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

Abstract

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 at1/2 of 40–60 msec and (ii) a slow process with a large amplitude and at1/2 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 apparentt1/2 of 20 msec. The slow phase brings about an increase of the internal Ca2+ concentration to 4 mM within its apparentt1/2 of 90 sec. The two phases have average rates of increase of internal free Ca2+ concentration, Δ [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.