N terminus is key to the dominant negative suppression of Ca(V)2 calcium channels: implications for episodic ataxia type 2.
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.
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Karen M
KM
Department of Neuroscience, Physiology, and Pharmacology, University College London, Gower Street, London WC1E 6BT, United Kingdom.
Heblich
Fay
F
Margas
Wojciech
W
Pratt
Wendy S
WS
Nieto-Rostro
Manuela
M
Chaggar
Kanchan
K
Sandhu
Kieran
K
Davies
Anthony
A
Dolphin
Annette C
AC
D020816:Amino Acid Motifs / Q000235:genetics; D019943:Amino Acid Substitution; D000818:Animals; D019556:COS Cells; D020864:Calcium Channels, N-Type / Q000235:genetics / Q000378:metabolism*; D002522:Chlorocebus aethiops; D004721:Endoplasmic Reticulum / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D020125:Mutation, Missense; D009865:Oocytes; D017434:Protein Structure, Tertiary / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D020754:Spinocerebellar Ataxias / Q000235:genetics / Q000378:metabolism*; D056811:Unfolded Protein Response; D014982:Xenopus laevis
D020864:Calcium Channels, N-Type; C503279:Cacna1b protein, rat