Figure 6. Fast kinetics of Cl⁻ release from the CHGB channels in vesicles.

(A) 300 mM KCl–loaded vesicles changed into 300 mM K-isethionate right before the assay. Time-lapsed light scattering was measured at 600 nm in a Fluoromax-4. Vesicles of different PLRs were prepared in the same batch. Adding DMSO triggered no signal, but adding 1.0 μM valinomycin did. (B) Time-lapsed light scattering (610 nm) measured in a stopped-flow system, by mixing equal volumes of vesicles and 2.0 μM valinomycin in buffer. 5% DMSO solution was added as negative control. Vesicles of PLR = 1:1,000 used to set a proper accelerating voltage for the PMT tube to have the broadest dynamic range. (C) Expanded region in the red box in panel A. (D) Expanded region in the red box in panel B. Data from the first 40 ms after the dead time of ∼2 ms for mixing are shown. (E) Average signals from the first 5-s data in (C) (black) and those from the first 40 ms in (D) (red) are plotted against [CHGB]. Fitting with Hill equations (solid lines) generated different Hill coefficients. Steady state data from four different datasets (n = 4; error bars are SD); Stopped-flow data from two sets of triplicate measurements (n = 6; error bars are SEM). (F) The data in panel E were plotted against the average number of CHGB monomers per 100-nm vesicle. The threshold for generating a significant signal is ∼4 monomers per vesicle for both measurements. (G) Titrating valinomycin for the light-scattering from CHGB vesicles in the stopped-flow system. The CHGB vesicles had PLR = 1:25,000. More vesicles than those in panel B were used to boost the signal, which also increased the baseline signal (buffer + DMSO). Three triplicate measurements were obtained (n = 9; error bars are SEM). (H) The normalized signal from panel G were plotted again [valinomycin] and fitted with a Hill equation to yield a Hill coefficient of ∼2.6.