Chelator Enhancement of Active Ca Transport

Much effort has been devoted to uncovering the biophysical mechanisms associated with energy-coupled active Ca transport. The issue presented here, which has received relatively little attention, concerns the mechanism(s) of physical delivery of calcium ions to their transport sites on the Ca-ATPase. Evidence suggests that if transport is below its maximum rate and free calcium is fixed, mobile Ca chelators (either native chelators such as calbindin and parvalbumin, or synthetic chelators such as EGTA) increase transport, both on intracellular- and plasma membrane-type calcium pumps (see, for example, Berman. 1982. J. Biol. Chem. 257:1953; Timmermans et al. 1995. J. Nutr. 125:1981S). These observations, however, oppose the theoretical expectation that Ca chelators should be unable to release calcium with enough speed to make much difference in the 'loading'' step of a 'Ca-starved' transport reaction. It must be concluded that we do not understand the mechanisms by which calcium transport sites become loaded or how chelators enhance a calcium loading/transport reaction. Such mechanisms, however. may be especially important for the transport activity of cells that must move large amounts of calcium, such as renal and intestinal epithelial cells and bone resorbing cells, or in certain types of myocytes and neurons that may need to transport calcium quickly into intracellular storage compartments. We have done experiments to examine the role of chelators in calcium transport in neurons. Microsomes isolated from rat brain accumulated [sup.45]Ca in a free (ionized) Ca concentration ([[Ca.sup.2+]])- and ATP-dependent manner. This accumulation was inhibited by thapsigargin, and was insensitive to mitochondrial inhibitors or to agents such as digitonin that selectively permeabilize plasma-membrane vesicles. Thus, these microsomes likely contain mainly endoplasmic reticular membranes. Adding the synthetic calcium chelator EGTA or the native Ca chelator parvalbumin increased the initial rate of uptake into the microsomes at a fixed [[Ca.sup.2+]]. The maximal uptake rate found in the presence of EGTA could also be achieved without chelator if [ [Ca.sup.2+]] was high enough. Spectrofluoremetric records of free [Ca.sup.2+] in microsome suspensions containing 1.5 [micro]M fura-2 also show that when 15 [micro]M EGTA is present, the microsomal Ca uptake rate is greater. A theoretical analysis suggests that chelator facilitation of calcium flux down a diffusion gradient to a 'Ca-starved' pump site is inadequate to explain these observations.