Kinetics of Chlorotetracycline Permeation in Fragmented, ATPase-Rich Sarcoplasmic Reticulum

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Abstract
Conditions are defined under which the fluorescent chelate probe chlorotetracycline (CTC) may be quantitatively used as a kinetic indicator of CA2+ transport by the membranes of the ATPase-rich fraction of sarcoplasmic reticulum (SR) isolated from rabbit skeletal muscle. The fluorescence enhancement accompanying Ca2+ transport results from binding of Ca-CTC complexes to the inside surface, a process requiring transport of CTC across the membrane. Slow permeation of the dye results in a time course of fluorescence mat is slower than the transport reaction. When the probe was added after attainment of maximally-accumulated Ca2+ the rate of fluorescence rise (t1/2 ≈ 30 sec) was on the same order as the rate when added before Ca2+ uptake had begun (t1/2 ≈ 35 sec). In both cases, the rate of the fluorescence was limited by the rate of transport of the probe. However, when A23187 was added to the SR following active Ca2+ accumulation in the presence of CTC, decreases in fluorescence with 1/2 values less than 3 sec were observed. CTC, therefore, does not need to exit the vesicle to show a decline in fluorescence on Ca2+ release. Experiments in which CTC influx and fluorescence increase were observed during the process of Ca2 + efflux showed that Ca2 + and CTC are not directly coupled in their respective translocations. The primary determinant of CTC influx and its final internal level is the internal Ca2 + level rather than the Ca2+ flux. The dependence of the rate of pH and divalent cations shows that CTC crosses membrane as the uncomplexed, neutral species and that its transport kinetics is first order. When CTC was added following active Ca2+ uptake at pH 5.8, the tlPz for dye transport was 5 sec compared to 30 sec and 26 sec for pH 6.8 and 7.8, respectively. CTC flux, defined as fluorescence amplitude/t, 2, at pH 6.8 was found to be increasingly retarded in the presence of increasing concentrations of added Ca2+ or Mg2+ so that the flux was proportional to the concentration of uncomplexed probe. The model developed for CTC trasnport predicts that (1) fluorescence halftime is independent of the total dye concentration but inversely proportional to the membrane concentration; (2) fluorescence amplitude is directly proportional to total dye concentration but bears a hyperbolic dependence on both the membrane concentration and the steady state level of free internal Ca%; (3) CTC flux is directly proportional to total dye concentration, the membrane concentration, and the internal Ca2+ concentration. These predictions were verified by experiment. The first-order rate constant for CTC permeation was calculated to be lop2 sec- I, and the ratio of membrane bound to free Ca-CTC complex within the vesicle was calculated to be about 600. The model predicts that after complete equilibration is achieved, the CTC fluorescence will show a hyperbolic saturation function of the internal Caw concentration. Two methods are discussed for the determination of the internal free Ca2+ concentration achieved by active transport using the CTC fluorescent response.