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Selective phosphorylation modulates the PIP2 sensitivity of the CaM–SK channel complex

Nature Chemical Biology volume 10, pages 753–759 (2014)Cite this article

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Abstract

Phosphatidylinositol bisphosphate (PIP2) regulates the activities of many membrane proteins, including ion channels, through direct interactions. However, the affinity of PIP2 is so high for some channel proteins that its physiological role as a modulator has been questioned. Here we show that PIP2 is a key cofactor for activation of small conductance Ca2+-activated potassium channels (SKs) by Ca2+-bound calmodulin (CaM). Removal of the endogenous PIP2 inhibits SKs. The PIP2-binding site resides at the interface of CaM and the SK C terminus. We further demonstrate that the affinity of PIP2 for its target proteins can be regulated by cellular signaling. Phosphorylation of CaM T79, located adjacent to the PIP2-binding site, by casein kinase 2 reduces the affinity of PIP2 for the CaM–SK channel complex by altering the dynamic interactions among amino acid residues surrounding the PIP2-binding site. This effect of CaM phosphorylation promotes greater channel inhibition by G protein–mediated hydrolysis of PIP2.

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Figure 1: PIP2 is an essential cofactor for activation of SK2 channels by Ca2+.
Figure 2: The PIP2-binding site resides at the interface of the CaM–SK complex.
Figure 3: Phosphorylation of CaM T79 by CK2 enhances inhibition of SK channels by PIP2.
Figure 4: The phosphomimetic CaM mutant T79D mimics the effect of phosphorylation by CK2.
Figure 5: Phosphorylation of T79 of CaM disrupts the interaction between PIP2 and its binding site and reduces the apparent affinity of PIP2 for the CaM–SK2 complex.
Figure 6: CaM T79 phosphorylation facilitates modulation of SK2 by PIP2 hydrolysis triggered by stimulation of a GPCR, hMR1.

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Acknowledgements

We thank J. Adelman, R. Horn, I. Levitan and S. Siegelbaum for their helpful discussions and comments on this work. We thank M. Eto for advice on the in vitro phosphorylation assay. We thank J.P. Bennett Jr. and L. O'Brien for sharing their mammalian cell patch clamp setup. We are grateful to H. Vaananen for technical assistance. We thank I.S. Ramsey for the HEK-293 stably transfected cell line with the muscarinic M1 receptor, the structural facility of the Kimmel Cancer Center of Thomas Jefferson University for access of equipment in initial protein crystal screening and initial in-house X-ray diffraction; staff at the Beamline facility (X6A) of the Brookhaven National labs for assistance with collection of X-ray diffraction data. The use of the National Synchrotron Light Source, Brookhaven National Laboratory, was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, under contract no. DE-AC02-98CH10886. This work was partly supported by grants 13SDG16150007 from the American Heart Association to M.Z., S10RR027411 from the US National Institutes of Health (NIH) to M.C., HL059949 and HL090882 from NIH to D.E.L. and R01MH073060 and R01NS39355 from NIH to J.F.Z.

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Authors and Affiliations

  1. Department of Molecular Physiology and Biophysics, Thomas Jefferson University, Philadelphia, Pennsylvania, USA

    Miao Zhang & Ji-Fang Zhang

  2. Department of Physiology and Biophysics, Virginia Commonwealth University School of Medicine, Richmond, Virginia, USA

    Miao Zhang, Xuan-Yu Meng, Meng Cui & Diomedes E Logothetis

  3. Department of Biochemistry & Molecular Biology, Thomas Jefferson University, Philadelphia, Pennsylvania, USA

    John M Pascal

  4. Farber Institute for Neurosciences, Thomas Jefferson University, Philadelphia, Pennsylvania, USA

    Ji-Fang Zhang

  5. Graduate Program in Neuroscience, Thomas Jefferson University, Philadelphia, Pennsylvania, USA

    Ji-Fang Zhang

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  1. Miao Zhang
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Contributions

M.Z., D.E.L. and J.-F.Z. designed the experiments. M.Z. performed electrophysiology and biochemistry experiments and carried them out. X.-Y.M. and M.C. performed the docking and MD simulations. M.Z., J.-F.Z. and J.M.P. performed experiments of X-ray crystallography. M.Z. and J.-F.Z. analyzed the data, made figures and wrote the first draft of the manuscript. All of the authors participated in revising the first draft into its final form.

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Correspondence to Diomedes E Logothetis or Ji-Fang Zhang.

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Zhang, M., Meng, XY., Cui, M. et al. Selective phosphorylation modulates the PIP2 sensitivity of the CaM–SK channel complex. Nat Chem Biol 10, 753–759 (2014). https://doi.org/10.1038/nchembio.1592

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