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Allostery in Ca2+ channel modulation by calcium-binding proteins

Nature Chemical Biology volume 10, pages 231–238 (2014)Cite this article

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Abstract

Distinguishing between allostery and competition among modulating ligands is challenging for large target molecules. Out of practical necessity, inferences are often drawn from in vitro assays on target fragments, but such inferences may belie actual mechanisms. One key example of such ambiguity concerns calcium-binding proteins (CaBPs) that tune signaling molecules regulated by calmodulin (CaM). As CaBPs resemble CaM, CaBPs are believed to competitively replace CaM on targets. Yet, brain CaM expression far surpasses that of CaBPs, raising questions as to whether CaBPs can exert appreciable biological actions. Here, we devise a live-cell, holomolecule approach that reveals an allosteric mechanism for calcium channels whose CaM-mediated inactivation is eliminated by CaBP4. Our strategy is to covalently link CaM and/or CaBP to holochannels, enabling live-cell fluorescence resonance energy transfer assays to resolve a cyclical allosteric binding scheme for CaM and CaBP4 to channels, thus explaining how trace CaBPs prevail. This approach may apply generally for discerning allostery in live cells.

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Figure 1: Potential mechanisms of CaM and CaBP interaction with calcium channels.
Figure 2: CaBP4 modulation and binding of CaV1.3 channels.
Figure 3: Chimeric channels reveal the importance of CaBP sites beyond the IQ domain.
Figure 4: CaBP4 and CaM simultaneously bind CaV1.3 channels.
Figure 5: Only one CaBP4 can bind per CaV1.3 channel.
Figure 6: Mechanism of CaV1.3 channel modulation by modest levels of CaBP4.

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Acknowledgements

We thank W. Yang for dedicated technical support and other members of the Ca2+ signals lab for valuable comments. H. Bazzazi generously made available the β2a–CaMWT construct. Y. Gao contributed the data analysis showing CaM, CaBP4 and CaBP1 gene expression from human prefrontal cortex. This work is supported by grants from the US National Institutes of Health (NIH) Heart, Lung, and Blood Institute MERIT Award (to D.T.Y.), NIH National Institute on Deafness and Other Communication Disorders (P.S.Y. and P. Fuchs), NIH National Institute of General Medical Sciences (GM08752 to P.S.Y.) and the NIH National Institute of Mental Health (M.B.J.).

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Authors and Affiliations

  1. Department of Biomedical Engineering, Calcium Signals Laboratory, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

    Philemon S Yang, Manu Ben Johny & David T Yue

  2. Department of Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

    David T Yue

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Contributions

P.S.Y. created mutant, chimeric and engineered channels. P.S.Y. and M.B.J. performed electrophysiology and FRET experiments and undertook extensive data analysis. M.B.J. performed molecular modeling. D.T.Y. supervised and helped conceive the project. All of the authors refined hypotheses, wrote the paper and created figures.

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Correspondence to David T Yue.

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The authors declare no competing financial interests.

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Supplementary Results, Supplementary Figures 1–12 and Supplementary Notes 1 and 2. (PDF 4031 kb)

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Yang, P., Johny, M. & Yue, D. Allostery in Ca2+ channel modulation by calcium-binding proteins. Nat Chem Biol 10, 231–238 (2014). https://doi.org/10.1038/nchembio.1436

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