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PI3K promotes voltage-dependent calcium channel trafficking to the plasma membrane

Nature Neuroscience volume 7, pages 939–946 (2004)Cite this article

Abstract

Phosphatidylinositol 3-kinase (PI3K) has been shown to enhance native voltage-dependent calcium channel (Cav) currents both in myocytes and in neurons; however, the mechanism(s) responsible for this regulation were not known. Here we show that PI3K promotes the translocation of GFP-tagged Cav channels to the plasma membrane in both COS-7 cells and neurons. We show that the effect of PI3K is mediated by Akt/PKB and specifically requires Cavβ2 subunits. The mutations S574A and S574E in Cavβ2a prevented and mimicked, respectively, the effect of PI3K/Akt-PKB, indicating that phosphorylation of Ser574 on Cavβ2a is necessary and sufficient to promote Cav channel trafficking.

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Figure 1: Effect of PI3Kγ-induced production of PIP3 on Cav1.2α1/β2a currents.
Figure 2: Relevance of Cav subunit composition for PI3Kγ effect on IBa.
Figure 3: Effect of PI3Kγ on Cav channel membrane localization.
Figure 4: Role of Akt/PKB in the PI3Kγ-induced enhancement of Cav expression at the membrane.
Figure 5: Effect of S574A mutation on the PI3Kγ and myr-Akt/PKB-induced regulation of Cav2.2 channels.
Figure 6: Effect of Cavβ2aS574E mutant on Cav2.2 channel expression at the membrane.
Figure 7: Effect of PI3Kα on PHGrp1-GFP and Cav channel membrane localization.
Figure 8: Effect of acute stimulation of PI3K on Cav channel localization in DRG neurons.

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Acknowledgements

We thank K. Chaggar, W.S. Pratt, J. Wratten and M. Nieto-Rostro for technical assistance and K. Page, A. Babich and N.S. Berrow for discussions. This work was supported by the Wellcome Trust and Medical Research Council (UK).

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Author notes

  1. Adrian J Butcher and Guillaume Halet: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Pharmacology, University College London, London, WC1E 6BT, UK

    Patricia Viard, Adrian J Butcher, Anthony Davies, Fay Heblich & Annette C Dolphin

  2. Department of Physiology, University College London, London, WC1E 6BT, UK

    Guillaume Halet

  3. Institut für Biochemie und Molekular Biologie II, Klinikum der Heinrich-Heine-Universität Düsseldorf, Düsseldorf, 40225, Germany

    Bernd Nürnberg

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Correspondence to Patricia Viard.

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Supplementary information

Supplementary Fig. 1

Nuclear staining with Hoechst 33342 of PHGrp1-GFP-labeled control and PI3Kγ-transfected cells. Subcellular localization of PHGrp1-GFP in control (a) and PI3Kγ-transfected cells (b). First panel: subcellular localization of PHGrp1-GFP. The intensity profiles below the image was taken at the position indicated by the red bar. Second panel: nucleus staining with Hoechst 33342. Third panel: overlay of the PHGrp1-GFP fluorescence image and nuclear staining. Scale bars: 10 μm (JPG 96 kb)

Supplementary Fig. 2

Effect of PI3Kγ and myr-Akt/PKB co-transfection on Cav2.2α1 subcellular localization. To ensure that the maximal PI3K effect was obtained, we used the membrane-targeted PI3Kγ-CAAX (p101-p110γ-CAAX), constitutively localized at the proximity of its lipid substrate39. (a) Subcellular distribution of PHGrp1-GFP in cells co-transfected with PI3Kγ-CAAX. The amount of PIP3–binding domain was increased at the plasma membrane. The arrowhead indicates lamellipodia which are generally associated with high lipid kinase activity. (b) Effect of PI3Kγ-CAAX and myr-Akt/PKB co-expression on the subcellular localization of GFP-Cav2.2α1/β2a channel. Profiles showing the fluorescence intensity along the line indicated in red are displayed below each cell. Scale bars: 10 μm. (c) Upper panel: example traces of Cav2.2α1/β2a IBa recorded in control and PI3Kγ-CAAX plus myr-Akt/PKB-co-transfected cells. Vertical and horizontal scales are 20 pA/pF and 100 ms, respectively. Lower panel: mean I-V relationship of Cav2.2α1/β2a channels in control (○) or PI3Kγ-CAAX and myr-Akt/PKB-co-transfected cells (●). Results are expressed as mean ± s.e.m of 7 cells each. (JPG 70 kb)

Both the localization of Cav2.2/β2a channels at the plasma membrane (b) and Cav2.2/ β2a IBa (c) were increased by simultaneous expression of myr-Akt/PKB and PI3Kγ-CAAX.

Supplementary Fig. 3

Immunodetection of total cell Cav2.2α1 in control and PI3Kγ-CAAX- plus myr-Akt/PKB-transfected cells. (a) Immunodetection of total cell Cav2.2 α 1 (arrowhead) in un-transfected, control and PI3Kγ-CAAX- plus myr-Akt/PKB-transfected cells. (b) Quantification of the relative amounts of total cell Cav2.2 α 1 co-expressed in control (open bars) and PI3Kγ-CAAX plus myr-Akt/PKB-transfected cells (closed bars). (JPG 19 kb)

Supplementary Fig. 4

Alignment of Cavβ C-terminus sequences. Identification of a unique putative consensus site for phosphorylation by Akt/PKB on Cavβ2a subunit, absent in Cavβ1b, Cavβ3 and Cavβ4 subunits. C-terminus sequences of Cavβ1b, Cavβ2a, Cavβ3, Cavβ4 and both Cavβ2aS574A and Cavβ2aS574E mutants. (GIF 28 kb)

Supplementary Video 1

Z-stack showing the membrane localization of GFP-Cav2.2α1/β2a channels in a PI3Kγ-transfected cell. In cells transfected with PI3Kγ, the strong plasma membrane localization of GFP-Cav2.2α1/β2a channels was visible independently of the focal plane of acquisition. (MOV 2349 kb)

Supplementary Video 2

Z-stack showing the membrane localization of GFP-Cav2.2α1/β2aS574E channels. The strong plasma membrane localization of GFP-Cav2.2α1/β2aS574E channels was visible independently of the focal plane of acquisition. (MOV 1813 kb)

Supplementary Methods (PDF 24 kb)

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Viard, P., Butcher, A., Halet, G. et al. PI3K promotes voltage-dependent calcium channel trafficking to the plasma membrane. Nat Neurosci 7, 939–946 (2004). https://doi.org/10.1038/nn1300

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