Localization of the Sodium-Taurocholate cotransporting polypeptide in membrane rafts and modulation of its activity by cholesterol in vitro.
BACKGROUND
The relevance of discrete localization of hepatobiliary transporters in specific membrane microdomains is not well known.


OBJECTIVE
To determine whether the Na+/taurocholate cotransporting polypeptide (Ntcp), the main hepatic sinusoidal bile salt transporter, is localized in specific membrane microdomains.


METHODS
Presence of Ntcp in membrane rafts obtained from mouse liver was studied by immunoblotting and immunofluorescence. HEK-293 cells stably transfected with rat Ntcp were used for in vitro studies. Expression, localization and function of Ntcp in these cells were assessed by immunoblotting, immunofluorescence and biotinylation studies and Na+ -dependent taurocholate uptake assays, respectively. The effect of cholesterol depletion/repletion assays on Ntcp function was also investigated.


RESULTS
Ntcp localized primarily to membrane rafts in in vivo studies and localized partially in membrane rafts in transfected HEK-293 cells. In these cells, membrane cholesterol depletion resulted in a shift of Ntcp localization into non-membrane rafts, which correlated with a 2.5-fold increase in taurocholate transport. Cholesterol repletion shifted back part of Ntcp into membrane rafts, and normalized taurocholate transport to values similar to control cells.


CONCLUSIONS
Ntcp localizes in membrane rafts and its localization and function are regulated by membrane cholesterol content. This may serve as a novel regulatory mechanism of bile salt transport in liver.
Molina
Héctor
H
Departamento de Gastroenterologia, Facultad de Medicina, Pontificia Universidad Católica de Chile, Marcoleta 367, 833-0024, Santiago, Chile.
Azocar
Lorena
L
Ananthanarayanan
Meenakshisundaram
M
Arrese
Marco
M
Miquel
Juan Francisco
JF
D000818:Animals; D001692:Biological Transport; D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006801:Humans; D008099:Liver / Q000378:metabolism; D008563:Membrane Lipids / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D029363:Organic Anion Transporters, Sodium-Dependent / Q000378:metabolism*; D051381:Rats; D027981:Symporters / Q000378:metabolism*
D008563:Membrane Lipids; D029363:Organic Anion Transporters, Sodium-Dependent; D027981:Symporters; C071908:sodium-bile acid cotransporter