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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 truncated domains on CaV2 channel expression.(A–C) Effect of the truncated domains on CaV2.1 currents. tsA-201 cells expressed full-length CaV2.1 with CaV3.1 Dom I–II as a control (black, control), EA2 (red) or EA2R57A, R59A (blue), together with α2δ-1 and β1b subunits. (A) Representative traces for the three conditions were evoked by 50 ms step depolarizations between − 50 and + 60 mV from a holding potential of − 80 mV. The currents are normalised to the cell capacitance. (B) Mean current-voltage relationships for CaV2.1 with CaV3.1 Dom I–II (black squares, control), CaV2.1 with EA2 (red circles) or CaV2.1 with EA2R57A, R59A (blue circles). (C) Bar chart of mean current densities (pA/pF) at + 5 mV ± SEM for CaV2.1 with CaV3.1 Dom I–II (black, control, n = 20), CaV2.1 with EA2 (red, n = 24) and CaV2.1 with EA2R57A, R59A (blue, n = 21). Statistical analysis *p < 0.05 vs control. (D–E) Effect of the truncated constructs on CaV2.2 HA cell surface expression. Neuro2A cells expressed full-length CaV2.2 with CaV3.1 Dom I–II, CaV2.2 Dom I–II or CaV2.2 Dom I–IIR52A, R54A, together with α2δ-1 and β1b subunits. (D) Three examples of confocal images of Neuro2A cells expressing CaV2.2 HA with CaV3.1 Dom I–II (top), CaV2.2 HA with CaV2.2 Dom I–II (middle) or CaV2.2 HA and CaV2.2 Dom I–IIR52A, R54A (bottom). Surface staining was obtained by using HA antibody in non-permeabilized cells. (E) Cell surface expression of CaV2.2 was quantified based on the HA signal. Bar chart of mean of cell surface expression ± SEM for CaV2.2 HA with CaV3.1 Dom I–II (black, control), CaV2.2 HA with CaV2.2 Dom I–II (red) or CaV2.2 HA with CaV2.2 Dom I–IIR52A, R54A (blue). The data were pooled from 4 independent experiments; more than 100 cells were included in the analysis for each condition. Statistical analysis: ***p < 0.001 vs control.
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f0010: Effect of truncated domains on CaV2 channel expression.(A–C) Effect of the truncated domains on CaV2.1 currents. tsA-201 cells expressed full-length CaV2.1 with CaV3.1 Dom I–II as a control (black, control), EA2 (red) or EA2R57A, R59A (blue), together with α2δ-1 and β1b subunits. (A) Representative traces for the three conditions were evoked by 50 ms step depolarizations between − 50 and + 60 mV from a holding potential of − 80 mV. The currents are normalised to the cell capacitance. (B) Mean current-voltage relationships for CaV2.1 with CaV3.1 Dom I–II (black squares, control), CaV2.1 with EA2 (red circles) or CaV2.1 with EA2R57A, R59A (blue circles). (C) Bar chart of mean current densities (pA/pF) at + 5 mV ± SEM for CaV2.1 with CaV3.1 Dom I–II (black, control, n = 20), CaV2.1 with EA2 (red, n = 24) and CaV2.1 with EA2R57A, R59A (blue, n = 21). Statistical analysis *p < 0.05 vs control. (D–E) Effect of the truncated constructs on CaV2.2 HA cell surface expression. Neuro2A cells expressed full-length CaV2.2 with CaV3.1 Dom I–II, CaV2.2 Dom I–II or CaV2.2 Dom I–IIR52A, R54A, together with α2δ-1 and β1b subunits. (D) Three examples of confocal images of Neuro2A cells expressing CaV2.2 HA with CaV3.1 Dom I–II (top), CaV2.2 HA with CaV2.2 Dom I–II (middle) or CaV2.2 HA and CaV2.2 Dom I–IIR52A, R54A (bottom). Surface staining was obtained by using HA antibody in non-permeabilized cells. (E) Cell surface expression of CaV2.2 was quantified based on the HA signal. Bar chart of mean of cell surface expression ± SEM for CaV2.2 HA with CaV3.1 Dom I–II (black, control), CaV2.2 HA with CaV2.2 Dom I–II (red) or CaV2.2 HA with CaV2.2 Dom I–IIR52A, R54A (blue). The data were pooled from 4 independent experiments; more than 100 cells were included in the analysis for each condition. Statistical analysis: ***p < 0.001 vs control.

Mentions: We next examined the suppressive effect of the truncated constructs on CaV2.1 and CaV2.2 channel function and trafficking. We first investigated the effect of the substitution of R57A, R59A in an EA2 mutant (CaV2.1-P1217fs), containing only the first two domains and the intracellular II–III loop; this mutant will be referred as EA2 throughout the paper. In all our studies, the equivalent first two domains of CaV3.1 were used as a negative control as this was shown previously not to cause dominant-negative inhibition of CaV2.2 channels (Page et al., 2010). This was also found in the present study for CaV2.1 as shown by the unchanged current densities at + 5 mV in the presence of either CaV3.1 Dom I–II (− 66.6 ± 8.0 pA/pF) or GFP-CAAX (− 75.3 ± 13.0 pA/pF) (data from Fig. 2C and Fig. 5C).


A Ca V 2.1 N-terminal fragment relieves the dominant-negative inhibition by an Episodic ataxia 2 mutant
Effect of truncated domains on CaV2 channel expression.(A–C) Effect of the truncated domains on CaV2.1 currents. tsA-201 cells expressed full-length CaV2.1 with CaV3.1 Dom I–II as a control (black, control), EA2 (red) or EA2R57A, R59A (blue), together with α2δ-1 and β1b subunits. (A) Representative traces for the three conditions were evoked by 50 ms step depolarizations between − 50 and + 60 mV from a holding potential of − 80 mV. The currents are normalised to the cell capacitance. (B) Mean current-voltage relationships for CaV2.1 with CaV3.1 Dom I–II (black squares, control), CaV2.1 with EA2 (red circles) or CaV2.1 with EA2R57A, R59A (blue circles). (C) Bar chart of mean current densities (pA/pF) at + 5 mV ± SEM for CaV2.1 with CaV3.1 Dom I–II (black, control, n = 20), CaV2.1 with EA2 (red, n = 24) and CaV2.1 with EA2R57A, R59A (blue, n = 21). Statistical analysis *p < 0.05 vs control. (D–E) Effect of the truncated constructs on CaV2.2 HA cell surface expression. Neuro2A cells expressed full-length CaV2.2 with CaV3.1 Dom I–II, CaV2.2 Dom I–II or CaV2.2 Dom I–IIR52A, R54A, together with α2δ-1 and β1b subunits. (D) Three examples of confocal images of Neuro2A cells expressing CaV2.2 HA with CaV3.1 Dom I–II (top), CaV2.2 HA with CaV2.2 Dom I–II (middle) or CaV2.2 HA and CaV2.2 Dom I–IIR52A, R54A (bottom). Surface staining was obtained by using HA antibody in non-permeabilized cells. (E) Cell surface expression of CaV2.2 was quantified based on the HA signal. Bar chart of mean of cell surface expression ± SEM for CaV2.2 HA with CaV3.1 Dom I–II (black, control), CaV2.2 HA with CaV2.2 Dom I–II (red) or CaV2.2 HA with CaV2.2 Dom I–IIR52A, R54A (blue). The data were pooled from 4 independent experiments; more than 100 cells were included in the analysis for each condition. Statistical analysis: ***p < 0.001 vs control.
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f0010: Effect of truncated domains on CaV2 channel expression.(A–C) Effect of the truncated domains on CaV2.1 currents. tsA-201 cells expressed full-length CaV2.1 with CaV3.1 Dom I–II as a control (black, control), EA2 (red) or EA2R57A, R59A (blue), together with α2δ-1 and β1b subunits. (A) Representative traces for the three conditions were evoked by 50 ms step depolarizations between − 50 and + 60 mV from a holding potential of − 80 mV. The currents are normalised to the cell capacitance. (B) Mean current-voltage relationships for CaV2.1 with CaV3.1 Dom I–II (black squares, control), CaV2.1 with EA2 (red circles) or CaV2.1 with EA2R57A, R59A (blue circles). (C) Bar chart of mean current densities (pA/pF) at + 5 mV ± SEM for CaV2.1 with CaV3.1 Dom I–II (black, control, n = 20), CaV2.1 with EA2 (red, n = 24) and CaV2.1 with EA2R57A, R59A (blue, n = 21). Statistical analysis *p < 0.05 vs control. (D–E) Effect of the truncated constructs on CaV2.2 HA cell surface expression. Neuro2A cells expressed full-length CaV2.2 with CaV3.1 Dom I–II, CaV2.2 Dom I–II or CaV2.2 Dom I–IIR52A, R54A, together with α2δ-1 and β1b subunits. (D) Three examples of confocal images of Neuro2A cells expressing CaV2.2 HA with CaV3.1 Dom I–II (top), CaV2.2 HA with CaV2.2 Dom I–II (middle) or CaV2.2 HA and CaV2.2 Dom I–IIR52A, R54A (bottom). Surface staining was obtained by using HA antibody in non-permeabilized cells. (E) Cell surface expression of CaV2.2 was quantified based on the HA signal. Bar chart of mean of cell surface expression ± SEM for CaV2.2 HA with CaV3.1 Dom I–II (black, control), CaV2.2 HA with CaV2.2 Dom I–II (red) or CaV2.2 HA with CaV2.2 Dom I–IIR52A, R54A (blue). The data were pooled from 4 independent experiments; more than 100 cells were included in the analysis for each condition. Statistical analysis: ***p < 0.001 vs control.
Mentions: We next examined the suppressive effect of the truncated constructs on CaV2.1 and CaV2.2 channel function and trafficking. We first investigated the effect of the substitution of R57A, R59A in an EA2 mutant (CaV2.1-P1217fs), containing only the first two domains and the intracellular II–III loop; this mutant will be referred as EA2 throughout the paper. In all our studies, the equivalent first two domains of CaV3.1 were used as a negative control as this was shown previously not to cause dominant-negative inhibition of CaV2.2 channels (Page et al., 2010). This was also found in the present study for CaV2.1 as shown by the unchanged current densities at + 5 mV in the presence of either CaV3.1 Dom I–II (− 66.6 ± 8.0 pA/pF) or GFP-CAAX (− 75.3 ± 13.0 pA/pF) (data from Fig. 2C and Fig. 5C).

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