot showing proteins immunoprecipitated with rat anti-HA antibody and immunoblotted with anti-CaV2.2. tsA-201 cells expressed CaV2.2 HA, α2δ-1 and β1b, together with CaV3.1 Dom I–II (lane 1), CaV2.2 Dom I–II (lane 2), CaV2.2 Dom I–II plus GFP-CAAX (lane 3) or CaV2.2 Dom I–II plus CaV2.2-(43-95)-CAAX (lane 4). This result is representative of 3 independent experiments. B: Western-blot showing 30 μg WCL of the same samples shown in A. C: Quantification of Western blots. Band intensities for CaV2.2 Dom I–II were measured using ImageJ and normalised for expression of full-length CaV2.2 HA for each condition and relative to the condition of CaV2.2 HA + CaV2.2 Dom I–II (lane 2 in A and B). Data are mean band intensities (+ SEM) from three co-immunoprecipitation experiments from three different transfections. Statistical analysis: ns = non-significant difference, **p < 0.01.
Mentions: Statistical analysis between groups was performed using one-way ANOVA with Bonferroni post-hoc test, using Graphpad Prism software. Statistical analysis between two groups (Fig. 4) was carried out using Student's t test.

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