EC50 None (nM) Chloride transport activities of the macrocyclic halogen bonding transporters 2-6 were determined using pH gradient dissipation assays in large unilamellar vesicles (LUVs). In these experiments, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine vesicles (POPC LUVs) were loaded with the pH-responsive fluorophore 8-hydroxypyrene-1,3,6-trisulfonate (HPTS) and buffered to pH 7.0 in NaCl solution. The ability of the transporter, added in an aliquot of DMSO at various concentrations, to dissipate a pH gradient generated by addition of a NaOH base pulse was determined by recording the change in the HPTS emission with time. At the end of the experiment, the detergent Triton X-100 was added to lyse the vesicles for calibration. For full details, see the ESI. Dose-response curves were then fitted to the Hill equation to determine the concentration to achieve half-maximal activity (EC50) (Figure ). Transport experiments were repeated in the presence of protonophore carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) to determine the relative rates of Cl -and H + /OH -transport by mediating rapid electrogenic H + transport, and therefore an overall coupled H + /Cl -symport process. In the absence of FCCP, transport occurs either via Cl -/OH -antiport (exchange), or the functionally equivalent H + /Cl - symport to maintain charge neutrality (Figure , left). Proton transport is unlikely to operate in the case of these halogen bonding transporters due to a lack of any acidic protons or basic nitrogen atoms (protonated triazole pKa <1), which would allow for reversible protonation states. In the case of a Cl ->OH -selective transporter, the transport of OH -ions is thus the rate-determining step in the antiport transport mechanism. Upon the addition of protonophore FCCP, Cl -transport becomes rate limiting and thus the selectivity (S) of Cl ->OH -of the transporter under the assay conditions can be determined from the ratio of activities (EC50) in the presence and absence of the protonophore.