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N terminus is key to the dominant negative suppression of Ca(V)2 calcium channels: implications for episodic ataxia
Karen M Page1, Fay Heblich, Wojciech Margas
1Department of Neuroscience, Physiology, and Pharmacology, University College London, Gower Street, London WC1E 6BT, United Kingdom.
Abstract:
Expression of the calcium channels Ca(V)2.1 and Ca(V)2.2 is markedly suppressed by co-expression with truncated constructs containing Domain I. This is the basis for the phenomenon of dominant negative suppression observed for many of the episodic ataxia type 2 mutations in Ca(V)2.1 that predict truncated channels. The process of dominant negative suppression has been shown previously to stem from interaction between the full-length and truncated channels and to result in downstream consequences of the unfolded protein response and endoplasmic reticulum-associated protein degradation. We have now identified the specific domain that triggers this effect. For both Ca(V)2.1 and Ca(V)2.2, the minimum construct producing suppression was the cytoplasmic N terminus. Suppression was enhanced by tethering the N terminus to the membrane with a CAAX motif. The 11-amino acid motif (including Arg(52) and Arg(54)) within the N terminus, which we have previously shown to be required for G protein modulation, is also essential for dominant negative suppression. Suppression is prevented by addition of an N-terminal tag (XFP) to the full-length and truncated constructs. We further show that suppression of Ca(V)2.2 currents by the N terminus-CAAX construct is accompanied by a reduction in Ca(V)2.2 protein level, and this is also prevented by mutation of Arg(52) and Arg(54) to Ala in the truncated construct. Taken together, our evidence indicates that both the extreme N terminus and the Arg(52), Arg(54) motif are involved in the processes underlying dominant negative suppression.
Insights
The N-terminal domain of calcium channels Ca(V)2.1 and Ca(V)2.2, specifically the Arg(52), Arg(54) motif, is crucial for dominant-negative suppression, impacting channel function and protein levels.
Area of Science:
- Neuroscience
- Molecular Biology
- Ion Channel Physiology
Background:
- Dominant-negative suppression of Ca(V)2.1 and Ca(V)2.2 calcium channels is linked to episodic ataxia type 2 mutations.
- This suppression involves interactions between full-length and truncated channels, leading to unfolded protein response and ER-associated degradation.
Purpose of the Study:
- To identify the specific domain responsible for dominant-negative suppression in Ca(V)2.1 and Ca(V)2.2 calcium channels.
- To elucidate the molecular mechanisms underlying this suppression, including the role of specific motifs and protein degradation pathways.
Main Methods:
- Co-expression of full-length and truncated calcium channel constructs (Ca(V)2.1, Ca(V)2.2).
- Utilized N-terminal constructs, CAAX motif tethering, and N-terminal tagging (XFP) to map the suppressive domain.
- Investigated the role of the Arg(52), Arg(54) motif through site-directed mutagenesis (Arg to Ala).
- Assessed effects on channel currents and protein levels, including ER-associated degradation.
Main Results:
- The cytoplasmic N terminus was identified as the minimal suppressive domain for both Ca(V)2.1 and Ca(V)2.2.
- Suppression was enhanced by CAAX motif tethering and required the Arg(52), Arg(54) motif.
- N-terminal tagging prevented suppression.
- Suppression of Ca(V)2.2 currents correlated with reduced protein levels, which was prevented by mutating Arg(52) and Arg(54).
Conclusions:
- The extreme N terminus, particularly the Arg(52), Arg(54) motif, is essential for dominant-negative suppression of Ca(V)2.1 and Ca(V)2.2 channels.
- This suppression mechanism involves direct interaction and potentially affects channel protein stability and degradation pathways.
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