Cell free expression and functional reconstitution of eukaryotic drug transporters.
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 (-).
Keller
Thorsten
T
Institute of Anatomy and Cell Biology, University of Würzburg, Koellikerstrasse 6, 97070 Würzburg, Germany.
Schwarz
Daniel
D
Bernhard
Frank
F
Dötsch
Volker
V
Hunte
Carola
C
Gorboulev
Valentin
V
Koepsell
Hermann
H
D015655:1-Methyl-4-phenylpyridinium / Q000378:metabolism; D000818:Animals; D050482:Catecholamine Plasma Membrane Transport Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002474:Cell-Free System; D007656:Ketoglutaric Acids / Q000378:metabolism; D027362:Organic Anion Transport Protein 1 / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D027701:Organic Cation Transport Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000075102:Organic Cation Transporter 2; D011510:Proteolipids / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D010130:p-Aminohippuric Acid / Q000378:metabolism
D050482:Catecholamine Plasma Membrane Transport Proteins; D007656:Ketoglutaric Acids; D027362:Organic Anion Transport Protein 1; D027701:Organic Cation Transport Proteins; D000075102:Organic Cation Transporter 2; D011510:Proteolipids; D011994:Recombinant Proteins; C493212:Slc22a1 protein, rat; C100590:Slc22a2 protein, rat; C421043:Slc22a6 protein, rat; C020485:proteoliposomes; D015655:1-Methyl-4-phenylpyridinium; D010130:p-Aminohippuric Acid