Intracellular Ca2+ levels modulate a multitude of vital cellular processes—including gene expression, cell viability, cell proliferation, cell motility, cell shape and volume regulation—thereby playing a key role in regulating cell responses to external signals. These dynamic changes in Ca2+ levels are regulated by ligand-gated and G-protein–coupled ion channels in the plasma membrane and by mobilization of Ca2+ from intracellular stores. The generation of cytosolic Ca2+ spikes and oscillations typically involves the coordinated release and uptake of Ca2+ from these stores, mediated by intracellular Ca2+ channels and their response to several second messengers such as Ca2+ itself, cyclic ADP ribose and inositol triphosphate.ref

This section includes several Molecular Probes reagents for studying Ca2+ regulation in live cells. Fluorescent nucleotides, including analogs of ATP, ADP, AMP, GTP and GDP, are described in Probes for Protein Kinases, Protein Phosphatases and Nucleotide-Binding Proteins—Section 17.3. Our GTP analogs may be particularly useful in the assay of G-protein–coupled receptors. Probes for Lipid Metabolism and Signaling—Section 17.4 discusses several selective phosopholipase substrates, as well as labeled ceramide and sphingomyelin probes.

Inositol Triphosphate Pathway

D-myo-1,4,5-Inositol Triphosphate and Caged D-myo-1,4,5-Inositol Triphosphate

We offer the potassium salt of D-myo-inositol 1,4,5-triphosphate (Ins 1,4,5-P3, I3716) for researchers investigating inositol triphosphate–dependent Ca2+ mobilization and signal transduction mechanisms.ref Cytoplasmic Ins 1,4,5-P3 is a potent intracellular second messenger that induces Ca2+ release from membrane-bound stores in many tissues. Our Ins 1,4,5-P3 is at least 99% pure, as determined by paper chromatography and by 1H NMR and 31P NMR.

NPE-caged Ins 1,4,5-P3 can be used to generate rapid and precisely controlled release of Ins 1,4,5-P3 in intact cells and is widely employed in studies of Ins 1,4,5-P3–mediated second messenger pathways.ref Our NPE-caged Ins 1,4,5-P3 (I23580) is a mixture of the physiologically inert, singly esterified P4 and P5 esters and does not contain the somewhat physiologically active P1 ester. NPE-caged Ins 1,4,5-P3 exhibits essentially no biological activity prior to photolytic release of the biologically active Ins 1,4,5-P3.

Fluorescent Heparin

Fluorescein-labeled heparin (H7482) is a useful tool for studying binding of this mucopolysaccharide in cells and tissues.ref In addition to its well-known anticoagulant activity,ref heparin binds to the Ins 1,4,5-P3 receptor and inhibits the biological cascade of events mediated by Ins 1,4,5-P3.ref Heparin also binds to thrombin ref and Alzheimer's tau protein,ref as well as to blood vessel–associated proteins such as laminin and fibronectin.ref Fluorescence polarization assays using fluorescein-labeled heparin as a tracer provide quantitative assessments of these binding interactions.ref Fluorescein-labeled heparin has also been used to assess the efficacy of transdermal delivery of heparin by pulsed current iontophoresis as a potential alternative to conventional subcutaneous injections.ref

Caged Ca2+ and Caged Ca2+ Chelators

Caged ions and caged chelators can be used to influence the ionic composition of both solutions and cells, particularly for ions such as Ca2+ that are present at low concentrations. The properties and uses of caged probes are described in Photoactivatable Reagents, Including Photoreactive Crosslinkers and Caged Probes—Section 5.3.

NP-EGTA: A Caged Ca2+ Reagent

Developed by Ellis-Davies and Kaplan, the photolabile chelator o-nitrophenyl EGTA (NP-EGTA) exhibits a high selectivity for Ca2+, a dramatic 12,500-fold decrease in affinity for Ca2+ upon UV illumination (its Kd increases from 80 nM to >1 mM) and a high photochemical quantum yield ref (~0.2). Furthermore, with a Kd for Mg2+ of 9 mM, NP-caged EGTA does not perturb physiological levels of Mg2+. We offer both the potassium salt (N6802) and the acetoxymethyl (AM) ester (N6803) of NP-EGTA. The NP-EGTA salt can be complexed with Ca2+ to generate a caged calcium complex that will rapidly deliver Ca2+ upon photolysis (Figure 17.2.1). The cell-permeant AM ester of NP-EGTA does not bind Ca2+ unless the AM esters are removed. It can potentially serve as a photolabile buffer in cells because, once converted to NP-EGTA by intracellular esterases, it will bind Ca2+ with high affinity until photolyzed with UV light. NP-EGTA has been used to measure the calcium buffering capacity of cells.ref


Figure 17.2.1 NP-EGTA (N6802) complexed with Ca2+. Upon illumination, this complex is cleaved to yield free Ca2+ and two iminodiacetic acid photoproducts. The affinity of the photoproducts for Ca2+ is ~12,500-fold lower than that of NP-EGTA.

DMNP-EDTA: A Caged Ca2+ Reagent

The first caged Ca2+ reagent described by Ellis-Davies and Kaplan was 1-(4,5-dimethoxy-2-nitrophenyl) EDTA (DMNP-EDTA, D6814), which they named DM-Nitrophen ref (now a trademark of Calbiochem-Novabiochem Corp.). Because its structure better resembles that of EDTA than EGTA, we named it as a caged EDTA derivative (Figure 17.2.2). Upon illumination, DMNP-EDTA's Kd for Ca2+ increases from 5 nM to 3 mM. Thus, photolysis of DMNP-EDTA complexed with Ca2+ results in a pulse of free Ca2+. Furthermore, DMNP-EDTA has significantly higher affinity for Mg2+ (Kd = 2.5 µM) than does NP-EGTA ref (Kd = 9 mM). The photolysis product's Kd for Mg2+ is ~3 mM, making DMNP-EDTA an effective caged Mg2+ source, in addition to its applications for photolytic Ca2+ release.ref Photorelease of Ca2+ has been shown to occur in <180 microseconds, with even faster photorelease of Mg2+.ref Two reviews by Ellis-Davies discuss the uses and limitations of DMNP-EDTA.ref


Figure 17.2.2 DMNP-EDTA (D6814) complexed with Ca2+. Upon illumination, this complex is cleaved to yield free Ca2+ and two iminodiacetic acid photoproducts. The affinity of the photoproducts for Ca2+ is ~600,000-fold lower than that of DMNP-EDTA.

Diazo-2: A Photoactivatable Ca2+ Knockdown Reagent

In contrast to NP-EGTA and DMNP-EDTA, diazo-2 (D3034) is a photoactivatable Ca2+ scavenger. Diazo-2, which was introduced by Adams, Kao and Tsien,ref is a relatively weak chelator (Kd for Ca2+ = 2.2 µM). Following flash photolysis at ~360 nm, however, cytosolic free Ca2+ rapidly binds to the diazo-2 photolysis product, which has a high affinity for Ca2+ (Kd = 73 nM). Microinjecting a relatively low concentration of fluo-3, fluo-4, or one of the Calcium Green or Oregon Green 488 BAPTA indicators (Fluorescent Ca2+ Indicators Excited with Visible Light—Section 19.3), along with a known quantity of diazo-2, permits measurement of the extent of depletion of cytosolic Ca2+ following photolysis.ref Intracellular loading of NP-EGTA, DMNP-EDTA and diazo-2 is best accomplished by patch pipette infusion with the carboxylate salt form of the caged compound added to the internal pipette solution at 1–10 mM. These reagents are increasingly being applied in vivo for controlled intervention in calcium-regulated fundamental processes in neurobiology ref and developmental biology.ref


Other Probes for Calcium Regulation

Thapsigargin and Fluorescent Thapsigargin

Thapsigargin is a naturally occurring sesquiterpene lactone isolated from the umbelliferous plant Thapsia garganica.ref This tumor promoter releases Ca2+ from intracellular stores by specifically inhibiting the sarcoplasmic reticulum Ca2+-ATPase ref (SERCA); it does not directly affect plasma membrane Ca2+-ATPases, Ins 1,4,5-P3 production or protein kinase C activity.ref

Thapsigargin is available in 1 mg units (T7458) and specially packaged in 20 vials containing 50 µg each (T7459). We have also prepared the green-fluorescent BODIPY FL thapsigargin (B7487) and red-fluorescent BODIPY TR-X thapsigargin (B13800). BODIPY FL thapsigargin has proven useful for imaging the intracellular localization of thapsigargin during store-operated calcium entry (SOCE) ref and for imaging SERCA depletion in injured sensory neurons.ref

Luminescent Calcium Analog

The trivalent lanthanide terbium (III), which is supplied as its chloride salt (T1247), is a luminescent analog of Ca2+ that can be used to study structure–function relationships in Ca2+-binding proteins such as calmodulin, oncomodulin, lactalbumin and ATPases.ref The long-lived luminescence of Tb3+ has also been use to probe Ca2+-binding sites of alkaline phosphatase,ref glutamine synthetase,ref integrins,ref protein kinase C ref and ryanodine-sensitive Ca2+ channels.ref Tb3+ reportedly binds most strongly to the I and II sites of calmodulin.ref

Data Table

Cat #MWStorageSolubleAbsECEmSolventNotes
D3034710.86F,D,LLpH >636918,000nonepH 7.21, 3, 4
D6814473.39D,LLDMSO3484200nonepH 7.21, 4, 5
H7482~18,000FF,D,LH2O493ND514pH 86, 7
I3716648.64F,DH2O<250 none  
I23580872.82FF,D,LLH2O2644200noneH2O1, 2, 8
N6802653.81FF,D,LLpH >62603500nonepH 7.21, 2, 4, 9
N6803789.70FF,D,LLDMSO2504200noneMeCN10, 11
T1247373.38DH2O2704700545H2O12, 13
T7458650.76F,DDMSO, EtOH<300 none  
T7459650.76F,DDMSO, EtOH<300 none  
  1. All photoactivatable probes are sensitive to light. They should be protected from illumination except when photolysis is intended.
  2. This compound has weaker visible absorption at >300 nm but no discernible absorption peaks in this region.
  3. The Ca2+ dissociation constant of diazo-2 is 2200 nM before photolysis and 73 nM after ultraviolet photolysis. The absorption spectrum of the photolysis product is similar to that of BAPTA (B1204).ref
  4. Abs and EC values determined in Ca2+-free solution (100 mM KCl, 10 mM EGTA, 10 mM MOPS, pH 7.2).
  5. Kd(Ca2+) increases from 5 nM to 3 mM after ultraviolet photolysis. Kd values determined in 130 mM KCl, 10 mM HEPES, pH 7.1.ref
  6. ND = not determined.
  7. This product is a multiply labeled bioconjugate. The number of labels per conjugate is indicated on the vial.
  8. Ultraviolet photolysis of I23580 generates I3716.
  9. Kd (Ca2+) increases from 80 nM to 1 mM after ultraviolet photolysis. Kd values determined in 100 mM KCl, 40 mM HEPES, pH 7.2.ref
  10. This product is intrinsically a liquid or an oil at room temperature.
  11. N6803 is converted to N6802 via hydrolysis of its acetoxymethyl ester (AM) groups.
  12. Absorption and luminescence of T1247 are extremely weak unless it is chelated. Data are for dipicolinic acid (DPA) chelate. The luminescence spectrum has secondary peak at 490 nm.
  13. MW is for the hydrated form of this product.