Cation-chloride cotransporters (CCCs) mediate the coupled, electroneutral symport of cations such as Na⁺ and/or K⁺ with chloride across membrane. Among CCCs family, K-Cl cotransporters (KCC1-KCC4) extrude intracellular Cl^- by the transmembrane K⁺ gradient. In humans, these KCCs play vital roles in the physiology of the nervous system and kidney. However, mechanisms underlying the KCCs specific properties remain poorly understood, partly because purification of membrane proteins is challenging. Here, we present the protocol for purifying the full-length KCC1 from HEK293F cells used in our recent publication (Liu et al., 2019). The procedure may be adapted for functional and structural studies.

Secondary active transporters reside in cell membranes transporting polar solutes like amino acids against steep concentration gradients, using electrochemical gradients of ions as energy sources. Commonly, ensemble-based measurements of radiolabeled substrate uptakes or transport currents inform on kinetic parameters of transporters. Here we describe a fluorescence-based functional assay for glutamate and aspartate transporters that provides single-transporter, single-transport cycle resolution using an archaeal elevator-type sodium and aspartate symporter GltPh as a model system. We prepare proteo-liposomes containing reconstituted purified GltPh transporters and an encapsulated periplasmic glutamate/aspartate-binding protein, PEB1a, labeled with donor and acceptor fluorophores. We then