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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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Effect of the N-terminus on CaV2.1 current and suppression by EA2.(A–C) Effect of the N-terminal constructs on CaV2.1/β1b/α2δ-1 currents. (A) Representative traces were evoked by 50 ms step depolarizations between − 50 and + 60 mV from a holding potential of − 80 mV for CaV2.1/α2δ-1/β1b currents, expressed together with GFP-CAAX (black, control), or CaV2.1-(46-100)-CAAX (blue). (B) Mean current-voltage relationships of CaV2.1 and GFP-CAAX (black squares, control, n = 13) or CaV2.1 and CaV2.1-(46-100)-CAAX (blue triangles, n = 12). (C) Mean current density at + 5 mV ± SEM. Statistical analysis: ns = non-significant difference. (D–E) Effect of the N-terminal constructs on the suppressive effect of EA2 on CaV2.1 currents. (D) Representative traces from tsA-201 cells transfected with CaV2.1/β1b/α2δ-1, together with CaV3.1 Dom I–II and GFP-CAAX (black, control), CaV2.1, EA2 and GFP-CAAX (red) or CaV2.1, EA2 and CaV2.1-(46-100)-CAAX (blue). (E) Mean current-voltage relationships for CaV2.1, CaV3.1 Dom I–II and GFP-CAAX (black squares, control, n = 25), CaV2.1 and EA2 plus GFP-CAAX (red circles, n = 24) or CaV2.1 and EA2 plus CaV2.1-(46-100)-CAAX (blue circles, n = 15). (F) Mean current density at + 5 mV ± SEM. Statistical analysis: **p < 0.01, ***p < 0.001.
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f0025: Effect of the N-terminus on CaV2.1 current and suppression by EA2.(A–C) Effect of the N-terminal constructs on CaV2.1/β1b/α2δ-1 currents. (A) Representative traces were evoked by 50 ms step depolarizations between − 50 and + 60 mV from a holding potential of − 80 mV for CaV2.1/α2δ-1/β1b currents, expressed together with GFP-CAAX (black, control), or CaV2.1-(46-100)-CAAX (blue). (B) Mean current-voltage relationships of CaV2.1 and GFP-CAAX (black squares, control, n = 13) or CaV2.1 and CaV2.1-(46-100)-CAAX (blue triangles, n = 12). (C) Mean current density at + 5 mV ± SEM. Statistical analysis: ns = non-significant difference. (D–E) Effect of the N-terminal constructs on the suppressive effect of EA2 on CaV2.1 currents. (D) Representative traces from tsA-201 cells transfected with CaV2.1/β1b/α2δ-1, together with CaV3.1 Dom I–II and GFP-CAAX (black, control), CaV2.1, EA2 and GFP-CAAX (red) or CaV2.1, EA2 and CaV2.1-(46-100)-CAAX (blue). (E) Mean current-voltage relationships for CaV2.1, CaV3.1 Dom I–II and GFP-CAAX (black squares, control, n = 25), CaV2.1 and EA2 plus GFP-CAAX (red circles, n = 24) or CaV2.1 and EA2 plus CaV2.1-(46-100)-CAAX (blue circles, n = 15). (F) Mean current density at + 5 mV ± SEM. Statistical analysis: **p < 0.01, ***p < 0.001.

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 the N-terminus on CaV2.1 current and suppression by EA2.(A–C) Effect of the N-terminal constructs on CaV2.1/β1b/α2δ-1 currents. (A) Representative traces were evoked by 50 ms step depolarizations between − 50 and + 60 mV from a holding potential of − 80 mV for CaV2.1/α2δ-1/β1b currents, expressed together with GFP-CAAX (black, control), or CaV2.1-(46-100)-CAAX (blue). (B) Mean current-voltage relationships of CaV2.1 and GFP-CAAX (black squares, control, n = 13) or CaV2.1 and CaV2.1-(46-100)-CAAX (blue triangles, n = 12). (C) Mean current density at + 5 mV ± SEM. Statistical analysis: ns = non-significant difference. (D–E) Effect of the N-terminal constructs on the suppressive effect of EA2 on CaV2.1 currents. (D) Representative traces from tsA-201 cells transfected with CaV2.1/β1b/α2δ-1, together with CaV3.1 Dom I–II and GFP-CAAX (black, control), CaV2.1, EA2 and GFP-CAAX (red) or CaV2.1, EA2 and CaV2.1-(46-100)-CAAX (blue). (E) Mean current-voltage relationships for CaV2.1, CaV3.1 Dom I–II and GFP-CAAX (black squares, control, n = 25), CaV2.1 and EA2 plus GFP-CAAX (red circles, n = 24) or CaV2.1 and EA2 plus CaV2.1-(46-100)-CAAX (blue circles, n = 15). (F) Mean current density at + 5 mV ± SEM. Statistical analysis: **p < 0.01, ***p < 0.001.
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f0025: Effect of the N-terminus on CaV2.1 current and suppression by EA2.(A–C) Effect of the N-terminal constructs on CaV2.1/β1b/α2δ-1 currents. (A) Representative traces were evoked by 50 ms step depolarizations between − 50 and + 60 mV from a holding potential of − 80 mV for CaV2.1/α2δ-1/β1b currents, expressed together with GFP-CAAX (black, control), or CaV2.1-(46-100)-CAAX (blue). (B) Mean current-voltage relationships of CaV2.1 and GFP-CAAX (black squares, control, n = 13) or CaV2.1 and CaV2.1-(46-100)-CAAX (blue triangles, n = 12). (C) Mean current density at + 5 mV ± SEM. Statistical analysis: ns = non-significant difference. (D–E) Effect of the N-terminal constructs on the suppressive effect of EA2 on CaV2.1 currents. (D) Representative traces from tsA-201 cells transfected with CaV2.1/β1b/α2δ-1, together with CaV3.1 Dom I–II and GFP-CAAX (black, control), CaV2.1, EA2 and GFP-CAAX (red) or CaV2.1, EA2 and CaV2.1-(46-100)-CAAX (blue). (E) Mean current-voltage relationships for CaV2.1, CaV3.1 Dom I–II and GFP-CAAX (black squares, control, n = 25), CaV2.1 and EA2 plus GFP-CAAX (red circles, n = 24) or CaV2.1 and EA2 plus CaV2.1-(46-100)-CAAX (blue circles, n = 15). (F) Mean current density at + 5 mV ± SEM. Statistical analysis: **p < 0.01, ***p < 0.001.
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