Cell-specific sorting of biogenic amine transporters expressed in epithelial cells

J Biol Chem. 1996 Jul 26;271(30):18100-6. doi: 10.1074/jbc.271.30.18100.

Abstract

We have utilized polarized epithelial cells stably expressing neurotransmitter transporters to analyze the sorting behavior of these membrane proteins. The transporters for serotonin (5-HT), dopamine (DA), and norepinephrine (NE) are expected to be present in situ in the most distal extremities of axonal membranes, where they terminate the action of their biogenic amine substrates. Both Madin-Darby canine kidney (MDCK) and LLC-PK1 cells were stably transfected with cDNAs encoding either the rat 5-HT transporter (SERT), the human NE transporter (NET), or the rat or human DA transporter (DAT). These cells were grown on permeable filter supports, and the transporters were localized by three independent techniques. Confocal immunofluorescence microscopy indicated that each of the transporters expressed in LLC-PK1 cells was sorted to the basolateral membrane, co-localizing with the Na+/K+-ATPase. In MDCK cells, however, DAT was located primarily on the apical surface, while SERT and NET were found on the basolateral membranes. Cell surface biotinylation using an impermeant biotinylating reagent confirmed the immunocytochemistry results. Thus, SERT and NET in MDCK cells were labeled more efficiently from the basolateral medium than the apical medium, and DAT in MDCK cells was labeled more efficiently from the apical side than the basolateral side. Transport measurements in transfected MDCK cells agreed with the immunocytochemistry and biotinylation results. These results suggest the existence of cell-specific mechanisms that discriminate between neurotransmitter transporters for surface expression and render unlikely any simple hypothesis that sorting mechanisms in neurons and epithelia are identical.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Biogenic Amines / metabolism*
  • Biological Transport
  • Carrier Proteins / genetics
  • Carrier Proteins / metabolism*
  • Cell Compartmentation*
  • Cell Membrane / metabolism*
  • Cell Polarity*
  • Cells, Cultured
  • Dogs
  • Dopamine / metabolism
  • Dopamine Plasma Membrane Transport Proteins
  • Epithelial Cells
  • Humans
  • Immunohistochemistry
  • Membrane Glycoproteins / genetics
  • Membrane Glycoproteins / metabolism
  • Membrane Transport Proteins*
  • Nerve Tissue Proteins*
  • Norepinephrine / metabolism
  • Norepinephrine Plasma Membrane Transport Proteins
  • Rats
  • Recombinant Proteins / metabolism
  • Serotonin / metabolism
  • Serotonin Plasma Membrane Transport Proteins
  • Symporters*

Substances

  • Biogenic Amines
  • Carrier Proteins
  • Dopamine Plasma Membrane Transport Proteins
  • Membrane Glycoproteins
  • Membrane Transport Proteins
  • Nerve Tissue Proteins
  • Norepinephrine Plasma Membrane Transport Proteins
  • Recombinant Proteins
  • SLC6A2 protein, human
  • SLC6A3 protein, human
  • SLC6A4 protein, human
  • Serotonin Plasma Membrane Transport Proteins
  • Slc6a2 protein, rat
  • Slc6a3 protein, rat
  • Slc6a4 protein, rat
  • Symporters
  • Serotonin
  • Dopamine
  • Norepinephrine