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The Cavβ subunit prevents RFP2-mediated ubiquitination and proteasomal degradation of L-type channels

Nature Neuroscience volume 14, pages 173–180 (2011)Cite this article

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Abstract

It is well established that the auxiliary Cavβ subunit regulates calcium channel density in the plasma membrane, but the cellular mechanism by which this occurs has remained unclear. We found that the Cavβ subunit increased membrane expression of Cav1.2 channels by preventing the entry of the channels into the endoplasmic reticulum–associated protein degradation (ERAD) complex. Without Cavβ, Cav1.2 channels underwent robust ubiquitination by the RFP2 ubiquitin ligase and interacted with the ERAD complex proteins derlin-1 and p97, culminating in targeting of the channels to the proteasome for degradation. On treatment with the proteasomal inhibitor MG132, Cavβ-free channels were rescued from degradation and trafficked to the plasma membrane. The coexpression of Cavβ interfered with ubiquitination and targeting of the channel to the ERAD complex, thereby facilitating export from the endoplasmic reticulum and promoting expression on the cell surface. Thus, Cavββ regulates the ubiquitination and stability of the calcium channel complex.

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Figure 1: Cavβ subunits increase surface and total expression of Cav1.2 channels.
Figure 2: Determinants of endoplasmic reticulum retention of intracelllular regions of HVA calcium channels.
Figure 3: MG132 and Cavβ both increase surface expression of Cav1.2 channels.
Figure 4: Cav1.2 channels interact with the ERAD protein complex in the absence of Cavβ.
Figure 5: The Cavβ subunit prevents ubiquitination of Cav1.2 channels.
Figure 6: Cav1.2 channels associate with the RING domain ubiquitin ligase RFP2.
Figure 7: Dominant-negative RFP2 increases barium current density, and enhances surface expression of Cav1.2 channels in hippocampal neurons.

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Acknowledgements

We thank J. Striessnig, S. Ferguson, J. Stutts, B. Schwappach, E. Bourinet and T. Snutch for cDNA constructs, and E. Stanley for help with the ICQ colocalization analysis. This work was supported by operating grants to G.W.Z. from the Heart and Stroke Foundation of Alberta, the Northwest Territories and Nunavut, and the Canadian Institutes for Health Research. G.W.Z. is a Scientist of the Alberta Heritage Foundation for Medical Research (AHFMR) and a Canada Research Chair in Molecular Neurobiology. H.Y. is supported by an AFHMR fellowship and B.S. is supported by an AHFMR studentship.

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  1. Christophe Altier and Agustin Garcia-Caballero: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Physiology and Pharmacology, Hotchkiss Brain Institute, University of Calgary, Calgary, Alberta, Canada

    Christophe Altier, Agustin Garcia-Caballero, Brett Simms, Haitao You, Lina Chen, Jan Walcher, H William Tedford, Tamara Hermosilla & Gerald W Zamponi

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Contributions

C.A., A.G.-C. and G.W.Z. designed the study and wrote the manuscript. G.W.Z. supervised the study. C.A., A.G.-C., B.S., H.Y. and L.C. performed experiments and data analysis. J.W. and H.W.T. contributed to molecular biology.

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Correspondence to Gerald W Zamponi.

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Altier, C., Garcia-Caballero, A., Simms, B. et al. The Cavβ subunit prevents RFP2-mediated ubiquitination and proteasomal degradation of L-type channels. Nat Neurosci 14, 173–180 (2011). https://doi.org/10.1038/nn.2712

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