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Cell-permeant caged InsP3 ester shows that Ca2+ spike frequency can optimize gene expression

Nature volume 392, pages 936–941 (1998)Cite this article

Abstract

Inositol 1,4,5-trisphosphate (InsP3) releases calcium from intracellular stores and triggers complex waves and oscillations in levels of cytosolic free calcium1,2,3,4,5,. To determine which longer-term responses are controlled by oscillations in InsP3 and cytosolic free calcium, it would be useful to deliver exogenous InsP3, under spatial and temporal control, into populations of unpermeabilized cells. Here we report the 15-step synthesis of a membrane-permeant, caged InsP3 derivative from myo-inositol. This derivative diffused into intact cells and was hydrolysed to produce a caged, metabolically stable InsP3 derivative. This latter derivative accumulated in the cytosol at concentrations of hundreds of micromolar, without activating the InsP3 receptor. Ultraviolet illumination uncaged an InsP3 analogue nearly as potent as real InsP3, and generated spikes of cytosolic free calcium, and stimulated gene expression via the nuclear factor of activated T cells6,7. The same total amount of InsP3 analogue elicited much more gene expression when released by repetitive flashes at 1-minute intervals than when released at 0.5- or ⩾2-minute intervals, as a single pulse, or as a slow sustained plateau. Thus, oscillations in cytosolic free calcium levels at roughly physiological rates maximize gene expression for a given amount of InsP3.

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Figure 1: Mode of action of the caged, membrane-permeant InsP3 derivative cmInsP3/PM (cmIP3/PM) (compound 1), the trapped caged molecule (cmInsP3 (cmIP3), compound 2), and 2,3-methoxymethylene-InsP3 (mInsP3, compound 3) released by photolysis.
Figure 2: Synthesis and biochemical characterization of cmInsP3/PM and its reaction products.
Figure 3: Cytosolic calcium transients evoked by photolysis of cmInsP3 in 1321N1 astrocytoma cells.
Figure 4: Expression of the β-lactamase reporter gene induced by uncaged cmInsP3/PM.
Figure 5: [Ca2+]c levels measured by Fluo-3 fluorescence and NF-AT-driven β-lactamase gene expression evoked by different temporal patterns of photolysing the same total amount of cmInsP3 in RBL cells.

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Acknowledgements

We thank E. A. Dennis for loan of facilities and S. R. Adams, A. Miyawaki, T. J. Rink and P. A. Negulescu for advice. This work was funded by the NIH and HHMI.

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Authors and Affiliations

  1. Departments of Pharmacology, University of California, San Diego, 9500 Gilman Drive, La Jolla, 92093-0647, California, USA

    Wen-hong Li, Juan Llopis & Roger Y. Tsien

  2. Department of Chemistry & Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, 92093-0647, California, USA

    Wen-hong Li & Roger Y. Tsien

  3. Department of Howard Hughes Medical Institute, University of California, San Diego, 9500 Gilman Drive, La Jolla, 92093-0647, California, USA

    Roger Y. Tsien

  4. Aurora Biosciences Corporation, 11010 Torreyana Road, San Diego, 92121, California, USA

    Michael Whitney & Gregor Zlokarnik

  5. Division of Biology 139-74, Beckman Institute, California Institute of Technology, Pasadena, 91125, California, USA

    Wen-hong Li

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Li, Wh., Llopis, J., Whitney, M. et al. Cell-permeant caged InsP3 ester shows that Ca2+ spike frequency can optimize gene expression. Nature 392, 936–941 (1998). https://doi.org/10.1038/31965

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