Rapid charge translocation by the cardiac Na+-Ca2+ exchanger after a Ca2+ concentration jump

The kinetics of Na+-Ca2+ exchange current after a cytoplasmic Ca2+ concentration jump (achieved by photolysis of DM-nitrophen) was measured in excised giant membrane patches from guinea pig or rat heart. Increasing the cytoplasmic Ca2+ concentration from 0.5 mu M to 100 mu M in the presence of 100 mM extracellular Na+ elicits an inward current that rises with a time constant tau(1) < 50 mu s and decays to a plateau with a time constant tau(2) = 0.65 +/- 0.18 ms (n = 101) at 21 degrees C, These current signals are suppressed by Ni2+ and dichlorobenzamil. No stationary current, but a transient inward current that rises with tau(1) < 50 mu s and decays with tau(2) = 0.28 +/- 0.06 ms (n = 53, T = 21 degrees C) is observed if the Ca2+ concentration jump is performed under conditions that promote Ca2+-Ca2+ exchange (i.e., no extracellular Na+, 5 mM extracellular Ca2+), The transient and stationary inward current is not observed in the absence of extracellular Ca2+ and Na+. The application of alpha-chymotrypsin reveals the influence of the cytoplasmic regulatory Ca2+ binding site on Ca2+-Ca2+ and forward Na+-Ca2+ exchange and shows that this site regulates both the transient and stationary current. The temperature dependence of the stationary current exhibits an activation energy of 70 kJ/mol for temperatures between 21 degrees C and 38 degrees C, and 138 kJ/mol between 10 degrees C and 21 degrees C. For the decay time constant an activation energy of 70 kJ/mol is observed in the Na+-Ca2+ and the Ca2+-Ca2+ exchange mode between 13 degrees C and 35 degrees C, The data indicate that partial reactions of the Na+-Ca2+ exchanger associated with Ca2+ binding and translocation are very fast at 35 degrees C, with relaxation time constants of about 6700 s(-1) in the forward Na+-Ca2+ exchange and about 12,500 s(-1) in the Ca2+-Ca2+ exchange mode and that net negative charge is moved during Ca2+ translocation, According to model calculations, the turnover number, however, has to be at least 2-4 times smaller than the decay rate of the transient current, and Na+ inward translocation appears to be slower than Ca2+ outward movement.