Subcellular localization of transporters along the rat blood-brain barrier and blood-cerebral-spinal fluid barrier by in vivo biotinylation

Neuroscience. 2008 Aug 13;155(2):423-38. doi: 10.1016/j.neuroscience.2008.06.015. Epub 2008 Jun 13.


Nutrient transporters and ABC efflux pumps at the blood-brain barrier are major determinants of drug penetration into the brain. Immunohistochemical analysis of transporter subcellular localization is challenging due to the close apposition of the luminal and abluminal microvessel plasma membranes. We employed in vivo perfusion of biotinylation reagent through rat brain microvessels to domain-specifically label proteins exposed on the microvessel luminal surface. Using this approach, we analyzed the luminal/abluminal localization of a number of blood-brain barrier transporters identified by quantitative PCR profiling as being highly expressed and enriched in rat brain endothelial cells compared with whole brain. We also examined the apical/basal-lateral distribution of transporters in the choroid plexus, a secondary site for transport of nutrients between the blood and CNS. We detected P-glycoprotein (Pgp) (Abcb1), ATP-binding cassette (Abc) g2, multidrug resistance protein (Mrp) 4 (Abcc4), glucose transporter 1 (Glut1) (Slc2a1), Lat1 (Slc7a5), and monocarboxylate transporter-1 (Mct1) (Slc16a1) on the luminal surface of rat cerebral microvessels by both immunofluorescence staining and Western blotting of in vivo biotinylated proteins. Mrp1 (Abcc1) appeared primarily abluminal by immunofluorescence staining, and was barely detectable in the biotinylated protein fraction. Organic anion transporter (Oat) 3 (Slc22a8), organic anion transporter polypeptide (Oatp) 2b1 (Slco2b1, Oatpb), and Mrp5 (Abcc5) were not detected on the luminal surface using either method, while Oatp1a4 (Slco1a4, Oatp2) appeared to partially localize to the microvessel lumen by immunofluorescence staining, but was not detected in the biotinylated protein fraction by Western blotting. Lat1, Mrp1 and Mrp4 were detected on the basal-lateral surface of lateral ventricle choroid plexus epithelial cells. Mrp5, Oct3 and Oatp2b1 (Oatpb) were detected in the ependymal cells lining the ventricle. We did not detect Pgp expression in choroid plexus by immunofluorescence staining. In vivo biotinylation provides a method for domain-specific labeling of luminal surface proteins within the capillaries of the blood-brain barrier, allowing for biochemical analysis of protein localization and facilitating optical discrimination of the luminal and abluminal endothelial surfaces.

MeSH terms

  • ATP Binding Cassette Transporter, Subfamily B, Member 1 / genetics
  • ATP Binding Cassette Transporter, Subfamily B, Member 1 / metabolism
  • ATP Binding Cassette Transporter, Subfamily G, Member 2
  • ATP-Binding Cassette Transporters / genetics
  • ATP-Binding Cassette Transporters / metabolism
  • Animals
  • Biotinylation
  • Blood-Brain Barrier / physiology*
  • Blood-Brain Barrier / ultrastructure
  • Blotting, Western
  • Cell Line
  • Cerebrovascular Circulation / physiology*
  • Choroid Plexus / blood supply
  • Choroid Plexus / metabolism*
  • Choroid Plexus / ultrastructure
  • Ependyma / blood supply
  • Ependyma / metabolism
  • Ependyma / ultrastructure
  • Gene Expression Profiling
  • Glucose Transporter Type 1 / genetics
  • Glucose Transporter Type 1 / metabolism
  • Humans
  • Kidney / cytology
  • Large Neutral Amino Acid-Transporter 1 / genetics
  • Large Neutral Amino Acid-Transporter 1 / metabolism
  • Male
  • Membrane Transport Proteins / genetics
  • Membrane Transport Proteins / metabolism*
  • Microcirculation / physiology
  • Microcirculation / ultrastructure
  • Monocarboxylic Acid Transporters / genetics
  • Monocarboxylic Acid Transporters / metabolism
  • Multidrug Resistance-Associated Proteins / genetics
  • Multidrug Resistance-Associated Proteins / metabolism
  • Rats
  • Rats, Sprague-Dawley
  • Symporters / genetics
  • Symporters / metabolism
  • Transfection


  • ATP Binding Cassette Transporter, Subfamily B, Member 1
  • ATP Binding Cassette Transporter, Subfamily G, Member 2
  • ATP-Binding Cassette Transporters
  • Abcc4 protein, rat
  • Abcg2 protein, rat
  • Glucose Transporter Type 1
  • Large Neutral Amino Acid-Transporter 1
  • Membrane Transport Proteins
  • Monocarboxylic Acid Transporters
  • Multidrug Resistance-Associated Proteins
  • Slc2a1 protein, rat
  • Symporters
  • monocarboxylate transport protein 1
  • multidrug resistance-associated protein 1