Purification and functional reconstitution of the rat organic cation transporter OCT1

Biochemistry. 2005 Sep 13;44(36):12253-63. doi: 10.1021/bi050676c.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 1-Methyl-4-phenylpyridinium / pharmacology
  • Animals
  • Catecholamine Plasma Membrane Transport Proteins / antagonists & inhibitors
  • Catecholamine Plasma Membrane Transport Proteins / genetics
  • Catecholamine Plasma Membrane Transport Proteins / isolation & purification*
  • Catecholamine Plasma Membrane Transport Proteins / metabolism*
  • Cell Line
  • Gene Expression
  • Proteolipids / drug effects
  • Proteolipids / genetics
  • Proteolipids / metabolism
  • Rats
  • Spodoptera
  • Substrate Specificity


  • Catecholamine Plasma Membrane Transport Proteins
  • Proteolipids
  • Slc22a1 protein, rat
  • proteoliposomes
  • 1-Methyl-4-phenylpyridinium