Measuring [Ca2+] in the endoplasmic reticulum with aequorin.

摘要: The photoprotein aequorin was the first probe used to measure specifically [Ca(2+)] inside lumen of endoplasmic reticulum ([Ca(2+)](ER)) intact cells and it provides values for steady-state [Ca(2+)](ER), around 500 microM, that closely match those obtained now by other procedures. Aequorin-based methods [Ca(2+)](ER) offer several advantages: (i) targeting is extremely precise; (ii) use low Ca(2+)-affinity allows covering a large dynamic range [Ca(2+)], from 10(-5) 10(-3)M; (iii) nearly insensitive changes in Mg(2+) or pH, has high signal-to-noise ratio calibration results made straightforward using simple algorithm; (iv) equipment required luminescence measurements cell populations low-cost. On negative side, this technique also some disadvantages: relatively amount emitted light makes difficult performing single-cell imaging studies; reconstitution with coelenterazine requires previous complete depletion Ca(2+) ER 1-2h, maneuver may result deleterious effects cells; because rate consumption at only brief experiments can be performed; expression ER-targeted transfection infection introduce appropriate DNA construct, alternatively stable clones. Choosing techniques will depend correct balance between these properties particular problem.