Transmembrane transport of fluoride studied by time-resolved emission spectroscopy

Here we present a new method to monitor fluoride transmembrane transport into liposomes using a europium(iii) complex. We take advantage of the long emission lifetime of this probe to measure the transport activity of a fluorescent transporter. The high sensitivity, selectivity, and versatility of the assay allowed us to study different types of fluoride transporters and unravel their mechanisms of action.

1. Comparison of the EuL1 assay and the ISE assay for the study of transmembrane transport of fluoride Table S1 Comparison of the assay presented in this work with the commonly used ISE assay [1][2][3][4] Criteria Definition Fluoride Selective Electrode The EuL1 assay presented here Sensitivity Fluoride gradient used 300 mM 3 mM

Versatility and Selectivity
Transport mechanisms studied Funiport F -/Clantiport F -/NO3antiport Funiport Versatility Study of highly lipophilic transporters, such as compound 3 No (only postinsertion) Yes (post-insertion and pre-incorporation) Mechanism Possibility to distinguish apparent and real transport of fluoride and to compare uniport and antiport mechanisms

S5
The hydrated lipid films were sonicated for ca. 30 s and stirred for 1 hour at room temperature to create a heterogeneous mixture of vesicles. The suspension was subjected to 10 freeze-thawing cycles to generate unilamellar vesicles, diluted to 1 mL with the same salt solution previously employed for the hydration of the lipid film (without the fluorescent probe) and extruded 29 times through a polycarbonate membrane (200 nm pore size) at room temperature. The large unilamellar vesicles (LUVs) suspension was eluted through a size exclusion column (Sephadex G-25) using the external salt solution as eluent to remove the external fluorescent probe. The resulting suspension was further diluted with the same salt solution to obtain the desired final lipids concentration of 0.4 mM (for the EuL1 assay) or 0.1 mM (for the HPTS assay) assuming no loss of lipids. Figure S1. Schematic representation of the conditions used to monitor the transport of fluoride using the EuL1 assay (a-d) and the transport of chloride (e) and hydroxide (f) using the HPTS assay.

DLS Analysis of the Large unilamellar Vesicles
DLS data from 4 different batches of LUVs (prepared as described in Section 2a) are plotted below. An average of 180 nm was found for the hydrodynamic diameter of the LUVs, as is common for liposomes after extrusion through a polycarbonate membrane with 200 nm pores. KCl and K + Gluconate -. After 600 seconds, a detergent (Triton X-100, 50 μL of 5% w/w in H2O) was added to lyse the liposomes. The normalised transport curves were obtained using the equation: Where Et is the emission intensity at time t, E0 is the emission intensity at t = 0, and Ef is the final emission intensity after the addition of the detergent.  The HF diffusion is related to the concentration of buffer used. When 10 mM HEPES (or higher) is used, complete equilibration of fluoride concentrations is reached. The use of 5 mM HEPES decreases the HF influx, but a significant initial increase of emission is still observed before reaching a plateau, due to the build-up of a pH gradient that stops the further HF diffusion into the liposomes. Further lowering the concentration of HEPES to 0.5 mM minimises the HF diffusion across the membrane in absence of transporters, while still allowing to control the pH of the buffer solution at the start of the experiments. For these reasons, a HEPES concentration of 0.5 mM was used for all experiments with the EuL1 assay.

Pulse concentration
The response of the probe [Eu.L 1 ] + upon increasing concentrations of fluoride is based on the strong coordination of this anion to the Eu 3+ cation. To avoid saturation of the probe (and thus a reduced sensitivity to changes in the fluoride concentration), but still ensuring a clear response of the [Eu.L 1 ] + probe, an experiment was performed to optimise the concentration of the NaF pulse added to the liposome suspension. Liposomes were prepared as described in Section 2a and transporter 2 was postinserted in the lipidic membrane of the liposomes via the addition of 5 µL of a 24 µM stock solution of 2 in methanol (to reach 0.01 mol%). Consecutive NaF pulses were added at intervals of circa 90 seconds to ensure completion of transport. Addition of 3 mM NaF results in a clear response while avoiding saturation, which is reached after addition of ~20 mM NaF. Given the results obtained, 3 mM Fwas used as pulse for all the experiments employing the EuL1 assay.

Probe response
In order to have an idea of the rate of equilibration of the emission intensity of the [Eu.L 1 ] + probe (without encapsulation in liposomes) upon addition of fluoride, a KF pulse (3 mM) was added to a solution containing the probe (50 nM in KCl 225 mM, HEPES 0.5 mM, pH 7). As shown in Figure S5, the increase of emission was imminent, and equilibration was reached within 5 seconds. This timescale of a few seconds is more likely to reflect the time it takes to fully mix the solutions in the cuvette than the time it takes for the probe to bind the fluoride anion. The observed time for equilibration in this experiment is shorter than the equilibration time in any of the transport experiments, where a datapoint is recorded every 8 seconds. Origin 2019b was used to fit the transport curves (average of three experiments, normalised), considering the data measured between 30 seconds (addition of NaF pulse) and 530 seconds (after which the data were removed).
To determine the half-life (t½), the normalised transport curves were fitted to a single exponential function (YldFert1): The half-life t½ was then calculated from fit parameter k using the equation: To obtain the initial rates of transport (I), the normalised curves were fitted to a double exponential function: Initial values for the fit parameters were used as follows: y = 1, a = 0.8, b = 0.05, c = 0.2, d = 0.01. The initial rate was calculated from fitted parameters a, b, c, and d using the equation: = +

S12
3.4 EC50 calculation in the EuL1 assay to monitor F -/Clantiport The transport curves for transporters 1 and 2 were measured at different concentrations. The EC50 values were determined via a Hill plot analysis of the emission intensity at 630 s (after 600 s of transport) plotted against the compound concentration. An EC50 of 0.009 mol% was found for 1 and 0.000014 mol% for 2. Figure S6. Hill plot analysis of the transport activity of compound 1 in the EuL1 assay to monitor F − /Cl − antiport upon addition of NaF (3 mM, after 30 s) and prior to lysis (after 630 s). The transporter was added as solution in methanol 3 minutes before the start of the measurement and concentrations are shown as transporter to lipids molar percentage. Error bars represent standard deviations from three experiments.

S13
3.5 EuL1 assay to monitor F -/NO3antiport In order to study the ability of the transporters to work as F -/NO3antiporters, the LUVs suspension was prepared as in Section 2c (NaNO3 is used instead of NaCl for preparation of the buffer) and the transport measurements were performed as described in Section 3.1.
The results in Figure S8 show activity for all the anion transporters. Although the conditions used encourage F -/NO3antiport, the observed response can be due to NO3 − /OH − antiport in combination with HF diffusion (as discussed in the main manuscript for the study of F − /Cl − antiport). This is demonstrated by the activity observed for monensin, which can be attributed to H + /Na + antiport combined with HF diffusion. In order to assess the effect of different cations on the fluoride transport activity of compounds 1-3, the LUVs suspension was prepared as in Section 2b (KCl was used instead of NaCl for the preparation of the buffer) and the transport measurements were performed as described in Section 3.1.
The results in Figure S9 show that the HF diffusion response in absence of a transport is higher in KCl compared to NaCl, but the trends observed for transporters 1-3 and the corresponding half-lives are similar (66 s -1 for 1, 13 s -1 for 2 and 103 s -1 for 3 in KCl) to the values obtained in NaCl (Section 3.3, Table S2). Thus, we concluded that the effect of the counter cation can be considered negligible.

EuL1 assay to monitor Funiport
In these F − uniport experiments, the readily transportable anions Cl − (or NO3 − ) are replaced by gluconate, whose high polarity impedes its transport. Furthermore, Na + is replaced by K + , which can be efficiently transported across the membrane by the cationophore valinomycin (see Section 2d). The data are shown in Figure S10. Transporter 3 (0.1 mol%) was active when tested in combination with valinomycin (0.02 mol%) and mild activity was recorded for 1 (0.1 mol%), while 2 was not able to perform F − uniport at the concentration (0.001 mol%) that gave clear transport in NaCl and NaNO3 solutions. Although the conditions studied aimed at observing F − uniport, a potential mechanism that could cause fluoride intake into the liposomes is OH − influx (or H + efflux) in combination with HF diffusion. This was proven using protonophore carbonyl cyanide 3-chlorophenylhydrazone (CCCP, 0.1 mol%), 8 which in combination with valinomycin showed good transport activity. For this reason, transporters 1, 2 and 3 were tested in a modified HPTS assay for monitoring OHuniport (refer to Section 5.6), but none of the compounds showed any significant activity. This suggests that the activity observed for 3 and 1 in Figure S10b is caused by actual Funiport.

Lifetime of compound 1 incorporated in liposomes
The excited-state lifetime of transporter 1 was studied using a LP920-K spectrometer from Edinburgh Instruments. The excitation source was a tunable Nd:YAG Laser NT342 Series from EKSPLA. The third harmonic (355 nm) was attenuated to 10 mJ/pulse and was used to excite the sample. Luminescence lifetime was measured using a PMT-900 detected with a minimum detector response width of 5 ns. The instrument response function (IRF) was used to correct the measured excited-state lifetime. An average of 30 laser pulses were used to record excited-state lifetimes. Origin was used to plot and fit the obtained data and to calculate the lifetime as described in Section 4.1.   In order to understand the effect of the addition of NaF on the internal pH of the vesicles during a typical experiment using the EuL1 assay to monitor the transport of fluoride, LUVs were prepared as in Section 2a, but HPTS was encapsulated instead of [Eu.L 1 ] + . Thus, the final system was composed of liposomes containing 1 mM HPTS and suspended in 225 mM NaCl, buffered at pH 7 using 0.5 mM HEPES and subjected to a 3 mM NaF pulse ( Figure S14). In absence of transporters, a fast decrease of pH was observed (from 7.20 to 6.75) upon addition of NaF. This acidification is caused by the diffusion of HF into the vesicles, leading to the build-up of a pH gradient. This gradient disappears upon lysis. In presence of transporter 2 (0.001 mol%) after a slight acidification caused by initial HF diffusion into the liposomes, rapid equilibration of pH was observed, caused by the efficient Cl − /OH − antiport activity of the transporter (see Sections 5.3 and 5.4). Vesicles with transporters 1 and 3 (0.1 mol%) also show initial acidification, followed by a pH equilibration slower than by 2. In the case of 3, which cannot work as Cl − /OH − antiporter (see Figure S16), the equilibration of pH is most likely caused by F−/Cl− antiport. In this case, after initial pH build-up caused by fast HF diffusion into the liposomes, the F − /Cl − antiport by 3 can induce a reverse HF diffusion (from inside to outside) to dissipate the pH gradient.

S20
To calibrate the response of intravesicular HPTS to pH, monensin (0.1 mol%) was added to 3 mL of a solution of vesicles containing 1 mM HPTS, 225 mM NaCl, 0.5 mM HEPES and buffered at pH 7. The pH of the sample was modified by adding HCl or NaOH and monitored using a pH-meter. The sample was stabilized (stirring, 25°C). Then the excitation spectrum was recorded using an Agilent Cary Eclipse fluorescence spectrometer (em: 511 nm, slits 5 nm, 5 nm).

General procedure for transport measurements with HPTS
Liposomes were prepared as described in Sections 2e and 2f. Quartz cuvettes containing 3.00 mL of the LUVs suspension with dye HPTS (1 mM ex: 403 nm and 455 nm, em: 511 nm) encapsulated were placed in the sample compartment of an Agilent Cary Eclipse fluorescence spectrometer equipped with a xenon flash lamp, a magnetic stirrer, and a temperature controller. Three transport experiments were run in parallel. If transporters 1 and 2, monensin or carbonyl cyanide 3-chlorophenylhydrazone (CCCP, serving as protonophore) were used, these were added in 5 µL of their stock solutions in methanol to reach the desired transporter to lipids ratio and the suspension was stirred for 3 minutes to stabilize the sample temperature to 25 ˚C. For the study of OH − /Cl − antiport and Cl − uniport, while the sample was stirring at 25 ˚C, a base pulse of NMDG (5 mM) was added to the sample to generate a pH gradient between the inside (pH 6.8) and the outside (pH 7.8) of the vesicles as prepared as in Section 2e. For the study of OH − uniport, the base pulse consisted of the addition of tetrabutylammonium hydroxide (TBAOH, 5 mM) to the vesicles prepared as in Section 2f. The rate of the pH gradient dissipation was monitored by recording the ratio between the fluorescence emissions of the protonated (excitation at 403 nm) and deprotonated (excitation at 455 nm) forms of HPTS (F455/F403). After 200 seconds, a detergent (Triton X-100, 50 μL of 5% w/w in H2O) was added to lyse the liposomes and dissipate the pH gradient. The ratio of the fluorescence intensities was normalised using the following equation: Where Rt is the fluorescence ratio at time t, R0 is the fluorescence ratio at t = 0, and Rf is the final fluorescence ratio after the addition of the detergent.

HPTS assay to monitor OH -/Clantiport and Cluniport
To test the ability of the transporters to work as OH -/Clantiporters, the HPTS assay was employed. LUV suspensions were prepared as described in Section 2e and results are shown in Figure S16.
In the absence of CCCP, the increase of F455/F403 is caused by the ability of the transporter to perform OH − /Cl − antiport. While transporter 1 showed mild transport activity when tested at 0.1 mol%, 2 was able to work as OH − /Cl − antiporter at very low concentration (0.0001 mol%, see also Figure S17). On the other hand, 3 did not show activity when tested at 0.1 mol%, as a result of its inability to work as OH − /Cl − antiporter.
In combination with protonophore CCCP (0.1 mol%), a slight increase of transport activity was recorded for 1, as a result of a slight selectivity for Cl − over OH − . The combination of 2 and CCCP did not trigger any difference on the rate of transport, indicating that the transport of OH − is not rate limiting. In contrast, when combined with CCCP, transporter 3 shows outstanding activity and it is clearly active as Cl − uniporter. Figure S16. Dissipation of a pH gradient mediated by transporters 1 (a), 2 (b) and 3 (c) monitored via the HPTS assay to monitor OH -/Clantiport (in absence of CCCP) and Cluniport (in presence of CCCP) upon addition of NMDG (5 mM, after 30 s) and prior to lysis (after 230 s). Liposomes prepared as in Section 2e. Transporters 1 and 2 and CCCP were added as solution in methanol 3 minutes before the start of the measurement. Transporter 3 was pre-incorporated in the membrane during the preparation of the liposomes. The transporters concentrations are shown as transporter to lipids molar percentage. Error bars represent standard deviations from three experiments.
The EC50 value for transporter 2 as OH − /Cl − antiporter was determined via a Hill plot analysis, performed with the transporter post-inserted at different concentrations and the normalised fluorescence ratio at 230 s (after 200 s of transport) plotted against the compound concentration ( Figure S17). An EC50 value of 0.00015 mol% was obtained.

Comparison of the EuL1 and HPTS assays
The similar conditions used in the EuL1 assay for the study of F − /Cl − antiport and in the HPTS assay for the study of OH − /Cl − antiport enabled the direct comparison of results from the two assays via plotting the normalised transport curves against time (230 s) together. This comparison is presented in Figure 4 of the main text and Figure  S18 shows the activity of 2 at 0.001 mol%, showing similar curves in both assays. However, as the curves from both assays reach equilibration rather fast at this concentration of 2, a lower concentration is used in Figure 4b, allowing to better assess the relative contributions of the different transport mechanisms to the response in the EuL1 assay.

HPTS assay to monitor OHuniport
In order to test the ability of the transporters to work as OH − uniporters, the HPTS assay was employed. LUV suspensions were prepared as described in Section 2f. This experiment was a crucial control experiment related to the ability of transporters 1 and 3 to work as F − uniporters discussed in Section 3.7. While the experiments are performed in the same way as described for the study of OH − /Cl − antiport (Section 5.3), some modifications are applied to the system used. The NMDGH + Cl − is replaced by sodium gluconate and NMDG is replaced by TBAOH. The free diffusion of the tetrabutylammonium cation (TBA + ) across the lipidic membrane balances the transport of OH -, while gluconate anions are too polar to diffuse or to be transported through the membrane. 9 As a result, the only mechanism that can generate a response in these conditions is the uniport of OH -(or H + ) mediated by the transporter. Figure S19 shows no significant transport by any of the compounds. This lack of activity in OHuniport observed for 1 and 3, proved that the response obtained in the EuL1 assay was caused by Funiport. On the other hand, compound 2 was unable to work as neither OHnor Funiporter at the concentration tested. Figure S19. Dissipation of a pH gradient mediated by transporters 1, 2 and 3 monitored via HPTS assay to monitor OHuniport upon addition of TBAOH (5 mM, after 30 s) and prior to lysis (after 230 s). Liposomes were prepared as in Section 2f. Transporters 1 and 2 were added as solution in methanol 3 minutes before the start of the measurement. Transporter 3 was pre-incorporated in the membrane during the preparation of the liposomes. The LUVs were lysed after 200 seconds from the addition of TBAOH. The transporter concentrations are shown as transporter to lipids molar percentage. Error bars represent standard deviations from three experiments.
A. Cataldo et al. Transmembrane Transport of Fluoride Studied by Time-Resolved Emission Spectroscopy. S26 6. Application of the time-resolved EuL1 assay for the study of bicarbonate transport The EuL1 assay can also be used for the study of HCO3transport, as described in reference 10. In this previous report, a regular fluorescence spectrometer with continuous light source was used. However, the time-resolved measurements as described for the study of Ftransport in the current work, can also be applied for HCO3transport studies. For instance, the concentration of prodigiosin in reference 10 was limited to 0.004 mol% (1:25000), because at higher concentrations the fluorescence of prodigiosin interfered with the emission of the [Eu.L 1 ] + . Thus, time-resolved measurements were applied to monitor the transport of HCO3by prodigiosin at higher concentration (0.04 mol%) and the results are given in Figure S20. These results show that a 10-fold higher concentration of prodigiosin (0.04 mol% instead of 0.004 mol%) does not lead to any faster increase of the HCO3concentration inside the liposomes. This indicates that CO2 diffusion is rate-limiting in the transport process and that prodigiosin transports HCO3via a combination of HCl transport and CO2 diffusion rather than HCO3 -/Clantiport, 10,11 even at a concentration of 0.04 mol%.