Measuring anion transport selectivity: a cautionary tale

pH-dependent liposomal assays are often used to determine anion selectivity in transmembrane anion transport experiments. We discuss the validity and limitations of these assays, and provide guidelines for their use to avoid misleading results.


S1 General
POPC was purchased from Corden Pharma GmbH.We have deliberately used the batches of POPC that contained ~1 mol% of free fatty acid impurities, to saturate the fatty acid-dependent H + transport pathway. 1 Fluorescence measurements were performed using an Agilent Cary Eclipse Fluorescence Spectrophotometer equipped with a stirrer plate and a temperature controller.The syntheses of compounds 1 2 , 2 3 , 3 4 , and 4 5 have been reported previously.

S2 Theoretical analysis of the "anion gradient assay"
We here derive the relationship between the anion permeability ratio (P !!P "# !⁄ ) and the pH gradient (ΔpH) for the equilibrium condition of the "anion gradient" assay (Fig. 1c) under the approximation that anion exchange is negligible.

S3 ISE assay
An ISE assay was conducted using POPC vesicles (mean diameter 200 nm) loaded with NaCl (100 mM) and suspended in NaNO3 (100 mM), mimicking the conditions in HPTS assays.The vesicles were prepared as follows.A chloroform solution of POPC was evaporated in a round-bottom flask and the lipid film formed was dried under vacuum for at least 6 h.Then, the lipid film was hydrated by vortexing with an internal solution of NaCl (100 mM) buffered at pH 7.0 with HEPES (10 mM).The lipid suspension was subjected to nine freeze/thaw cycles and then extruded 25 times through a 200 nm polycarbonate membrane.The unentrapped NaCl was removing by a Sephadex G-25 column using an external solution of NaNO3 (100 mM) buffered at pH 7.0 with 10 mM HEPES as the eluent.A NaCl in /NaNO3 out vesicle stock suspension (with POPC concentration of ~10 mM) was then obtained.
For each test, the vesicle stock suspension was diluted using the external solution to obtain a 5 mL sample containing 1 mM of POPC.A DMSO solution (10 µL) of an anion transporter was added to the vesicle suspension to initiate the anion exchange process.Cl − efflux as appearance of Cl − in the external solution was monitored using a chloride ISE over 30 minutes.At the end of the experiment, the vesicles were lysed with detergent (50 μL of 11% (w%) Triton X-100 in 7 : 1 (v/v) H2O-DMSO) to release all Cl − and calibrate Cl − efflux to 100%.

S4.2 HPTS calibration
A calibration has been completed for HPTS assays conducted using NaCl in vesicles.The pH of the external NaCl (100 mM) solution with 10 mM HEPES was adjusted from 5.

S4.3 "Dual gradient" assay
Following vesicle preparation according to the general procedure, the NaCl-containing vesicles were diluted using NaX (100 mM) external solutions to obtain NaCl in /NaX out vesicles suspended in 2.

S4.4 "pH gradient" assay
Following vesicle preparation according to the general procedure, the NaX-containing vesicles were diluted using NaX (100 mM) external solutions to obtain NaX in /NaX out vesicles suspended in 2.

Eq. S6
where Rt is the fluorescence ratio at time t, R0 is the fluorescence ratio at time 0, and Rf is the final fluorescence ratio obtained by curve fitting to the single exponential decay function.Where necessary (e.g., for slow ClO4 -transport facilitated by transporter 4), the assay was run for up to 1 hour to ensure precise determination of the Rf value.
We have previously shown that the I460 / I403 value, instead of the pHin, is proportional to the amount of H + efflux. 7Therefore, here for the purpose of indicating the progress of membrane transport, the I460 / I403 values were not converted to pHin values.

S4.4.1 Comparison of HPTS and ISE assays
To mimic the conditions of the ISE assay, a POPC concentration of 1 mM was used here instead of 0.1 mM used in most HPTS experiments.
The data from the HPTS assay were overlayed with the ISE data (Fig. S1) to compare the rates of pH gradient dissipation and Cl -/NO3 -exchange, shown in Fig. S4 below.

S.4.6 "Competitive" assay
Following vesicle preparation according to the general procedure, the NaX-containing vesicles were diluted using NaY (100 mM) external solutions to obtain NaX in /NaY out vesicles suspended in 2.5 mL samples containing 0.1 mM of POPC.The samples were stirred at 298 K.
No base pulse was added so no pH gradient was initially present.An anion transporter (added in 5 μL of DMSO) was added at time 0 to induce pHin changes.The pHin was monitored by the ratiometric fluorescence response of HPTS I460 / I403 (λex = 460 nm, λem = 510 nm divided by λex = 403 nm, λem = 510 nm).
Transporter 4 has been previously tested under this assay 8 giving a selectivity sequence consistent with that obtained based on ΔpHmax comparison under NaCl in /NaX out "anion gradient" assay (Fig. 3d).
To further demonstrate the robustness of the "competitive" NaX in /NaY out assay, we used this assay to re-investigate the I -vs ClO4 -selectivity of transporters 1-3.Under NaClO4 in /NaI out condition (Fig. S9), transporter 1 induced a pHin increase whereas transporters 2 and 3 induced pHin decrease (with stronger responses than the DMSO baseline).The results indicate the ClO4 -> I -selectivity of transporter 1, and the opposite I -> ClO4 -selectivity of transporters 2 and 3, consistent with the ΔpHmax comparison under the NaCl in /NaX out "anion gradient" assay (Fig. 3).The NaI in /NaClO4 out condition, however, failed to generate a significant response for all transporters (Fig. S10).The cause for the lack of responses under this combination is currently unclear and is possibly related to complex interactions of the membrane with hydrophobic I -and ClO4 -ions 9 leading to transport activity inhibition.These results highlight the benefit of using both NaX in /NaY out and NaY in /NaX out conditions when comparing two anions X -and Y -. -2-oleoyl-sn-glycero-3-phosphocholine
Fig. S2Calibration for the HPTS assay using NaCl in vesicles.
5 mL samples containing 0.1 or 1.0 mM of POPC.The samples were stirred at 298 K.A base pulse of NaOH (25 μL of 0.5 M NaOH solution, final concentration 5 mM) was added to the vesicle samples to generate a pH gradient with pH 7.0 inside and pH 8.0 outside vesicles.Subsequently, an anion transporter (added in 5 μL of DMSO) was added at time 0 and the rate of the pH gradient dissipation was monitored by the ratiometric fluorescence response of HPTS I460 / I403 (λex = 460 nm, λem = 510 nm divided by λex = 403 nm, λem = 510 nm).The I460 / I403 data were converted to fractional fluorescence (If) values using the following equation:  ; = " % ." ." / ." .

Fig. S7 "
Fig.S7"pH gradient assay" for transporter 4 originally added at 0.0004 mol% under NaNO3 in /NaNO3 out (blue) and NaI in /NaI out (purple) conditions.After transporter addition, the vesicles were isolated, resuspended in NaCl, and stirred overnight to convert to the NaCl in /NaCl out condition.NaOH (5 mM) was added to initiate the membrane transport.The initial steep increase of HPTS fluorescence ratio due to response from membrane-bound or unentrapped HPTS was removed from the data.Initial conditions of kinetic study: In: NaCl (100 mM), HPTS (1 mM), HEPES (10 mM), pH 7.0; Out: NaCl (100 mM), HEPES (10 mM), pH 8.0.[POPC] = 0.1 mM.Error bars represent standard deviations from two runs.