Dominant-negative calcium channel suppression by truncated constructs involves a kinase implicated in the unfolded protein response.
Expression of the calcium channel Ca(V)2.2 is markedly suppressed by coexpression with truncated constructs of Ca(V)2.2. Furthermore, a two-domain construct of Ca(V)2.1 mimicking an episodic ataxia-2 mutation strongly inhibited Ca(V)2.1 currents. We have now determined the specificity of this effect, identified a potential mechanism, and have shown that such constructs also inhibit endogenous calcium currents when transfected into neuronal cell lines. Suppression of calcium channel expression requires interaction between truncated and full-length channels, because there is inter-subfamily specificity. Although there is marked cross-suppression within the Ca(V)2 calcium channel family, there is no cross-suppression between Ca(V)2 and Ca(V)3 channels. The mechanism involves activation of a component of the unfolded protein response, the endoplasmic reticulum resident RNA-dependent kinase (PERK), because it is inhibited by expression of dominant-negative constructs of this kinase. Activation of PERK has been shown previously to cause translational arrest, which has the potential to result in a generalized effect on protein synthesis. In agreement with this, coexpression of the truncated domain I of Ca(V)2.2, together with full-length Ca(V)2.2, reduced the level not only of Ca(V)2.2 protein but also the coexpressed alpha2delta-2. Thapsigargin, which globally activates the unfolded protein response, very markedly suppressed Ca(V)2.2 currents and also reduced the expression level of both Ca(V)2.2 and alpha2delta-2 protein. We propose that voltage-gated calcium channels represent a class of difficult-to-fold transmembrane proteins, in this case misfolding is induced by interaction with a truncated cognate Ca(V) channel. This may represent a mechanism of pathology in episodic ataxia-2.
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Karen M
KM
Department of Pharmacology, University College London, London WC1E 6BT, United Kingdom.
Heblich
Fay
F
Davies
Anthony
A
Butcher
Adrian J
AJ
Leroy
Jerôme
J
Bertaso
Federica
F
Pratt
Wendy S
WS
Dolphin
Annette C
AC
D000818:Animals; D015153:Blotting, Western; D015220:Calcium Channels / Q000378:metabolism*; D009865:Oocytes / Q000201:enzymology; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D017510:Protein Folding; D012333:RNA, Messenger; D014981:Xenopus; D019892:eIF-2 Kinase / Q000235:genetics / Q000378:metabolism*
D015220:Calcium Channels; D012333:RNA, Messenger; D019892:eIF-2 Kinase