Cell Free Expression and Functional Reconstitution of Eukaryotic Drug Transporters

Abstract

Polyspecific organic cation and anion transporters of the SLC22 protein family are critically involved in absorption and excretion of drugs. To elucidate transport mechanisms, functional and biophysical characterization of purified transporters is required and tertiary structures must be determined. Here, we synthesized rat organic cation transporters OCT1 and OCT2 and rat organic anion transporter OAT1 in a cell free system in the absence of detergent. We solubilized the precipitates with 2% 1-myristoyl-2-hydroxy- sn-glycero-3-[phospho- rac-(1-glycerol)] (LMPG), purified the transporters in the presence of 1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) or octyl glucoside, and reconstituted them into proteoliposomes. From 1 mL reaction vessels 0.13-0.36 mg of transporter proteins was purified. Thus, from five to ten 1 mL reaction vessels sufficient protein for crystallization was obtained. In the presence of 1% LMPG and 0.5% CHAPS, OCT1 and OAT1 formed homo-oligomers but no hetero-oligomers. After reconstitution of OCT1, OCT2, and OAT1 into proteoliposomes, similar Michaelis-Menten K m values were measured for uptake of 1-methyl-4-phenylpyridinium and p-aminohippurate (PAH (-)) by the organic cation and anion transporters, respectively, as after expression of the transporters in cells. Using the reconstituted system, evidence was obtained that OAT1 operates as obligatory and electroneutral PAH (-)/dicarboxylate antiporter and contains a low-affinity chloride binding site that stimulates turnover. PAH (-) uptake was observed only with alpha-ketoglutarate (KG (2-)) on the trans side, and trans-KG (2-) increased the PAH (-) concentration in voltage-clamped proteoliposomes transiently above equilibrium. The V max of PAH (-)/KG (2-) antiport was increased by Cl (-) in a manner independent of gradients, and PAH (-)/KG (2-) antiport was independent of membrane potential in the absence or presence of Cl (-).

The Large Extracellular Loop of Organic Cation Transporter 1 Influences Substrate Affinity and Is Pivotal for Oligomerization

“…To make this assumption a priority is not justified since evidence has been provided that OATs and OCTs form homo-oligomers. Hong and co-workers (11) showed that human OAT1 (hOAT1) forms oligomers in cultured cells and kidney, and we provided evidence that rOat1, rOct1, and rOct2 form homo-oligomers in detergent solution (12). A recent study suggests that the sixth transmembrane ␣-helix (TMH) of hOAT1 comprises a contact region for oligomerization and that surface expression of hOAT1 was reduced when oligomerization of hOAT1 was disturbed (13).…”

“…Cell-free Expression of Transport Proteins-Cell-free expression was performed in the continuous exchange mode using bacterial extracts from Escherichia coli strain A19 as described (12,19). Reaction mixture and feeding mixture were composed as described (19).…”

“…Affinity Purification of Transporters-Purification was performed as described previously (12). Briefly, for purification on Ni 2ϩ -NTA-agarose, 1 ml of LMPG supernatant was mixed with 11 ml of Tris buffer containing 10 mM imidazole plus 1% (w/v) CHAPS and Ni 2ϩ -NTA-agarose and incubated for 1 h at 4°C.…”

“…Coprecipitation of His-tagged and FLAG-tagged TransportersCoprecipitation was performed as described (12). LMPG supernatants (100 l of each containing 20 g of in vitro expressed His-tagged or FLAG-tagged transporter, 2% LMPG, and 10 mM imidazole) were mixed and diluted with 200 l of Tris buffer containing 1% CHAPS and 10 mM imidazole (final concentration of 1% LMPG and 0.5% CHAPS).…”

“…2-(trimethylammonium)ethyl methanethiosulfonate bromide (MTSET) was purchased from Toronto Research Chemical (Ontario, Canada). All other chemicals were obtained as described earlier (12,14).…”