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Enzyme-inhibitor-like tuning of Ca2+ channel connectivity with calmodulin

A Corrigendum to this article was published on 01 April 2010

This article has been updated

Abstract

Ca2+ channels and calmodulin (CaM) are two prominent signalling hubs1 that synergistically affect functions as diverse as cardiac excitability2, synaptic plasticity3 and gene transcription4. It is therefore fitting that these hubs are in some sense coordinated, as the opening of CaV1–2 Ca2+ channels are regulated by a single CaM constitutively complexed with channels5. The Ca2+-free form of CaM (apoCaM) is already pre-associated with the isoleucine–glutamine (IQ) domain on the channel carboxy terminus, and subsequent Ca2+ binding to this ‘resident’ CaM drives conformational changes that then trigger regulation of channel opening6. Another potential avenue for channel–CaM coordination could arise from the absence of Ca2+ regulation in channels lacking a pre-associated CaM6,7. Natural fluctuations in CaM concentrations might then influence the fraction of regulable channels and, thereby, the overall strength of Ca2+ feedback. However, the prevailing view has been that the ultrastrong affinity of channels for apoCaM ensures their saturation with CaM8, yielding a significant form of concentration independence between Ca2+ channels and CaM. Here we show that significant exceptions to this autonomy exist, by combining electrophysiology (to characterize channel regulation) with optical fluorescence resonance energy transfer (FRET) sensor determination of free-apoCaM concentration in live cells9. This approach translates quantitative CaM biochemistry from the traditional test-tube context into the realm of functioning holochannels within intact cells. From this perspective, we find that long splice forms of CaV1.3 and CaV1.4 channels include a distal carboxy tail10,11,12 that resembles an enzyme competitive inhibitor that retunes channel affinity for apoCaM such that natural CaM variations affect the strength of Ca2+ feedback modulation. Given the ubiquity of these channels13,14, the connection between ambient CaM levels and Ca2+ entry through channels is broadly significant for Ca2+ homeostasis. Strategies such as ours promise key advances for the in situ analysis of signalling molecules resistant to in vitro reconstitution, such as Ca2+ channels.

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Figure 1: Distal carboxy tail of Ca V 1.4 weakens Ca 2+ regulation of channels.
Figure 2: Provisional evidence for competition.
Figure 3: Live-cell holochannel biochemistry proves competition.
Figure 4: Molecular interactions and biology of competitive inhibitory tuning.

Change history

  • 01 April 2010

    Nature 463, 968–972 (2010) In the Acknowledgements section of this Letter, the work was incorrectly listed as being funded in part by the US National Institute of Neurological Disorders and Stroke. This work was in fact funded by the US National Institute on Deafness and Other Communication Disorders.

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Acknowledgements

We thank M. Tadross, I. Dick and members of the Calcium Signals Laboratory for comments; M. Tadross for data-acquisition software; D. J. Black and A. Persechini for BSCaMIQ and neuromodulin complementary DNA; J. McRory and T. Snutch for human α1F cDNA; J. Streissnig for human α1D cDNA; and V. Wu for earlier foundational experiments. This work is supported by grants from the US National Institute of Mental Health, the US National Heart, Lung, and Blood Institute and the US National Institute of Neurological Disorders and Stroke.

Author Contributions X.L. devised and refined experimental design, carried out all phases of the experiments and performed extensive data analysis. P.S.Y. consulted on initial molecular biology approaches, constructed certain channels with ICDI point mutations and contributed importantly to CaV1.4 expression strategies and electrophysiological characterization. W.Y. conducted FRET experiments, undertook molecular biology and extensively managed technical aspects of the project. D.T.Y. conceived, refined and oversaw the experiments, performed FRET experiments, analysed data and wrote the manuscript. All authors commented on and edited the manuscript.

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Correspondence to David T. Yue.

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This file contains Supplementary Information and Data including Supplementary Figures S1.1- S1.5, S2.2-S2.3, S3.2, S3.4-S3.6 & S4.2 with Legends and Supplementary References. (PDF 3710 kb)

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Liu, X., Yang, P., Yang, W. et al. Enzyme-inhibitor-like tuning of Ca2+ channel connectivity with calmodulin. Nature 463, 968–972 (2010). https://doi.org/10.1038/nature08766

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