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Title of Journal: Mol Cell Biochem

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Abbravation: Molecular and Cellular Biochemistry

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Publisher

Springer US

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DOI

10.1007/978-3-642-11638-4_4

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ISSN

1573-4919

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Expression and analysis of two novel rat organic c

Authors: Katie M Bennett Jun Liu Courtney Hoelting James Stoll
Publish Date: 2011/02/27
Volume: 352, Issue: 1-2, Pages: 143-154
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Abstract

The organic cation transporter OCT SLC22 family is a family of polyspecific transmembrane proteins that are responsible for the uptake or excretion of many cationic drugs toxins and endogenous metabolites in a variety of tissues Many of the OCTs have been previously characterized but there are a number of orphan genes whose functions remain unknown In this study two novel rat SLC22 genes SLC22A17 BOCT1 and SLC22A23 BOCT2 were cloned and characterized Northern blot analysis showed that BOCT1 and BOCT2 mRNA was expressed in a wide variety of tissues BOCT1 was strongly expressed in brain primary neurons and brain endothelial cells with highest expression in choroid plexus BOCT2 was also abundantly expressed in brain as well as in liver To characterize the products of these genes BOCT1 cDNA was isolated from a rat blood–brain barrier cDNA library and BOCT2 cDNA was isolated from rat brain capillary and from cultured neurons using PCR techniques Plasmids expressing BOCT1 and BOCT2 were transfected into HEK293 cells as were control cDNAs for OCT1 and OCTN2 Recombinant cell surface protein was verified by western blot and fluorescence microscopy Transport activity of BOCT1 and BOCT2 was evaluated using radioisotope uptake assays The OCT1 and OCTN2expressing cells transported the canonical substrates 1methyl4phenylpyridinium MPP+ and carnitine respectively However BOCT1 and BOCT2expressing cells did not show transport activity for these substrates or a number of other SLC22 substrates These novel family members have a nonconserved amino terminus relative to other OCTs that may preclude typical SLC22 transport functionWe would like to thank Laura Ngo Kelly Fine Jeff Smith and Christi Srader for technical support The authors would also like to thank Wesley M Williams for assistance with microvessel purification This research was funded by the National Institute of General Medical Sciences R15 GM070578 JS and the Achievement Rewards for College Scientists ARCS scholarship foundation KMB

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