Cell-permeant caged lnsP_3 ester shows that Ca~(2+) spike frequency can optimize gene expression

摘要

Inositol 1,4,5-trisphosphate (InsP_3) releases calcium from intra-cellular stores and triggers complex waves and oscillations in levels of cytosolic free calcium. To determine which longer-term responses are controlled by oscillations in InsP_3 and cytosolic free calcium, it would be useful to deliver exogenous InsP_3, under spatial and temporal control, into populations of unper-meabilized cells. Here we report the 15-step synthesis of a membrane-permeant, caged InsP_3 derivative from myo-inositol. This derivative diffused into intact cells and was hydrolysed to produce a caged, metabolically stable InsP_3 derivative. This latter derivative accumulated in the cytosol at concentrations of hundreds of micromolar, without activating the InsP_3 receptor. Ultraviolet illumination uncaged an InsP_3 analogue nearly as potent as real InsP_3, and generated spikes of cytosolic free calcium, and stimulated gene expression via the nuclear factor of activated T cells. The same total amount of InsP_3 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 InsP_3.