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The Journal of Neuroscience, November 1, 2001, 21(21):8495-8504

Dominant-Negative Synthesis Suppression of Voltage-Gated Calcium Channel Ca_v2.2 Induced by Truncated Constructs

Ayesha Raghib, Federica Bertaso, Anthony Davies, Karen M. Page, Alon Meir, Yuri Bogdanov, and Annette C. Dolphin

Department of Pharmacology, University College London, London WC1E6BT, United Kingdom

Voltage-gated calcium channel 1 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_v2.2 (1B) of its coexpression with truncated constructs of Ca_v2.2. Domains I-II or domains III-IV, when expressed individually, together with the accessory subunits 1b and 2-1, 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_v2.2, 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_v2.2 and expression of Ca_v2.2 protein was almost abolished. Suppression does not involve sequestration of the Ca_v subunit, because loss of GFP-Ca_v2.2 expression also occurred in the absence of  subunit, and the effect of domain I-II or domain I could not be mimicked by the cytoplasmic I-II loop of Ca_v2.2. It requires transmembrane segments, because the isolated Ca_v2.2 N terminus did not have any effect. Our results indicate that the mechanism of suppression of Ca_v2.2 by truncated constructs containing domain I involves inhibition of channel synthesis, which may represent a role of endogenously expressed truncated Ca_v isoforms.

Key words: calcium channel; truncation; expression; suppression; protein synthesis; GFP

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Copyright © 2001 Society for Neuroscience  0270-6474/01/21218495-10$05.00/0

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