Purification and functional reconstitution of the rat organic cation transporter OCT1
- PMID: 16142924
- DOI: 10.1021/bi050676c
Purification and functional reconstitution of the rat organic cation transporter OCT1
Abstract
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.
Similar articles
-
Cell free expression and functional reconstitution of eukaryotic drug transporters.Biochemistry. 2008 Apr 15;47(15):4552-64. doi: 10.1021/bi800060w. Epub 2008 Mar 25. Biochemistry. 2008. PMID: 18361503
-
High-affinity cation binding to organic cation transporter 1 induces movement of helix 11 and blocks transport after mutations in a modeled interaction domain between two helices.Mol Pharmacol. 2008 Jan;73(1):50-61. doi: 10.1124/mol.107.040170. Epub 2007 Oct 16. Mol Pharmacol. 2008. PMID: 17940192
-
Identification of cysteines in rat organic cation transporters rOCT1 (C322, C451) and rOCT2 (C451) critical for transport activity and substrate affinity.Am J Physiol Renal Physiol. 2007 Sep;293(3):F767-79. doi: 10.1152/ajprenal.00106.2007. Epub 2007 Jun 13. Am J Physiol Renal Physiol. 2007. PMID: 17567940
-
Subtype-specific affinity for corticosterone of rat organic cation transporters rOCT1 and rOCT2 depends on three amino acids within the substrate binding region.Mol Pharmacol. 2005 May;67(5):1612-9. doi: 10.1124/mol.104.008821. Epub 2005 Jan 20. Mol Pharmacol. 2005. PMID: 15662045
-
The organic cation transporters rOCT1 and hOCT2 are inhibited by cGMP.J Membr Biol. 2002 Oct 1;189(3):237-44. doi: 10.1007/s00232-002-1023-7. J Membr Biol. 2002. PMID: 12395288
Cited by
-
Substrates and Inhibitors of Organic Cation Transporters (OCTs) and Plasma Membrane Monoamine Transporter (PMAT) and Therapeutic Implications.Handb Exp Pharmacol. 2021;266:119-167. doi: 10.1007/164_2021_516. Handb Exp Pharmacol. 2021. PMID: 34495395
-
Functional inhibition of Oct leads to HNF4α upregulation.Exp Ther Med. 2021 Apr;21(4):349. doi: 10.3892/etm.2021.9780. Epub 2021 Feb 11. Exp Ther Med. 2021. PMID: 33732322 Free PMC article.
-
Na(+)-D-glucose cotransporter SGLT1 is pivotal for intestinal glucose absorption and glucose-dependent incretin secretion.Diabetes. 2012 Jan;61(1):187-96. doi: 10.2337/db11-1029. Epub 2011 Nov 28. Diabetes. 2012. PMID: 22124465 Free PMC article.
-
The large extracellular loop of organic cation transporter 1 influences substrate affinity and is pivotal for oligomerization.J Biol Chem. 2011 Oct 28;286(43):37874-86. doi: 10.1074/jbc.M111.289330. Epub 2011 Sep 6. J Biol Chem. 2011. PMID: 21896487 Free PMC article.
-
High-level expression in Saccharomyces cerevisiae enables isolation and spectroscopic characterization of functional human adenosine A2a receptor.J Struct Biol. 2007 Aug;159(2):166-78. doi: 10.1016/j.jsb.2007.05.001. Epub 2007 May 16. J Struct Biol. 2007. PMID: 17591446 Free PMC article.
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Molecular Biology Databases