Controlling transmembrane ion transport via photo-regulated carrier mobility

Stimuli-responsive transmembrane ion carriers allow for targeted and controllable transport activity, with potential applications as therapeutics for channelopathies and cancer, and in fundamental studies into ion transport phenomena. These applications require OFF–ON activation from a fully inactive state which does not exhibit background activity, but this remains challenging to achieve with synthetic transport systems. Here we introduce a novel mechanism for photo-gating mobile ion carriers, which involves modulating the mobility of the carriers within the lipid bilayer membrane. By appending a membrane-targeting anchor to the carrier using a photo-cleavable linker, the carrier's ion transport activity is fully switched off by suppressing its ability to shuttle between the two aqueous-membrane interfaces of the bilayer. The system can be reactivated rapidly in situ within the membrane by photo-triggered cleavage of the anchor to release the mobile ion carrier. This approach does not involve direct functionalization of the ion binding site of the carrier, and so does not require the de novo design of novel ion binding motifs to implement the photo-caging of activity. This work demonstrates that controlling the mobility of artificial transport systems enables precise control over activity, opening up new avenues for spatio-temporally targeted ionophores.


Materials and Methods
All reagents and solvents were purchased from commercial sources and used without further purification. Lipids were purchased from Avanti polar lipids and used without further purification. Where necessary, solvents were dried by passing through an MBraun MPSP-800 column and degassed with nitrogen. Column chromatography was carried out on Merck® silica gel 60 under a positive pressure of nitrogen. Where mixtures of solvents were used, ratios are reported by volume. TEA was distilled and stored over potassium hydroxide. NMR spectra were recorded on a Bruker AVIII 400, Bruker AVII 500 (with He cryoprobe) and Bruker AVIIIHD 500 spectrometers. Chemical shifts are reported as δ values in ppm. Mass spectra were carried out on an Agilent 6120 bench-top single quadrupole, a Waters LCT Premier XE benchtop (oa-TOF) and a Thermo Exactive High-Resolution Orbitrap FTMS spectrometer. Fluorescence spectroscopic data were recorded using a Horiba Duetta fluorescence spectrophotometer, equipped with Peltier temperature controller and stirrer. Experiments were conducted at 25 °C unless otherwise stated. Vesicles were prepared as described below using Avestin "LiposoFast" extruder apparatus, equipped with polycarbonate membranes with 200 nm pores. GPC purification of vesicles was carried out using GE Healthcare PD-10 desalting columns prepacked with Sephadex G-25 medium.

1 H NMR Titration Experiments
Anion binding constants were determined by 1 H NMR titrations using a Bruker AVIII 500 spectrometer at 500 MHz and 298 K. The initial sample volumes were 0.5 mL, at a concentration of 1 mM of host dissolved in acetone-d6 or 2.5% D2O-acetone-d6 (v/v). Known volumes of the anion guest, as the tetrabutylammonium (TBA) salt in the same solvent were added to the host and the spectra were recorded after each addition. The chemical shift perturbations of the host spectra were monitored as a function of guest concentration. The spectra were referenced to the residual solvent peak. The data was analysed using a global fit procedure using the Bindfit program, 9,10 using non-linear least squares analysis to obtain the best fit between observed and calculated chemical shifts for the 1:1 binding stoichiometry. The association of guest and host was fast on the NMR timescale.     Figure S36. Chloride binding isotherms for 2 in acetone-d6 (left) and in 2.5% D2O-acetone-d6 (right). Experimental data shown by symbols. In each case the binding affinity was too strong to be determined (>10 4 M -1 ).

Photocleavage Experiments
A 2 mM sample of each anchored carrier in DMSO was subjected to irradiation at 365 nm using an LED (irradiation intensity 1.3 W) and followed by 1 H NMR after increasing irradiation time intervals.    The conversion to the free carrier was determined as follows: t1 was calculated by fitting the data to an exponential decay function (equation S1) which was then used to derive a half-life of 1-A and 2-A using equation S2. The results are presented in Table S1.

Vesicle Preparation
A thin film of lipid (1-palmitoyl-2-oleoyl-sn-3-phosphatidylcholine (POPC) or dipalmitoyl phosphatidylcholine (DPPC)) was formed by evaporating a chloroform solution of the lipid under reduced pressure on a rotary evaporator (40 °C) and then under high vacuum for 6 hours. Then the lipid film was hydrated by vortexing with 1 mL of the prepared internal buffer solution. The lipid suspension was then subjected to 5 freeze-thaw cycles using liquid nitrogen and a water bath (40°C) followed by extrusion 19 times through a polycarbonate membrane (pore size 200 nm). Extrusion was performed at 50°C in the case of DPPC lipids. Extra-vesiclular components were removed by size exclusion chromatography on a Sephadex G-25 column with 100 mM NaCl, 10 mM HEPES, pH 7.0. Final conditions: LUVs (2.5 mM lipid); inside 100 mM NaCl, 10 mM HEPES, 1 mM HPTS, pH 7.0; outside: 100 mM NaCl, 10 mM HEPES, pH 7.0. Vesicles for the sodium gluconate assay were prepared by the same procedure, substituting NaCl for NaGluconate in the buffer solution The HPTS Assay 11,12 In a typical experiment, the LUVs containing HPTS (25 μL, final lipid concentration 31 μM) were added to buffer (1950 μL of 100 mM NaCl, 10 mM HEPES, pH 7.0) at 25°C under gentle stirring. A pulse of NaOH (20 μL, 0.5 M) was added at 40 secs to initiate the experiment. At 100 s the test transporter (various concentrations, typically in 5 μL DMSO) was added, followed by detergent (25 μL of Triton X-100 in 7:1 (v/v) H2O-DMSO) at 300 secs to lyse the vesicles for calibration. The fluorescence emission was monitored at λem = 510 nm (λex = 460/405 nm). For each compound, Hill plots were fitted to atleast 7, and up to 10 data points spanning the required concentration range, and each individual concentration was repeated at-least twice and averaged.
The fractional fluorescence intensity (Irel) was calculated using Equation S3, where Rt is the fluorescence ratio at time t, R0 is the fluorescence ratio at time 0, and Rd is the fluorescence ratio after the addition of detergent.
The fractional fluorescence intensity (Irel) at 288 s just prior to lysis, defined as the fractional activity y, was plotted as a function of the ionophore concentration (x / µM). Hill coefficients (n) and EC50 values were calculated by fitting to the Hill equation (S4) where y0 is the fractional activity in the absence of transporter, ymax is the fractional activity in with excess transporter, x is the transporter concentration in the cuvette. Hill plots were fitted to at-least 8 data points spanning the required concentration range.

HPTS assay following photo-irradiation
The HPTS assay was modified to allow for in-situ irradiation of the anchored carriers, and carried out as follows: the desired concentration of the anchored carriers in DMSO (5 μL) are added to the lipid suspension (POPC LUVs containing 1 mM HPTS, 100 mM internal and external NaCl, buffered with 10 mM HEPES at pH 7.0) and stirred for 1 minute to allow for incorporation into the bilayer. The sample was then irradiated with 365 nm UV light using an LED irradiation intensity (~1.3 W). The transport assay was initiated with a base pulse of NaOH (20 μL, 0.5 M) and the change in ratiometric emission (em = 510 nm; λex = 460/405 nm) was monitored over time, before calibration with Triton-X100.
Normalisation procedure. Under the standard HPTS assay condition (in which the base pulse is added prior to the transporter), the initial instantaneous jump in fluorescence intensity observed upon addition of NaOH (due to external HPTS absorbed to the vesicle surface, reporting on the increase in external pH) occurs prior to the carrier addition, and is removed by normalisation. Under the experimental conditions used here for the in-situ irradiation conditions (in which the carrier is added to the membrane prior to the base pulse), this instantaneous jump happens simultaneously with transport. For normalisation, the intensity recorded immediately after the addition of the base pulse is therefore subtracted, according to the Equation S3, where R0 is the fluorescence ratio immediately after the base pulse addition ( Figure S42). The subsequent increase in Irel reports on the increase in pH inside the vesicles due to carrier mediated transport. Hill analysis was performed as usual with the Irel value immediately prior to vesicle lysis.   13 The self-quenching dye 5(6)-Carboxyfluorescein (CF) was loaded into POPC LUVs to investigate the effect of the transporter upon vesicle integrity. POPC LUVs were prepared with an internal solution of 10 mM NaCl, 10 mM HEPES and 50 mM CF buffered to pH 7.0 and external buffer of 107 mM NaCl, 10 mM HEPES, pH 7.0. Each transport experiment was carried out as follows: the CF-containing POPC vesicles were suspended in the external buffer (1995 µL, LUV concentration 31 µM) at 25 °C and gently stirred. At 50 s, the test transporter (in 5 µL DMSO) was added. The assay was calibrated at 250 s with Triton X-100 detergent (40 µL, 7:1 (v/v) H2O-DMSO). The time-dependent change in fluorescence intensity (λex = 492 nm, λem = 517 nm) was monitored, and normalised according to Equation S5

Carboxyfluorescein Leakage Assay
where I0 = It before transporter addition, Imax = It after lysis.

HPTS Assay with DPPC lipids
The HPTS assay was carried out using the same procedure as described above, however, the POPC LUVs were replaced with dipalmitoyl phosphatidylcholine (DPPC) LUVs. These lipids are extruded at 45 °C.