使用基于水母发光蛋白的探针测定哺乳动物细胞中的Ca2+

Anna Tosatto; Rosario Rizzuto; Cristina  Mammucari

doi:10.21769/BioProtoc.2155

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Peer-reviewed

Ca²⁺ Measurements in Mammalian Cells with Aequorin-based Probes

使用基于水母发光蛋白的探针测定哺乳动物细胞中的Ca2+

AT Anna Tosatto

RR Rosario Rizzuto email

CM Cristina Mammucari email

发布: 2017年03月05日第7卷第5期 DOI: 10.21769/BioProtoc.2155 浏览次数: 7881

评审: Nicoletta CordaniPia GiovannelliHsin-Yi Chang

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Cover of EMBO Molecular Medicine, featuring study using the protocol.

May 2016

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Abstract

Aequorin is a Ca²⁺ sensitive photoprotein suitable to measure intracellular Ca²⁺ transients in mammalian cells. Thanks to recombinant cDNAs expression, aequorin can be specifically targeted to various subcellular compartments, thus allowing an accurate measurement of Ca²⁺ uptake and release of different intracellular organelles. Here, we describe how to use this probe to measure cytosolic Ca²⁺ levels and mitochondrial Ca²⁺ uptake in mammalian cells.

Keywords: Ca²⁺ (Ca²⁺)

Aequorin (水母发光蛋白)

Probes (探针)

Luminescence (发光)

Background

Aequorin is a 21 kDa photoprotein isolated from jellyfish Aequorea victoria that emits blue light in the presence of Ca²⁺. In its active form the photoprotein includes an apoprotein and a covalently bound prosthetic group, called coelenterazine. The apoprotein contains four helix-loop-helix ‘EF-hand’ domains, three of which are Ca²⁺-binding sites. These domains confer to the protein a particular globular structure forming the hydrophobic core cavity that accommodates the coelenterazine. When Ca²⁺ ions bind to the three high affinity EF-hand sites, coelenterazine is irreversibly oxidized to coelenteramide, with a concomitant release of CO₂ and emission of light (Head et al., 2000).

Aequorin began to be widely used when the cDNA encoding the photoprotein was cloned, thus opening the way to recombinant expression. In particular, recombinant aequorin can be expressed not only in the cytoplasm, but also in single intracellular compartments by including specific targeting sequences in the engineered cDNAs (Hartl et al., 1989). To expand the range of Ca²⁺ sensitivity that can be monitored, point mutations in the EF-hand motives that lower the affinity for Ca²⁺ have been introduced (Granatiero et al., 2014a and 2014b). Reconstitution of an active recombinant aequorin in living cells is obtained by simple addition of coelenterazine into the medium. Coelenterazine is highly hydrophobic and permeates cell membranes of various cell types. Different coelenterazine analogues have been synthetized and are now commercially available.

Materials and Reagents

Round glass coverslips 12 mm (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 1014355112NR15 ) - sterile upon autoclave cycle
24-well plate (Corning, Costar^®, catalog number: 3524 )
MDA-MB-231 cell line
MDA-MB-468 cell line
BT-549 cell line
Aequorin-expressing plasmids (Brini, 2008)
Gelatin
Collagen
Coelenterazine 0.5 mM stock solution, in methanol (IS Chemical Technology, catalog number: I14-2266 ) - aliquot in 50 μl aliquots. Store at -80 °C. Save it from light
Agonist (e.g., ATP, Histamine, Bradykinin, Caffeine, Carbachol, Glutamate)
Note: In this protocol example 100 µM ATP is used.
Milli-Q water
Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S9888 )
Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P5405 )
Magnesium chloride hexahydrate (MgCl₂·6H₂O) (Sigma-Aldrich, catalog number: M2670 )
Potassium dihydrogen phosphate (KH₂PO₄) (Sigma-Aldrich, catalog number: NIST200B )
Magnesium sulfate heptahydrate (MgSO₄·7H₂O) (Sigma-Aldrich, catalog number: M2773 )
HEPES (Sigma-Aldrich, catalog number: H3375 )
Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: 71687 )
Calcium chloride (CaCl₂) (Sigma-Aldrich, catalog number: 449709 )
Glucose (Sigma-Aldrich, catalog number: G8270 )
Digitonin (Sigma-Aldrich, catalog number: D5628 or D141 )
Krebs-Ringer modified buffer (KRB) (see Recipes)
Digitonin lysis solution (see Recipes)

Equipment

Windows-based computer
Perfusion chamber (Elettrofor)
Low noise photomultiplier (Hamamatsu Photonics K. K., model: H7360-01)
Peristaltic pump (Gilson’s MINIPULS^® 3)
Water bath (temperature-controlled)
Photon-counting unit (Hamamatsu Photonics K. K., model: C8855-01 )

Note: Equipment is assembled as depicted in Figure 1.

Figure 1. Equipment. A. Fully equipped aequorinometer is composed of: (1) computer, (2) perfusion chamber, (3) photomultiplier, (4) peristaltic pump, (5) water bath, (6) photon-counting unit. B. During the experiment, the perfusion chamber is placed in close proximity to the photomultiplier, protected from light.

Procedure

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Tosatto, A., Rizzuto, R. and Mammucari, C. (2017). Ca²⁺ Measurements in Mammalian Cells with Aequorin-based Probes. Bio-protocol 7(5): e2155. DOI: 10.21769/BioProtoc.2155.