Calcium Regulation—Section 17.2

Page Contents

• Inositol Triphosphate Pathway
  
• Caged Ca²⁺ and Caged Ca²⁺ Chelators
  
• Other Probes for Calcium Regulation
• Data Table
• Ordering Information

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.

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.

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. 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. 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. In addition to its well-known anticoagulant activity, heparin binds to the Ins 1,4,5-P₃ receptor and inhibits the biological cascade of events mediated by Ins 1,4,5-P₃. Heparin also binds to thrombin and Alzheimer's tau protein, as well as to blood vessel–associated proteins such as laminin and fibronectin. Fluorescence polarization assays using fluorescein-labeled heparin as a tracer provide quantitative assessments of these binding interactions. 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.

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 K_d increases from 80 nM to >1 mM) and a high photochemical quantum yield (~0.2). Furthermore, with a K_d 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.

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 (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 K_d 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⁺ (K_d = 2.5 µM) than does NP-EGTA (K_d = 9 mM). The photolysis product's K_d for Mg²⁺ is ~3 mM, making DMNP-EDTA an effective caged Mg²⁺ source, in addition to its applications for photolytic Ca²⁺ release. Photorelease of Ca²⁺ has been shown to occur in <180 microseconds, with even faster photorelease of Mg²⁺. Two reviews by Ellis-Davies discuss the uses and limitations of DMNP-EDTA.

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, is a relatively weak chelator (K_d 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⁺ (K_d = 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. 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 and developmental biology.

Thapsigargin and Fluorescent Thapsigargin

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

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) and for imaging SERCA depletion in injured sensory neurons.

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. The long-lived luminescence of Tb³⁺ has also been use to probe Ca²⁺-binding sites of alkaline phosphatase, glutamine synthetase, integrins, protein kinase C and ryanodine-sensitive Ca²⁺ channels. Tb³⁺ reportedly binds most strongly to the I and II sites of calmodulin.