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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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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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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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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DOI: https://doi.org/10.1038/nchembio.1592
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