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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 N-terminally mutated CaV2 channel expression.(A-C) Effect of the truncated constructs on CaV2.1R57A, R59A currents. (A) tsA-201 cells were co-transfected with full-length CaV2.1R57A, R59A/α2δ-1/β1b and CaV3.1 Dom I–II (control, black), EA2 (red) or EA2R57A, R59 (blue). Representative traces 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 CaV2.1R57A, R59A and CaV3.1 Dom I–II (black squares, control), CaV2.1R57A, R59A and EA2 (red circles) or CaV2.1R57A, R59A and EA2R57A, R59A (blue circles). (C) Mean current densities (pA/pF) at + 5 mV ± SEM for CaV2.1R57A, R59A with CaV3.1 Dom I–II (black, control, n = 10), CaV2.1R57A, R59A with EA2 (red, n = 18) or CaV2.1R57A, R59A with EA2R57A, R59A (blue, n = 13). Statistical analysis: *p < 0.05, ***p < 0.001 vs control. (D–E) Effect of the truncated constructs on CaV2.2R52A, R54A HA cell surface expression. Neuro2A cells expressed CaV2.2R52A, R54A HA/α2δ-1/β1b with CaV3.1 Dom I–II (control), CaV2.2 Dom I–II or CaV2.2 Dom I–IIR52A, R54A. (D) Three examples of confocal images CaV2.2R52A, R54A HA with CaV3.1 Dom I–II (top), CaV2.2R52A, R54A HA with CaV2.2 Dom I–II (middle) or CaV2.2R52A, R54A HA with CaV2.2 Dom I–IIR52A, R54A (bottom). (E) Cell surface expression was quantified based on the HA signal. The data represent mean ± SEM for CaV2.2R52A, R54A HA with CaV3.1 Dom I–II (black, control), CaV2.2R52A, R54A HA with CaV2.2 Dom I–II (red) or CaV2.2R52A, R54A 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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f0015: Effect of truncated domains on N-terminally mutated CaV2 channel expression.(A-C) Effect of the truncated constructs on CaV2.1R57A, R59A currents. (A) tsA-201 cells were co-transfected with full-length CaV2.1R57A, R59A/α2δ-1/β1b and CaV3.1 Dom I–II (control, black), EA2 (red) or EA2R57A, R59 (blue). Representative traces 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 CaV2.1R57A, R59A and CaV3.1 Dom I–II (black squares, control), CaV2.1R57A, R59A and EA2 (red circles) or CaV2.1R57A, R59A and EA2R57A, R59A (blue circles). (C) Mean current densities (pA/pF) at + 5 mV ± SEM for CaV2.1R57A, R59A with CaV3.1 Dom I–II (black, control, n = 10), CaV2.1R57A, R59A with EA2 (red, n = 18) or CaV2.1R57A, R59A with EA2R57A, R59A (blue, n = 13). Statistical analysis: *p < 0.05, ***p < 0.001 vs control. (D–E) Effect of the truncated constructs on CaV2.2R52A, R54A HA cell surface expression. Neuro2A cells expressed CaV2.2R52A, R54A HA/α2δ-1/β1b with CaV3.1 Dom I–II (control), CaV2.2 Dom I–II or CaV2.2 Dom I–IIR52A, R54A. (D) Three examples of confocal images CaV2.2R52A, R54A HA with CaV3.1 Dom I–II (top), CaV2.2R52A, R54A HA with CaV2.2 Dom I–II (middle) or CaV2.2R52A, R54A HA with CaV2.2 Dom I–IIR52A, R54A (bottom). (E) Cell surface expression was quantified based on the HA signal. The data represent mean ± SEM for CaV2.2R52A, R54A HA with CaV3.1 Dom I–II (black, control), CaV2.2R52A, R54A HA with CaV2.2 Dom I–II (red) or CaV2.2R52A, R54A 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 used full-length channel constructs carrying the arginine-to-alanine substitutions (CaV2.1R57A, R59A and CaV2.2R52A, R54A), and assessed the dominant-negative effect of the truncated constructs. As shown in Fig. 3A–C, co-expression of the EA2 mutant significantly reduced CaV2.1R57A, R59A current by 60% (control: − 93.5 ± 15.0 pA/pF; EA2: − 37.5 ± 6.3 pA/pF), indicating that the full-length channel still interacts with the EA2 mutant despite the fact that its own arginine motif was disrupted. However there was still a dominant-negative effect when CaV2.1R57A, R59A was co-expressed with EA2R57A, R59A (− 61.1 ± 6.4 pA/pF), albeit reduced compared to the EA2 condition (Fig. 3B). It is of interest that CaV2.1R57A, R59A exhibited larger currents than wild-type CaV2.1 (Fig. 3C, compared to Fig. 2C), suggesting that there may be a tonic inhibitory effect that is relieved by mutating the N-terminus.


A Ca V 2.1 N-terminal fragment relieves the dominant-negative inhibition by an Episodic ataxia 2 mutant
Effect of truncated domains on N-terminally mutated CaV2 channel expression.(A-C) Effect of the truncated constructs on CaV2.1R57A, R59A currents. (A) tsA-201 cells were co-transfected with full-length CaV2.1R57A, R59A/α2δ-1/β1b and CaV3.1 Dom I–II (control, black), EA2 (red) or EA2R57A, R59 (blue). Representative traces 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 CaV2.1R57A, R59A and CaV3.1 Dom I–II (black squares, control), CaV2.1R57A, R59A and EA2 (red circles) or CaV2.1R57A, R59A and EA2R57A, R59A (blue circles). (C) Mean current densities (pA/pF) at + 5 mV ± SEM for CaV2.1R57A, R59A with CaV3.1 Dom I–II (black, control, n = 10), CaV2.1R57A, R59A with EA2 (red, n = 18) or CaV2.1R57A, R59A with EA2R57A, R59A (blue, n = 13). Statistical analysis: *p < 0.05, ***p < 0.001 vs control. (D–E) Effect of the truncated constructs on CaV2.2R52A, R54A HA cell surface expression. Neuro2A cells expressed CaV2.2R52A, R54A HA/α2δ-1/β1b with CaV3.1 Dom I–II (control), CaV2.2 Dom I–II or CaV2.2 Dom I–IIR52A, R54A. (D) Three examples of confocal images CaV2.2R52A, R54A HA with CaV3.1 Dom I–II (top), CaV2.2R52A, R54A HA with CaV2.2 Dom I–II (middle) or CaV2.2R52A, R54A HA with CaV2.2 Dom I–IIR52A, R54A (bottom). (E) Cell surface expression was quantified based on the HA signal. The data represent mean ± SEM for CaV2.2R52A, R54A HA with CaV3.1 Dom I–II (black, control), CaV2.2R52A, R54A HA with CaV2.2 Dom I–II (red) or CaV2.2R52A, R54A 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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f0015: Effect of truncated domains on N-terminally mutated CaV2 channel expression.(A-C) Effect of the truncated constructs on CaV2.1R57A, R59A currents. (A) tsA-201 cells were co-transfected with full-length CaV2.1R57A, R59A/α2δ-1/β1b and CaV3.1 Dom I–II (control, black), EA2 (red) or EA2R57A, R59 (blue). Representative traces 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 CaV2.1R57A, R59A and CaV3.1 Dom I–II (black squares, control), CaV2.1R57A, R59A and EA2 (red circles) or CaV2.1R57A, R59A and EA2R57A, R59A (blue circles). (C) Mean current densities (pA/pF) at + 5 mV ± SEM for CaV2.1R57A, R59A with CaV3.1 Dom I–II (black, control, n = 10), CaV2.1R57A, R59A with EA2 (red, n = 18) or CaV2.1R57A, R59A with EA2R57A, R59A (blue, n = 13). Statistical analysis: *p < 0.05, ***p < 0.001 vs control. (D–E) Effect of the truncated constructs on CaV2.2R52A, R54A HA cell surface expression. Neuro2A cells expressed CaV2.2R52A, R54A HA/α2δ-1/β1b with CaV3.1 Dom I–II (control), CaV2.2 Dom I–II or CaV2.2 Dom I–IIR52A, R54A. (D) Three examples of confocal images CaV2.2R52A, R54A HA with CaV3.1 Dom I–II (top), CaV2.2R52A, R54A HA with CaV2.2 Dom I–II (middle) or CaV2.2R52A, R54A HA with CaV2.2 Dom I–IIR52A, R54A (bottom). (E) Cell surface expression was quantified based on the HA signal. The data represent mean ± SEM for CaV2.2R52A, R54A HA with CaV3.1 Dom I–II (black, control), CaV2.2R52A, R54A HA with CaV2.2 Dom I–II (red) or CaV2.2R52A, R54A 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 used full-length channel constructs carrying the arginine-to-alanine substitutions (CaV2.1R57A, R59A and CaV2.2R52A, R54A), and assessed the dominant-negative effect of the truncated constructs. As shown in Fig. 3A–C, co-expression of the EA2 mutant significantly reduced CaV2.1R57A, R59A current by 60% (control: − 93.5 ± 15.0 pA/pF; EA2: − 37.5 ± 6.3 pA/pF), indicating that the full-length channel still interacts with the EA2 mutant despite the fact that its own arginine motif was disrupted. However there was still a dominant-negative effect when CaV2.1R57A, R59A was co-expressed with EA2R57A, R59A (− 61.1 ± 6.4 pA/pF), albeit reduced compared to the EA2 condition (Fig. 3B). It is of interest that CaV2.1R57A, R59A exhibited larger currents than wild-type CaV2.1 (Fig. 3C, compared to Fig. 2C), suggesting that there may be a tonic inhibitory effect that is relieved by mutating the N-terminus.

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