| pubmed-article:10650997 | pubmed:abstractText | Whole-cell patch-clamp recordings were made to examine the effects of the Ca2+ chelators EGTA and BAPTA on the biophysical properties of voltage-operated Ca2+ currents in bovine adrenal chromaffin cells. Ca2+ currents in the presence of either EGTA or BAPTA over a concentration range of 0.1-60 mM were recorded under otherwise identical conditions. Analysis of current-voltage relationships yielded unexpected differences in several important parameters such as the voltage dependence of activation, kinetics, slope, and reversal potential, which seemed to be unrelated to the Ca(2+)-binding properties of these chelators. Increasing concentrations of BAPTA augmented the peak Ca2+ current amplitude while current amplitudes in the presence of EGTA remained constant over the entire concentration range tested. Increasing concentrations of BAPTA shifted the voltage sensitivity of Ca2+ currents by about 15 mV towards positive voltages. EGTA, over the same concentration range, did not affect the voltage sensitivity. The shift in voltage sensitivity observed with BAPTA was unrelated to its faster Ca92+)-binding kinetics, as it was also observed when substituting Ca2+ with Ba2+ as the charge carrier. The mechanism by which BAPTA affects Ca2+ channel voltage dependence also seems unrelated to kinase-mediated modulation of Ca2+ channels, since the protein-kinase-C- (PKC-) specific drugs bisindolylmaleimide and phorbol ester (PMA) neither mimicked nor prevented the action of BAPTA. The less specific kinase inhibitor staurosporine, however, augmented Ca2+ currents similarly to BAPTA, but without affecting the voltage sensitivity. The BAPTA-mediated shift in voltage sensitivity was partially suppressed by non-hydrolysable analogs of GTP (GDP[beta-S] and GTP[gamma-S]). Lowering [Mg2+]i mimicked the BAPTA-induced shift in voltage sensitivity and prevented further shifts in voltage sensitivity by BAPTA. The results demonstrate that BAPTA and EGTA, despite their similarities in terms of Ca2+ buffering, have disparate effects on the voltage dependence of Ca2+ channels and careful selection of the chelator is required to quantitatively assess Ca2+ currents. | lld:pubmed |
