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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.

No MeSH data available.


Related in: MedlinePlus

Effect of the N-terminal construct on CaV2.1R57A, R59A current and suppression by EA2.(A) Representative traces of the currents evoked by CaV2.1R57A, R59A in presence of GFP-CAAX (black) or CaV2.1-(46-100)-CAAX (blue). (B) Mean current-voltage relationships of CaV2.1 and GFP-CAAX (black squares, n = 11) or CaV2.1 and CaV2.1-(46-100)-CAAX (blue triangles, n = 12). (C) Mean current density at + 5 mV for the two conditions. Statistical analysis: ns = non-significant difference. (D–F) Effect of the N-terminal construct on the suppressive effect of EA2 on CaV2.1R57A, R59A current. (D) Representative traces of tsA-201 cells transfected with CaV2.1R57A, R59A, CaV3.1 Dom I–II and GFP-CAAX for the control condition (black) or CaV2.1 and EA-2 in the presence of GFP-CAAX (red), or CaV2.1-(46-100)-CAAX (blue). (E) Mean current-voltage relationships of CaV2.1, CaV3.1 Dom I–II and GFP-CAAX (black squares, n = 14), CaV2.1, EA2 and GFP-CAAX (red circles, n = 26) or CaV2.1, EA2 and CaV2.1-(46-100)-CAAX (blue circles, n = 21). (F) Mean current density at + 5 mV ± SEM for CaV2.1, CaV3.1 Dom I–II and GFP-CAAX (black, n = 10), CaV2.1, EA2 and GFP-CAAX (red, n = 27) or CaV2.1, EA2 and CaV2.1-(46-100)-CAAX (blue, n = 23). Statistical analysis: **p < 0.01, ns = non-significant difference.
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f0035: Effect of the N-terminal construct on CaV2.1R57A, R59A current and suppression by EA2.(A) Representative traces of the currents evoked by CaV2.1R57A, R59A in presence of GFP-CAAX (black) or CaV2.1-(46-100)-CAAX (blue). (B) Mean current-voltage relationships of CaV2.1 and GFP-CAAX (black squares, n = 11) or CaV2.1 and CaV2.1-(46-100)-CAAX (blue triangles, n = 12). (C) Mean current density at + 5 mV for the two conditions. Statistical analysis: ns = non-significant difference. (D–F) Effect of the N-terminal construct on the suppressive effect of EA2 on CaV2.1R57A, R59A current. (D) Representative traces of tsA-201 cells transfected with CaV2.1R57A, R59A, CaV3.1 Dom I–II and GFP-CAAX for the control condition (black) or CaV2.1 and EA-2 in the presence of GFP-CAAX (red), or CaV2.1-(46-100)-CAAX (blue). (E) Mean current-voltage relationships of CaV2.1, CaV3.1 Dom I–II and GFP-CAAX (black squares, n = 14), CaV2.1, EA2 and GFP-CAAX (red circles, n = 26) or CaV2.1, EA2 and CaV2.1-(46-100)-CAAX (blue circles, n = 21). (F) Mean current density at + 5 mV ± SEM for CaV2.1, CaV3.1 Dom I–II and GFP-CAAX (black, n = 10), CaV2.1, EA2 and GFP-CAAX (red, n = 27) or CaV2.1, EA2 and CaV2.1-(46-100)-CAAX (blue, n = 23). Statistical analysis: **p < 0.01, ns = non-significant difference.

Mentions: Since we found in this study that the EA2 mutant still exerted a dominant-negative effect on CaV2.1R57A, R59A (Fig. 3A–C), we next examined whether it was possible to perturb this suppressive effect by co-expressing CaV2.1-(46-100)-CAAX. We first showed that this construct did not have a direct effect on CaV2.1R57A, R59A currents (control: 114.0 ± 28.0 pA/pF; CaV2.1-(46-100)-CAAX: 110.0 ± 20.0 pA/pF; Fig. 7A–C). Furthermore, the dominant-negative effect of EA2 on CaV2.1R57A, R59A currents was not significantly reversed by CaV2.1-(46-100)-CAAX (control: 109.9 ± 19.2 pA/pF; EA2 + GFP-CAAX: − 36.8 ± 6.0; EA2 + CaV2.1-(46-100)-CAAX: − 65.0 ± 8.8 pA/pF; Fig.7D–F). This likely indicates that the suppressive effect of EA2 on CaV2.1R57A, R59A is more robust, and less easily disrupted.


A Ca V 2.1 N-terminal fragment relieves the dominant-negative inhibition by an Episodic ataxia 2 mutant
Effect of the N-terminal construct on CaV2.1R57A, R59A current and suppression by EA2.(A) Representative traces of the currents evoked by CaV2.1R57A, R59A in presence of GFP-CAAX (black) or CaV2.1-(46-100)-CAAX (blue). (B) Mean current-voltage relationships of CaV2.1 and GFP-CAAX (black squares, n = 11) or CaV2.1 and CaV2.1-(46-100)-CAAX (blue triangles, n = 12). (C) Mean current density at + 5 mV for the two conditions. Statistical analysis: ns = non-significant difference. (D–F) Effect of the N-terminal construct on the suppressive effect of EA2 on CaV2.1R57A, R59A current. (D) Representative traces of tsA-201 cells transfected with CaV2.1R57A, R59A, CaV3.1 Dom I–II and GFP-CAAX for the control condition (black) or CaV2.1 and EA-2 in the presence of GFP-CAAX (red), or CaV2.1-(46-100)-CAAX (blue). (E) Mean current-voltage relationships of CaV2.1, CaV3.1 Dom I–II and GFP-CAAX (black squares, n = 14), CaV2.1, EA2 and GFP-CAAX (red circles, n = 26) or CaV2.1, EA2 and CaV2.1-(46-100)-CAAX (blue circles, n = 21). (F) Mean current density at + 5 mV ± SEM for CaV2.1, CaV3.1 Dom I–II and GFP-CAAX (black, n = 10), CaV2.1, EA2 and GFP-CAAX (red, n = 27) or CaV2.1, EA2 and CaV2.1-(46-100)-CAAX (blue, n = 23). Statistical analysis: **p < 0.01, ns = non-significant difference.
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f0035: Effect of the N-terminal construct on CaV2.1R57A, R59A current and suppression by EA2.(A) Representative traces of the currents evoked by CaV2.1R57A, R59A in presence of GFP-CAAX (black) or CaV2.1-(46-100)-CAAX (blue). (B) Mean current-voltage relationships of CaV2.1 and GFP-CAAX (black squares, n = 11) or CaV2.1 and CaV2.1-(46-100)-CAAX (blue triangles, n = 12). (C) Mean current density at + 5 mV for the two conditions. Statistical analysis: ns = non-significant difference. (D–F) Effect of the N-terminal construct on the suppressive effect of EA2 on CaV2.1R57A, R59A current. (D) Representative traces of tsA-201 cells transfected with CaV2.1R57A, R59A, CaV3.1 Dom I–II and GFP-CAAX for the control condition (black) or CaV2.1 and EA-2 in the presence of GFP-CAAX (red), or CaV2.1-(46-100)-CAAX (blue). (E) Mean current-voltage relationships of CaV2.1, CaV3.1 Dom I–II and GFP-CAAX (black squares, n = 14), CaV2.1, EA2 and GFP-CAAX (red circles, n = 26) or CaV2.1, EA2 and CaV2.1-(46-100)-CAAX (blue circles, n = 21). (F) Mean current density at + 5 mV ± SEM for CaV2.1, CaV3.1 Dom I–II and GFP-CAAX (black, n = 10), CaV2.1, EA2 and GFP-CAAX (red, n = 27) or CaV2.1, EA2 and CaV2.1-(46-100)-CAAX (blue, n = 23). Statistical analysis: **p < 0.01, ns = non-significant difference.
Mentions: Since we found in this study that the EA2 mutant still exerted a dominant-negative effect on CaV2.1R57A, R59A (Fig. 3A–C), we next examined whether it was possible to perturb this suppressive effect by co-expressing CaV2.1-(46-100)-CAAX. We first showed that this construct did not have a direct effect on CaV2.1R57A, R59A currents (control: 114.0 ± 28.0 pA/pF; CaV2.1-(46-100)-CAAX: 110.0 ± 20.0 pA/pF; Fig. 7A–C). Furthermore, the dominant-negative effect of EA2 on CaV2.1R57A, R59A currents was not significantly reversed by CaV2.1-(46-100)-CAAX (control: 109.9 ± 19.2 pA/pF; EA2 + GFP-CAAX: − 36.8 ± 6.0; EA2 + CaV2.1-(46-100)-CAAX: − 65.0 ± 8.8 pA/pF; Fig.7D–F). This likely indicates that the suppressive effect of EA2 on CaV2.1R57A, R59A is more robust, and less easily disrupted.

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