Rapid kinetic study of the passive permeability of a Ca2+-ATPase rich fraction of the sarcoplasmic reticulum

Summary A method was developed for the study of divalent cation transport events on the time scale of 20 msec or longer. Passive Ca²⁺ equilibration across the membranes of the Ca²⁺-ATPase rich fraction of sarcoplasmic reticulum (SR) was studied. The method makes use of the divalent cation sensitivity of the surface binding of the fluorescent probe 1-anilino-8-naphthalenesulfonate (ANS^–). Binding to the inside and outside surfaces is distinguished in fluorescent stopped-flow experiments. The surface binding reactions of the probe are faster than the time resolution of the instrument (ca. 3 msec), while binding reactions requiring transport across the membrane could be resolved. In Ca²⁺ influx experiments, the time course of fluorescent enhancement was monitored following a Ca²⁺ jump. The kinetics of Ca²⁺ efflux were studied by pre-equilibrating Ca²⁺ across the membrane, removing the external Ca²⁺ with an EGTA jump, and observing the time course of the fluorescence decrease. Rapid transport of ANS^– (coupled to K⁺) was ensured by the addition of valinomycin. Two processes of Ca²⁺ influx were observed: (i) a rapid process with small fluorescent amplitudes and at _1/2 of 40–60 msec and (ii) a slow process with a large amplitude and at _1/2 of 70–100 sec. The rates and extents of the two phases were quantitated in terms of the rates and extents of change in the Ca²⁺ concentration in the SR lumen. The slow phase accounted for a larger change, in the internal free Ca²⁺ concentration than did the first phase. For the influx of 10 mM Ca²⁺, the rapid phase raises the internal Ca²⁺ concentration to ca. 1 mM within its apparentt _1/2 of 20 msec. The slow phase brings about an increase of the internal Ca²⁺ concentration to 4 mM within its apparentt _1/2 of 90 sec. The two phases have average rates of increase of internal free Ca²⁺ concentration, Ca] _i /sec of ca. 50 mM/sec and ca. 0.02 mM/sec, respectively. The Ca²⁺ influx rates increased with increasing KCl concentration and with increasing external Ca²⁺ concentration.Two phases of Ca²⁺ efflux were observed. The amplitudes and rates were analyzed and the fast phase was shown to account for more Ca²⁺ movement than the slow phase. The rate of the fast phase was greatly increased by increasing the K⁺ concentration. The rate of the slow phase efflux decreased with increasing Ca²⁺ concentration in the external medium. The concentration for half-maximal inhibition was 4 M, a value close to the dissociation constant of the high affinity site on the Ca²⁺-ATPase.The above constitutes a body of circumstantial evidence that the passive Ca²⁺ permeability observed is mediated by the Ca²⁺-ATPase, acting as a Ca²⁺ for 2 K⁺ exchanger. The fast phases are explained as a partial turnover of the pump in the steady state. The slow rate is explained by a preference of the ion binding translocator site of the carrier for an outward orientation.The ANS^– technique was applied to the monovalent cation permeability of the Ca²⁺-ATPase rich SR and the results of other studies were corroborated and extended. The interaction of valinomycin with intrinsic permeability mechanisms of the SR was considered.