Finally, we turned to a chloride co-transport or "dual host" assay, inspired by work reported by Moore et al., which allows the cooperative action of two uniport processes to be assessed (Figure (a), Section S2.4). We re-designed this assay to avoid the use of fluorescent probes, and instead used a chloride ion selective electrode to provide a readout of ion transport. 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) vesicles were prepared containing 489 mM KCl, and suspended in 167 mM Na2SO4, (the ionic strength was maintained at 500 mM; all solutions were buffered to pH 7.2 with 5 mM sodium phosphate salts). Upon the addition of potential transporters, chloride efflux was monitored using a chloride selective electrode. We reasoned that any observed chloride efflux upon addition of the potential transporters would most likely be the result from an overall K + /Cl -efflux process, which would avoid the build-up of an unfavourable charge gradient. We tested SSAs 1-3 in isolation, and in combination with previously reported anionophore 4, which is a known anion antiporter but cannot mediate K + /C l-symport alone. We thus hoped to investigate whether the SSAs could mediate K + uniport, which would lead to cooperative chloride efflux in combination with anionophore 4. Initially, potential transporters 1-4 were prepared as stock solutions in DMSO. The experiment was initialised by the addition of anionophore 4 (1 mol% w.r.t. lipid), and this was followed by the addition of either DMSO (as a negative control) or SSAs 1-3 (10 mol% w.r.t. lipid). The activity of the SSAs alone was assessed by adding DMSO followed by the SSA. As expected, the addition of anionophore 4 alone resulted in minimal chloride efflux (Figure ). Likewise, the addition of SSA 1 alone produced minimal chloride efflux (Figure ). However, when SSA 1 and anionophore 4 were added in combination, significant chloride efflux was observed (Figure ). Importantly, the total chloride efflux was more than the sum of the efflux mediated by 1 and 4 independently. We thus concluded that 1 and 4 function together in a cooperative fashion to mediate an overall K + /Cl -efflux process, and therefore that SSA 1 can function as a K + uniporter.