The Cationophore Coupled Transport Assay and Initial Rate Constant Calculations Synthetic vesicles (200 nm) were made using the same initial process as described for the Cl -/NO3 -exchange assay, except the vesicles were made to contain an internal solution of KCl (300 mM) or KF (300 mM) and were suspended in an external solution of K-Gluc (300 mM). Both the internal and external solutions were buffered to pH 7.2 using HEPES (10 mM). Depending on which transport method was being assessed, either the ionophore Vln, to study electrogenic transport, or the ionophore Mon, to study electroneutral transport (0.1 mol%, 10 μL, as DMSO solutions) were added to the system of vesicles (1 mM, with a final assay volume of 5 mL) at t = -30 s. Fresh DMSO solutions of each tetralactam (0.05 mol% or 0.0005 mol% for 3c) were made, and 10 μL was added to the assay system at t = 0 s to initiate anion transport. Each transport experiment continued until t = 300 s, where Triton X-100 (50 μL, 10% v/v) was added to lyse the vesicles. Each experiment continued until t = 420 s to record the value 100% Cl -efflux, using a Cl -ISE, or 100% F -efflux, using a F -ISE, which was necessary to calibrate the recorded data. Each transport experiment was performed at least twice to obtain standard errors. A control experiment was also performed using pure DMSO to ensure no transport was being observed without transporters. Note that if F -transport is being tested, Vln is the only ionophore that can be added to assess electrogenic F -uniport; if Mon is added to try and analyse H + /F -co-transport, uncontrolled HF transport will occur. The calibrated anion efflux (%) data recorded for each transporter was then graphed as a function of time before being fitted to a two-phase exponential decay function (Equation ) to derive initial rate constants (kini, s -1 ).