Ca2+ concentrations

Fig. 4.1.5 The time course of aequorin luminescence measured with various concentrations of Ca2+. Calcium acetate solution (5 ml) was added to 10 pi of aequorin solution to give the final Ca2+ concentrations of 10 2 M (A), 10-4 M (B), 10-5 M (C), 10 6 M (D), and 10 7 M (E) at 25°C. The dashed line (F) represents the light emitted following the addition of deionized distilled water that had been redistilled in quartz. The concentration of EDTA derived from the aequorin sample was 10 7 M (final cone.). From Shimomura et al., 1963b, with permission from John Wiley Sons Ltd.

The interaction between aequorin and a chelator must be carefully considered when estimating Ca2+ concentrations with aequorin in a calcium buffer containing EDTA or EGTA. This is particularly crucial when using a common calcium buffer system that contains a constant total concentration of a chelator in the buffer solutions of various Ca2+ concentrations in such a buffer system, a buffer of lower Ca2+ concentration contains a higher concentration of the free form of the chelator, resulting in an increased inhibition. 

Relationship between Ca2+ concentration and luminescence intensity. In the measurement of Ca2+ concentration with aequorin, the calibration of the relationship between Ca2+ concentration and luminescence intensity is essential. However, the application of this relationship is complicated by the chelator used to set the Ca2+ concentration, for the reason noted above. To minimize the complication, we used only a minimum amount of EDTA to protect aequorin in the measurements to obtain the relationship between Ca2+ -concentration and light intensity, and plotted the data as shown in Fig. 4.1.7 (Shimomura and Johnson, 1976). The concentration of EDTA was... 

Fig. 4.1.8 Influence of various calcium chelators on the relationship between Ca2 " concentration and the luminescence intensity of aequorin, at 23-25°C (panel A) in low-ionic strength buffers (I < 0.005) and (panel B) with 150 mM KC1 added. Buffer solutions (3 ml) of various Ca2+ concentrations, pH 7.05, made with or without a calcium buffer was added to 2 pi of 10 pM aequorin solution containing 10 pM EDTA. The calcium buffer was composed of the free form of a chelator (1 or 2mM) and various concentrations of the Ca2+-chelator (1 1) complex to set the Ca2+ concentrations (the concentration of free chelator was constant at all Ca2+ concentrations). The curves shown are obtained with 1 mM MOPS (A), 1 mM gly-cylglycine ( + ), 1 mM citrate (o), 1 mM EDTA plus 2mM MOPS ( ), 1 mM EGTA plus 2 mM MOPS ( ), 2 mM NTA plus 2 mM MOPS (V), and 2 mM ADA plus 2 mM MOPS (A). In the chelator-free buffers, MOPS and glycylglycine, Ca2+ concentrations were set by the concentration of calcium acetate. Reproduced with permission, from Shimomura and Shimomura, 1984. the Biochemical Society.

Fig. 4.1.14 Relationship between Ca2+ concentration and the initial light intensity of various recombinant semisynthetic aequorins and w-aequorin J (a semisynthetic natural aequorin made from isoform J). The curve number corresponds to the number of semisynthetic aequorin used in Table 4.1.4. A sample aequorin (3 (Ag) was in 3 ml of calcium-buffer solution containing 1 mM total EGTA, 100 mM KC1,1 mM Mg2+ and 1 mM MOPS (pH 7.0), at 23-24°C. From Shimomura etal., 1993a, with permission from Elsevier.

The second procedure is to measure the luminescence intensities at various Ca2+ concentrations and plot log (light intensity) against —log [Ca2+] for each aequorin. Examples of this method are shown in Fig. 4.1.14. This method provides more detailed information on the sensitivity of each aequorin. Generally, an increase in Ca2+ sensitivity shifts the curve to the left. 

Short (typically lasting no more than a few seconds) increases in cytosolic Ca2+ concentration that periodically interrupts the stable resting level. Many Ca2+ signals are delivered to cells as frequency-coded Ca2+ spikes. 

A typically prolonged transient increase in intracellular Ca2+ concentration, which is detected by a Ca2+ indicator. The increased Ca2+ levels are due to Ca2+... 

DMD and BMD DMD and BMD are caused by the absence or deficiency of dystrophin a membrane-associated protein, resulting in increased Ca2+ concentration in muscle, loss of Ca2+ homeostasis, and inappropriate calpain activity36... 

The plasma membrane Na+/Ca2+ exchanger is a high-capacity and low affinity ionic transporter that exchanges three Na+ ions for one Ca2+ ion. When intracellular Ca2+ concentrations [Ca2+]i rise and the... 

This subfamily including the large- (BKCa), intermediate-(IKca), and small-conductance (SKCa) Ca2+-activated K + channels are activated by increases in intracellular free Ca2+ concentration. The opening of DCca and SKCa channels are less voltage-dependent, whereas the activation of BKCa channel has steep voltage sensitivity. 

Dantrolene is an antidote for MH, which inhibits the CICR activity. Action of dantrolene may be isoform specific (less effective on RyR2), Ca2+-dependent (more effective at a lower Ca2+ concentration), and temperature-dependent (more effective at 37°C than at 25°C). 

S100A1 is the most abundant in the myocardium but is also expressed in brain and other tissues. S100A1 was found to stimulate Ca2+-induced Ca2+-release (CICR) in skeletal muscle terminal cisternae. In the presence of nanomolar Ca2+-concentrations, S100A1 binds to the ryanodine receptor increasing its channel open probability, and was shown to enhance SR Ca2+-release and contractile performance. Several animal models (over expressing S100A1 or S100A1-deficient mice) have... 

Figure 10 illustrates the precipitation boundary for a commercial CI2 LAS. The boundary divides the LAS /Ca2+ concentration ranges into regions of clear... 

GEN Kass, SK Duddy, GA Moore, S Orrenius. (1992). 2,5-Di(ferf-butyl)-l,4-benzohydroquinone rapidly elevates cytosolic Ca2+ concentration by mobilizing the inositol 1,4,5-trisphosphate-sensitive Ca2+ pool. J Biol Chem 264 15192-15198. 

As the Ca2+ concentration is lowered, the rate of the uncatalyzed hydrolysis decreases more rapidly than the catalyzed process, producing an increase in the rate acceleration ratio. For example, as the ratio of Ca2+ to cycloheptaamylose is decreased from 1 1 to 0.33 1, the rate acceleration for the hydrolysis of diphenyl pyrophosphate increases from 4.4 to 27. At lower Ca2+ concentrations, catalysis by the cycloamyloses was described as absolute catalysis since the rate of the spontaneous hydrolysis could not be measured. The term absolute catalysis is misleading, however, since the uncatalyzed rate, no matter how slow, must be finite. 

---