Manipulation of [Ca2

Calcium buffers and ionophores are frequently used to lower or raise intracellular or extracellular calcium. The purpose of these protocols is to study Ca2+-dependent cellular processes (Kao, 1994).

EGTA is highly selective for binding Ca2+ over Mg2+, and because of this it is the most commonly used Ca2+ buffer. However, the Ca2+-binding activity by EGTA is very pH-dependent when used at physiological pH. This is because at these pH values EGTA exists primarily as protonated species (H2EGTA2-) (highest pKas 8.90 and 9.52). Upon binding Ca2+, 2 H+ will be released, suggesting that this reaction should have very steep pH dependence. In fact, a drop in pH from 7.2 to 7.1 changes the Kd(Ca) of EGTA by a factor of approximately 1.6. Acidification of the medium by high levels of EGTA was proposed to be responsible for the postulated requirement of extracellular Ca2+ for invasion of host cells by tachyzoites (Lovett and Sibley, 2003).

Tsien developed an analog of EGTA in which the methylene links between oxygen and nitrogen atoms were replaced with benzene rings to yield a compound called BAPTA (Tsien, 1980). This compound has a considerable lower pH sensitivity than EGTA at physiological pH values, since its highest pKas are 5.47 and 6.36 (Tsien, 1980). Because of this, BAPTA is a less troublesome Ca2+ buffer to use - although it is significantly more costly than EGTA.

To study the correlation of a biological process with changes in intracellular calcium ([Ca2+]j), it is useful to be able to block the change in [Ca2+]j with a calcium chelator. The easiest way to introduce extra Ca2+ buffering capacity into cells is by loading them with BAPTA/AM, the ester form of BAPTA (Vieira and Moreno, 2000). Cells can be loaded with AM esters of BAPTA and a calcium indicator simultaneously, since the same conditions can be used (Kao, 1994). This BAPTA buffering method has been widely used to understand the role of calcium in microneme secretion by T. gondii (Carruthers and Sibley, 1999), conoid extrusion (Mondragon and Frixione, 1996), gliding motility (Wetzel et al., 2004) and invasion (Vieira and Moreno, 2000). It is important to load the cells with BAPTA analogs unable to chelate Ca2+, as a control that the effect of BAPTA is because of Ca2+ chelation and not because of toxicity. Analogs such as 'half-BAPTA' (Vieira and Moreno, 2000) or D-BAPTA (Saoudi et al., 2004) can be used, although half-BAPTA is not currently available. It is important to know that BAPTA can display side effects. It has a potent microtubule depolymerizing effect, and decreases the ATP pool of the cells (Saoudi et al., 2004). It is also important to provide controls showing that the concentrations of BAPTA-AM used are able to chelate intracellular Ca2+ (Vieira and Moreno, 2000).

The ionophores Br-A23187 and ionomycin form lipid-soluble complexes with divalent metal cations and increase the permeability of biological membranes to Ca2+. There are significant differences in the properties of both ionophores that should be considered when using them in an experiment. The ability of Ca2+ transport by both ionophores is pH-dependent, and this pH-dependence differs (Liu and Hermann, 1978). Transport of Ca2+ by Br-A23187 is best at pH 7.5, whereas Ca2+ transport by ionomycin does not reach a maximum until pH 9.5. In addition, iono-mycin has better selectivity for Ca2+ over Mg2+, whereas Br-A23187 shows no preference for one cation over the other (Liu and Hermann, 1978). Both ionophores are inefficient in mediating Ca2+ transport at low Ca2+ concentrations. Br-A23187 (or ionomycin) should be used instead of A23187 for fluorescence microscopy, since A23187 is fluorescent.

Figure 10.1 shows a typical tracing with fura-2-loaded T. gondii tachyzoites in suspension in a buffer containing 1 mM EGTA. Under these conditions, fluorescence changes reflect Ca2+ movements from intracellular calcium stores. The addition of

FIGURE 10.1 Effect of ionomycin and nigericin on [Ca2+]i of tachyzoites. Tachyzoites were loaded with fura2. Ionomycin (ION; 1pM) or nigericin (NIG; 1 pM) were added where indicated. Trace a shows the initial addition of ION followed by NIG. Trace b shows the initial addition of NIG followed by ION. Reproduced with permission from Moreno and Zhong (1996), Biochem. J. 313, 655-659.

FIGURE 10.1 Effect of ionomycin and nigericin on [Ca2+]i of tachyzoites. Tachyzoites were loaded with fura2. Ionomycin (ION; 1pM) or nigericin (NIG; 1 pM) were added where indicated. Trace a shows the initial addition of ION followed by NIG. Trace b shows the initial addition of NIG followed by ION. Reproduced with permission from Moreno and Zhong (1996), Biochem. J. 313, 655-659.

ionomycin shows a large increase in intracellular calcium, indicating that calcium is released from an intracellular compartment with neutral pH into the cytosol (endoplasmic reticulum). When nigericin is added alter ionomycin, a second increase in cytosolic Ca2+ occurs due to its release from an acidic compartment (acidocalcisomes). Similar results are observed if the order of additions is reversed (Figure 10.1). Nigericin is a potassium/proton exchanger, and because of this property it alkalin-izes acidic compartments by allowing protons to be released in exchange for potassium. As a consequence, a postulated proton/calcium exchanger takes protons back and releases Ca2+ into the cytosol.

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