Potential-sensitive response mechanism of diS-C₃-(5) in biological membranes

Summary

The potential-sensitive response mechanism of 3,3′-dipropylthiodicarbocyanine iodide (diS-C₃-(5)) was examined based on our previous model of diS-C₃-(5) interaction with brush border membrane vesicles (BBMV) in the absence of a membrane potential. The model contained binding (6 msec), reorientation (30 msec), dimerization (<10 nsec), and translocation (1 sec) reaction steps (Cabrini & Verkman, 1986.J. Membrane Biol. 90:163–175). Transmembrane potentials (ψ) were induced in BBMV by K⁺ gradients and valinomycin. Steady-state diS-C₃-(5) fluorescence (excitation 622 nm, emission 670 nm) increased linearly with ψ. The reorientation and translocation reaction steps were resolved by the stopped-flow technique as a biexponential decrease in fluorescence following mixture of diS-C₃-(5) with BBMV at varying ψ. The fractional amplitude of the faster exponential increased from 0.36 to 0.73 with increasing ψ (−17 to 87 mV); the time constant for the faster exponential (30–35 msec) was independent of ψ. There were single exponential kinetics (0.5–1.5 sec) for diS-C₃-(5) fluorescence response to a rapid (<2 msec) change in ψ in the absence of a diS-C₃-(5) concentration gradient. These results, and similar findings in placental brush border vesicles, red cell vesicles, and phosphatidylcholine vesicles, support a translocation mechanism for diS-C₃-(5) response, where induced membrane potentials drive diS-C₃-(5) redistribution between sites at the inner and outer membrane leaflets, with secondary effects on diS-C₃-(5) dimerization and solution/membrane partitioning. Fluorescence lifetime and dynamic depolarization measurements showed no significant change in diS-C₃-(5) rotational characteristics or in the polarity of the diS-C₃-(5) environment with changes in ψ. Based on the experimental results, a mathematical model is developed to explain the quantitative changes in diS-C₃-(5) fluorescence which accompany changes in ψ at arbitrary dye/lipid ratios.