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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.


Diagram of the possible mechanism for involvement of the N-terminus in dominant-negative suppression.(A) The four domains of CaV2 channels are shown in light blue, orange, green and red. The N-terminus is shown interacting with an intramolecular docking site on the left, whereas on the right interaction is reduced by mutation of the RAR motif (**). (B) The two domains of the truncated constructs are shown in dark blue and brown. On the right the truncated construct has a mutated RAR (**). (C) The N-terminus of the truncated construct can interact with the docking site on the full-length channel, initiating aggregation. This interaction is reduced by mutation of the RAR motif. (D) The free N-terminus interacts with the docking site on the full-length channel reducing the ability of the truncated channel to interact with this site.
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f0045: Diagram of the possible mechanism for involvement of the N-terminus in dominant-negative suppression.(A) The four domains of CaV2 channels are shown in light blue, orange, green and red. The N-terminus is shown interacting with an intramolecular docking site on the left, whereas on the right interaction is reduced by mutation of the RAR motif (**). (B) The two domains of the truncated constructs are shown in dark blue and brown. On the right the truncated construct has a mutated RAR (**). (C) The N-terminus of the truncated construct can interact with the docking site on the full-length channel, initiating aggregation. This interaction is reduced by mutation of the RAR motif. (D) The free N-terminus interacts with the docking site on the full-length channel reducing the ability of the truncated channel to interact with this site.

Mentions: We postulate that the N-terminus is normally involved in intramolecular docking with another part of the same channel, to form correctly folded and functional channels, and the RAR motif is involved in this interaction (Fig. 9A). This intramolecular docking is also involved in G-protein–mediated inhibition of the CaV2 channels (Page et al., 1998, Canti et al., 1999). We propose that in the truncated channels this intramolecular interaction cannot occur, because the channel structure is incomplete and misfolded (Fig. 9B), with the consequence that the exposed N-terminus of the truncated channel is able to participate in competition for the N-terminal docking site on the full-length channel, to form a deleterious intermolecular interaction (Fig. 9B, C). When the RAR motif is mutated in the N-terminus of the truncated channels, it therefore reduces their ability to interact with docking site on the full length channel (Fig. 9B, C).


A Ca V 2.1 N-terminal fragment relieves the dominant-negative inhibition by an Episodic ataxia 2 mutant
Diagram of the possible mechanism for involvement of the N-terminus in dominant-negative suppression.(A) The four domains of CaV2 channels are shown in light blue, orange, green and red. The N-terminus is shown interacting with an intramolecular docking site on the left, whereas on the right interaction is reduced by mutation of the RAR motif (**). (B) The two domains of the truncated constructs are shown in dark blue and brown. On the right the truncated construct has a mutated RAR (**). (C) The N-terminus of the truncated construct can interact with the docking site on the full-length channel, initiating aggregation. This interaction is reduced by mutation of the RAR motif. (D) The free N-terminus interacts with the docking site on the full-length channel reducing the ability of the truncated channel to interact with this site.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4940211&req=5

f0045: Diagram of the possible mechanism for involvement of the N-terminus in dominant-negative suppression.(A) The four domains of CaV2 channels are shown in light blue, orange, green and red. The N-terminus is shown interacting with an intramolecular docking site on the left, whereas on the right interaction is reduced by mutation of the RAR motif (**). (B) The two domains of the truncated constructs are shown in dark blue and brown. On the right the truncated construct has a mutated RAR (**). (C) The N-terminus of the truncated construct can interact with the docking site on the full-length channel, initiating aggregation. This interaction is reduced by mutation of the RAR motif. (D) The free N-terminus interacts with the docking site on the full-length channel reducing the ability of the truncated channel to interact with this site.
Mentions: We postulate that the N-terminus is normally involved in intramolecular docking with another part of the same channel, to form correctly folded and functional channels, and the RAR motif is involved in this interaction (Fig. 9A). This intramolecular docking is also involved in G-protein–mediated inhibition of the CaV2 channels (Page et al., 1998, Canti et al., 1999). We propose that in the truncated channels this intramolecular interaction cannot occur, because the channel structure is incomplete and misfolded (Fig. 9B), with the consequence that the exposed N-terminus of the truncated channel is able to participate in competition for the N-terminal docking site on the full-length channel, to form a deleterious intermolecular interaction (Fig. 9B, C). When the RAR motif is mutated in the N-terminus of the truncated channels, it therefore reduces their ability to interact with docking site on the full length channel (Fig. 9B, C).

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.