Pre-steady state electrogenic events of Ca2+/H+ exchange and transport by the Ca2+-ATPase

Native or recombinant SERCA (sarco(endo)plasmic reticulum Ca2+ ATPase) was adsorbed on a solid supported membrane and then activated with Ca2+ and ATP concentration jumps through rapid solution exchange. The resulting electrogenic events were recorded as electrical currents flowing along the external circuit. Current transients were observed following Ca2+ jumps in the absence of ATP and following ATP jumps in the presence of Ca2+. The related charge movements are attributed to Ca2+ reaching its binding sites in the ground state of the enzyme (E-1) and to its vectorial release from the enzyme phosphorylated by ATP (E2P). The Ca2+ concentration and pH dependence as well as the time frames of the observed current transients are consistent with equilibrium and pre-steady state biochemical measurements of sequential steps within a single enzymatic cycle. Numerical integration of the current transients recorded at various pH values reveal partial charge compensation by H+ in exchange for Ca2+ at acidic (but not at alkaline) pH. Most interestingly, charge movements induced by Ca2+ and ATP vary over different pH ranges, as the protonation probability of residues involved in Ca2+/H+ exchange is lower in the E-1 than in the E2P state. Our single cycle measurements demonstrate that this difference contributes directly to the reduction of Ca2+ affinity produced by ATP utilization and results in the countertransport of two Ca2+ and two H+ within each ATPase cycle at pH 7.0. The effects of site-directed mutations indicate that Glu-771 and Asp-800, within the Ca2+ binding domain, are involved in the observed Ca2+/H+ exchange.