Measuring [Ca2+] in the endoplasmic reticulum with aequorin

Abstract

SummaryThe photoprotein aequorin was the first probe used to measure specifically the [Ca 2+ ] inside the lumen of the endoplasmic reticulum ([Ca 2+ ] ER ) of intact cells and it provides values for the steady-state [Ca 2+ ] ER , around 500 M, that closely match those obtained now by other procedures. Aequorin-based methods to measure [Ca 2+ ] ER offer several advantages: (i) targeting of the probe is extremely precise; (ii) the use of low Ca 2+ -affinity aequorin allows covering a large dynamic range of [Ca 2+ ], from 10 −5 to 10 −3 M; (iii) aequorin is nearly insensitive to changes in Mg 2+ or pH, has a high signal-to-noise ratio and calibration of the results in [Ca 2+ ] is made straightforward using a simple algorithm; and (iv) the equipment required for luminescence measurements in cell populations is simple and low-cost. On the negative side, this technique has also some disadvantages: (i) the relatively low amount of emitted light makes difficult performing single-cell imaging studies; (ii) reconstitution of aequorin with coelenterazine requires previous complete depletion of Ca 2+ of the ER for 1-2 h, a maneuver that may result in deleterious effects in some cells; (iii) because of the high rate of aequorin consumption at steady-state [Ca 2+ ] ER , only relatively brief experiments can be performed; and (iv) expression of ER-targeted aequorin requires previous transfection or infection to introduce the appropriate DNA construct, or alternatively the use of stable cell clones. Choosing aequorin or other techniques to measure [Ca 2+ ] ER will depend of the correct balance between these properties in a particular problem. © 2002 Elsevier Science Ltd. All rights reserved. HISTORICAL BACKGROUNDAlthough the ER has been known to be the main intracellular Ca 2+ store for nearly 20 years, monitoring the free [Ca 2+ ] in the ER ([Ca 2+ ] ER ) has proven to be a difficult task. The first studies using low-affinity fluorescent indicators required cell permeabilization to release the cytosolic dye and could not avoid compartmentalization of the dye into other organelles [1][2][3]. Apart of that, selectivity of these probes over Mg 2+ is poor and calibration was highly uncertain. On the other side, use of aequorin to measure [Ca 2+ ] ER has required solving two difficult technical problems: getting specific targeting of the probe into the ER and modifying the Ca 2+ -affinity of the probe to reach the adequate [Ca 2+ ] range.

Peroxisomes as Novel Players in Cell Calcium Homeostasis

“…As previously reported (22), the addition of targeting motifs to the C terminus of aequorin could modify its stability as well as its Ca 2ϩ -dependent luminescence emission. Accordingly, the light emission from lysates of CHO cells expressing the peroxAEQs was measured upon reconstitution of the probes in the presence of various Ca 2ϩ -buffered solutions at neutral pH.…”

Bcl-2-mediated alterations in endoplasmic reticulum Ca 2+ analyzed with an improved genetically encoded fluorescent sensor

“…Importantly, these characteristics enable the monitoring of Ca 2ϩ oscillations within the ER. Additionally, the cameleon is not consumed by Ca 2ϩ as aequorin is, and therefore the instantaneous signal depends only on Ca 2ϩ and not the entire past history of Ca 2ϩ in the cell (1 oscillations are controlled by a host of factors, including the level of Ca 2ϩ in the ER and capacitative calcium entry, both of which are altered in Bcl-2-overexpressing cells (9,11,26). However, our data show that, in addition to altering the resting level and leak rate of [Ca 2ϩ ] ER , Bcl-2 overexpression can also modify fundamental Ca 2ϩ signaling processes such as the nature of IP 3 -induced Ca 2ϩ oscillations.…”

“…T o date, researchers have attempted to use a number of strategies to deduce the role of [Ca 2ϩ ] ER in signaling processes, but all possess shortcomings (1,2). Conventional methods use cytosolic small-molecule Ca 2ϩ indicators to infer that an observed signal likely originated from the endoplasmic reticulum (ER).…”

The Endoplasmic Reticulum of Dorsal Root Ganglion Neurons Contains Functional TRPV1 Channels

“…Aequorin photoluminescence was measured as described previously (32), and calibrations in [Ca 2ϩ ] were done using the constant values published before (32,33). All of the measurements were performed at 22°C.…”