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

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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.