Purification and functional reconstitution of the rat organic cation transporter OCT1.
The rat organic cation transporter rOCT1 with six histidine residues added to the C-terminus was expressed in Sf9 insect cells, and expression of organic cation transport was demonstrated. To purify rOCT1 protein, Sf9 cells were lysed with 1% (w/v) CHAPS [3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate], centrifuged, and subjected to sequential affinity chromatography using lentil-lectin Sepharose and nickel(II)-charged nitrilotriacetic acid-agarose. This procedure yielded approximately 70 microg of purified rOCT1 protein from 10 standard culture plates. Using a freeze-thaw procedure, purified rOCT1 was reconstituted into proteoliposomes formed from phosphatidylcholine, phosphatidylserine, and cholesterol. Proteoliposomes exhibited uptake of [3H]-1-Methyl-4-phenylpyridinium ([3H]MPP) that was inhibited by quinine and stimulated by an inside-negative membrane potential. MPP uptake was saturable with an apparent K(m) of 30 +/- 17 microM. MPP uptake (0.1 microM) was inhibited by tetraethylammonium, tetrabutylammonium, and tetrapentylammonium with IC50 values of 197 +/- 11, 19 +/- 1, and 1.8 +/- 0.03 microM, respectively. With membrane potential clamped to 0 mV using valinomycin in the presence of 100 mM potassium on both sides of the membrane, uptake of 0.1 microM MPP was trans stimulated 3-fold by 2.5 mM intracellular choline, and efflux of 0.1 microM MPP was trans stimulated 4-fold by 9.5 mM extracellular choline. The data show that rOCT1 is capable and sufficient to mediate transport of organic cations. The observed trans stimulation under voltage-clamp conditions shows that rOCT1 operates as a transporter rather than a channel. Purification and reconstitution of functional active rOCT1 protein is an important step toward the biophysical characterization and crystallization.
Keller
Thorsten
T
Institute of Anatomy and Cell Biology, University of Würzburg, Koellikerstrasse 6, 97070 Würzburg, Germany.
Elfeber
Marco
M
Gorboulev
Valentin
V
Reiländer
Helmut
H
Koepsell
Hermann
H
D015655:1-Methyl-4-phenylpyridinium / Q000494:pharmacology; D000818:Animals; D050482:Catecholamine Plasma Membrane Transport Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D002460:Cell Line; D015870:Gene Expression; D011510:Proteolipids / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D051381:Rats; D018411:Spodoptera; D013379:Substrate Specificity
D050482:Catecholamine Plasma Membrane Transport Proteins; D011510:Proteolipids; C493212:Slc22a1 protein, rat; C020485:proteoliposomes; D015655:1-Methyl-4-phenylpyridinium