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Abstract

The kinetics of N⁺-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 microM in the presence of 100 mM extracellular Na⁺ elicits an inward current that rises with a time constant tau₁ < 50 microseconds and decays to a plateau with a time constant tau₂ = 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 tau₁ < 50 microseconds and decays with tau₂ = 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 Ca(²⁺)-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.