RGD Reference Report - Cell free expression and functional reconstitution of eukaryotic drug transporters. - Rat Genome Database

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Cell free expression and functional reconstitution of eukaryotic drug transporters.

Authors: Keller, T  Schwarz, D  Bernhard, F  Dotsch, V  Hunte, C  Gorboulev, V  Koepsell, H 
Citation: Keller T, etal., Biochemistry. 2008 Apr 15;47(15):4552-64. doi: 10.1021/bi800060w. Epub 2008 Mar 25.
RGD ID: 7243886
Pubmed: PMID:18361503   (View Abstract at PubMed)
DOI: DOI:10.1021/bi800060w   (Journal Full-text)

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 (-).



Gene Ontology Annotations    Click to see Annotation Detail View

Molecular Function

  
Object SymbolSpeciesTermQualifierEvidenceWithNotesSourceOriginal Reference(s)
Slc22a6Ratantiporter activity  IDA  RGD 
Slc22a6Ratchloride ion binding  IDA  RGD 
Slc22a1Ratidentical protein binding  IPISlc22a1 (Rattus norvegicus)homodimerization and homooligomerizationRGD 
Slc22a6Ratidentical protein binding  IPISlc22a6 (Rattus norvegicus)homodimerization and homooligomerizationRGD 
Slc22a1Ratorganic cation transmembrane transporter activity  IDA  RGD 
Slc22a2Ratorganic cation transmembrane transporter activity  IDA  RGD 

Objects Annotated

Genes (Rattus norvegicus)
Slc22a1  (solute carrier family 22 member 1)
Slc22a2  (solute carrier family 22 member 2)
Slc22a6  (solute carrier family 22 member 6)


Additional Information