N Terminus Is Key to the Dominant Negative Suppression of CaV2 Calcium Channels: IMPLICATIONS FOR EPISODIC ATAXIA TYPE 2

Expression of the calcium channels CaV2.1 and CaV2.2 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 CaV2.1 that predict truncated...

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Veröffentlicht in:The Journal of biological chemistry 2010, Vol.285 (2), p.835-844
Hauptverfasser: Page, Karen M, Heblich, Fay, Margas, Wojciech, Pratt, Wendy S, Nieto-Rostro, Manuela, Chaggar, Kanchan, Sandhu, Kieran, Davies, Anthony, Dolphin, Annette C
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Sprache:eng
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Zusammenfassung:Expression of the calcium channels CaV2.1 and CaV2.2 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 CaV2.1 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 CaV2.1 and CaV2.2, 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 Arg⁵² and Arg⁵⁴) 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 CaV2.2 currents by the N terminus-CAAX construct is accompanied by a reduction in CaV2.2 protein level, and this is also prevented by mutation of Arg⁵² and Arg⁵⁴ to Ala in the truncated construct. Taken together, our evidence indicates that both the extreme N terminus and the Arg⁵², Arg⁵⁴ motif are involved in the processes underlying dominant negative suppression.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M109.065045