2.4.2. Cation Transport Experiments

The fast filter method was used for data collection. 100 μL of stock vesicle solution was suspended in 1.85 mL of the corresponding bu ffer (10 mM PBS pH 6.4 containing 100 mM of the analyzed cation) and placed into a quartz fluorimetric cell. The emission of HPTS at 510 nm was monitored at two excitation wavelengths (403 and 460 nm) simultaneously. 0.464 mM lipid was used in each experiment. The final concentration of the compound is 0.06 mM for 12.9 mol% of compound/lipid ratio. This concentration is high enough for crown ethers to assemble as channels in lipid bilayers, assuming the insertion is complete. At 20 s, 29 μL of aqueous NaOH (0.5 M) was added, and macrocyclic compound added at 50 s, detergent was added after 340 s, and the measurement lasted another 50 s. For experiments involving FCCP, the compound was added at 40 s, and the tested macrocyclic compound was added at 60 s. The lysis started at 350 s for 50 s to finish the measurements this time. Experiments without FCCP (from 50 s to 340 s) are compared to experiments with FCCP (from 60 s to 350 s). The extent of transport was monitored using the ratio of the emission intensities of HPTS at 460 and 403 nm (I460 and I403). We calculated the first-order initial rate constant from the slopes of the plot of ln ([H+]in − [H+]out) versus time, where [H+]in and [H+]out are the intravesicular and the extravesicular proton concentrations, respectively. The [H+]out was assumed to remain constant during the experiment (pH 7.4), while [H+]in values were calculated for each point from HPTS emission intensity using the equation pH = 1.1684 × log(I460/I403) + 6.9807. The initial and final pH are pH at *t* = 0 s and at the end of the experiment, respectively. In the picture, I refer to the intensity at 460 nm, and I0 refers to intensity at 403 nm. To calculate EC50 and Hill coe fficient *n*, we used the fractional activity Y. Y was calculated for each curve using the normalized value of I460/I403 (just before lysis of the vesicles). We expressed Y as a function of time, and we performed fittings using a 2-parameter equation, which is, Hill equation: Y = Yblank + (Ymax − Yblank)/(1 + (EC50/[C])n), from which Yblank is the fractional activity from the experiment of blank and Ymax is the largest fractional activity from the same series of experiments.
