To identify the mechanism by which RCOO -anions diffuse into liposomes without an anion transporter, we conducted experiments using liposomes with different salt solutions. For these experiments, benzoate and 4-hydroxybenzoate were used, because they also displayed spontaneous diffusion across the lipid membrane, are present in their anionic form >99.5% at pH 7 (Figure ), and they quench lucigenin more effectively than the aliphatic carboxylates. Accordingly, two liposome solutions with 0.8 mM lucigenin in 225 mM NaNO3 were tested. The first solution contained 5 mM HEPES at pH 7, while the second had no buffer. We observed the slow diffusion of carboxylates through the lipid membrane in both samples (Figures and). This was the case even in the non-buffered liposome solutions, where the global pH became basic after adding the anion pulse (pH 8.1-8.5). It should be noted that pH values ≤ 8.5 do not significantly affect the fluorescence of lucigenin. Subsequently, NaNO3 was replaced by Na2SO4, and a new liposome solution with 0.8 mM lucigenin in 112.5 mM Na2SO4 was prepared. Under these conditions, benzoate and 4-hydroxybenzoate addition gave pH values of 8.2-8.4. After a rapid small drop in fluorescence intensity, which was larger for 4-hydroxybenzoate than for benzoate, the intensity remained nearly constant thereafter, indicating no diffusion (Figure ). To obtain further insight into these results, the experiments were repeated with lucigenin replaced by 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium (HPTS). HPTS is a pH-sensitive dye which can be used to monitor the internal pH inside vesicles by monitoring its ratiometric fluorescence intensity (λex = 403 and 455 nm, λem = 510 nm). Accordingly, liposome solutions were prepared with 0.1 mM HPTS in 112.5 mM Na2SO4, 5 mM HEPES, pH 7 or 225 mM NaNO3, 5 mM HEPES, pH 7. Once again, benzoate and 4-hydroxybenzoate were added to the liposomes. The HPTS experiments showed that the carboxylate pulse instantly increased the acidity inside liposomes, after which the pH remained constant over time, both in liposomes suspended in NaNO3 or Na2SO4 (Figure and). However, this pH decrease was significantly more pronounced for the samples in the NaNO3 solution. As no variations in pH were observed outside liposomes upon addition of the carboxylates, a pH gradient is induced. These experiments thus suggest that the carboxylates diffuse across the lipid membrane along with protons, as carboxylic acids. Combining the results of the lucigenin and HPTS experiments, we hypothesise that carboxylates cross the lipid membrane through a three-step mechanism. Step 1: The formation of small amounts of carboxylic acid, which diffuse freely across the lipid membrane. Step 2: The deprotonation of the carboxylic acid inside the liposomes, leading to the formation of free carboxylate and a drop of the pH, which increases the fraction of protonated nitrate (HNO3). Step 3: The efflux of HNO3 to decrease the pH gradient, followed by its rapid dissociation to recover the NO3 -anion (Figure ). This process allows the slow carboxylic acid diffusion to continue over time. In contrast, only the first steps can take place in Na2SO4 solution, as HSO4 -cannot cross the membrane spontaneously. Consequently, the potential gradient cannot be dissipated, limiting the entry of RCOOH molecules (Figure ). In order to demonstrate the diffusion of HNO3 across liposomal membranes at neutral pH, we prepared liposomes with internal solutions of 0.1 mM HPTS in 225 mM NaNO3, 5 mM HEPES at pH 7, and an external solution of in 112.5 mM Na2SO4, 5 mM HEPES at pH 7. We then monitored the sample by following the excitation band at 450 nm over time. The 450 nm band displayed a slight increase in intensity, which was attributed to the basification of the liposome interior due to HNO3 efflux (Figure and Figure ). Conversely, liposomes with an internal solution of 0.1 mM HPTS in 112.5 mM Na2SO4, 5 mM HEPES at pH 7, with an external solution of 225 mM NaNO3, 5 mM HEPES at pH 7 showed acidification of the liposome interior, in this case attributed to HNO3 influx (Figure and Figure ). To discard any potential buffer effect of the NaNO3 and Na2SO4 salts themselves, aliquots of 1 M HCl were added to both salt solutions and similar pH values were measured in both solutions (Table ).