Dominant-negative synthesis suppression of voltage-gated calcium channel Cav2.2 induced by truncated constructs.
Voltage-gated calcium channel alpha1 subunits consist of four domains (I-IV), each with six transmembrane segments. A number of truncated isoforms have been identified to occur as a result of alternative splicing or mutation. We have examined the functional consequences for expression of full-length Ca(v)2.2 (alpha1B) of its coexpression with truncated constructs of Ca(v)2.2. Domains I-II or domains III-IV, when expressed individually, together with the accessory subunits beta1b and alpha2delta-1, did not form functional channels. When they were coexpressed, low-density whole-cell currents and functional channels with properties similar to wild-type channels were observed. However, when domain I-II, domain III-IV, or domain I alone were coexpressed with full-length Ca(v)2.2, they markedly suppressed its functional expression, although at the single channel level, when channels were recorded, there were no differences in their biophysical properties. Furthermore, when it was coexpressed with either domain I-II or domain I, the fluorescence of green fluorescent protein (GFP)-Ca(v)2.2 and expression of Ca(v)2.2 protein was almost abolished. Suppression does not involve sequestration of the Ca(v)beta subunit, because loss of GFP-Ca(v)2.2 expression also occurred in the absence of beta subunit, and the effect of domain I-II or domain I could not be mimicked by the cytoplasmic I-II loop of Ca(v)2.2. It requires transmembrane segments, because the isolated Ca(v)2.2 N terminus did not have any effect. Our results indicate that the mechanism of suppression of Ca(v)2.2 by truncated constructs containing domain I involves inhibition of channel synthesis, which may represent a role of endogenously expressed truncated Ca(v) isoforms.
Raghib
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Department of Pharmacology, University College London, London WC1E6BT, United Kingdom.
Bertaso
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F
Davies
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Page
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KM
Meir
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Bogdanov
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Y
Dolphin
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D000818:Animals; D019556:COS Cells; D020864:Calcium Channels, N-Type / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression / Q000187:drug effects*; D005799:Genes, Dominant; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D018408:Patch-Clamp Techniques; D017434:Protein Structure, Tertiary / Q000502:physiology; D021122:Protein Subunits; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000494:pharmacology*; D014162:Transfection
D020864:Calcium Channels, N-Type; D008164:Luminescent Proteins; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins