Document Detail


Regulation of renal organic anion transporter 3 (SLC22A8) expression and function by the integrity of lipid raft domains and their associated cytoskeleton.
MedLine Citation:
PMID:  23615001     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
BACKGROUND/AIMS: In humans and rodents, organic anion transporter 3 (Oat3) is highly expressed on the basolateral membrane of renal proximal tubules and mediates the secretion of exogenous and endogenous anions. Regulation of Oat3 expression and function has been observed in both expression system and intact renal epithelia. However, information on the local membrane environment of Oat3 and its role is limited. Lipid raft domains (LRD; cholesterol-rich domains of the plasma membrane) play important roles in membrane protein expression, function and targeting. In the present study, we have examined the role of LRD-rich membranes and their associated cytoskeletal proteins on Oat3 expression and function.
METHODS: LRD-rich membranes were isolated from rat renal cortical tissues and from HEK-293 cells stably expressing human OAT3 (hOAT3) by differential centrifugation with triton X-100 extraction. Western blots were subsequently analyzed to determine protein expression. In addition, the effect of disruption of LRD-rich membranes was examined on functional Oat3 mediated estrone sulfate (ES) transport in rat renal cortical slices. Cytoskeleton disruptors were investigated in both hOAT3 expressing HEK-293 cells and rat renal cortical slices.
RESULTS: Lipid-enriched membranes from rat renal cortical tissues and hOAT3-expressing HEK-293 cells showed co-expression of rOat3/hOAT3 and several lipid raft-associated proteins, specifically caveolin 1 (Cav1), β-actin and myosin. Moreover, immunohistochemistry in hOAT3-expressing HEK-293 cells demonstrated that these LRD-rich proteins co-localized with hOAT3. Potassium iodide (KI), an inhibitor of protein-cytoskeletal interaction, effectively detached cytoskeleton proteins and hOAT3 from plasma membrane, leading to redistribution of hOAT3 into non-LRD-rich compartments. In addition, inhibition of cytoskeleton integrity and membrane trafficking processes significantly reduced ES uptake mediated by both human and rat Oat3. Cholesterol depletion by methyl-β-cyclodextrin (MβCD) also led to a dose dependent reduction Oat3 expression and ES transport by rat renal cortical slices. Moreover, the up-regulation of rOat3-mediated transport seen following insulin stimulation was completely prevented by MβCD.
CONCLUSION: We have demonstrated that renal Oat3 resides in LRD-rich membranes in proximity to cytoskeletal and signaling proteins. Disruption of LRD-rich membranes by cholesterol-binding agents or protein trafficking inhibitors altered Oat3 expression and regulation. These findings indicate that the integrity of LRD-rich membranes and their associated proteins are essential for Oat3 expression and function.
Authors:
Chutima Srimaroeng; Jennifer Perry Cecile; Ramsey Walden; John B Pritchard
Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Intramural     Date:  2013-04-26
Journal Detail:
Title:  Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology     Volume:  31     ISSN:  1421-9778     ISO Abbreviation:  Cell. Physiol. Biochem.     Publication Date:  2013  
Date Detail:
Created Date:  2013-05-28     Completed Date:  2013-12-17     Revised Date:  2014-05-07    
Medline Journal Info:
Nlm Unique ID:  9113221     Medline TA:  Cell Physiol Biochem     Country:  Switzerland    
Other Details:
Languages:  eng     Pagination:  565-78     Citation Subset:  IM    
Copyright Information:
Copyright © 2013 S. Karger AG, Basel.
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MeSH Terms
Descriptor/Qualifier:
Actins / metabolism
Animals
Biological Transport / drug effects
Caveolin 1 / metabolism
Cytoskeleton / metabolism
Estrone / analogs & derivatives,  metabolism
HEK293 Cells
Humans
Insulin / pharmacology
Kidney Tubules, Proximal / cytology
Male
Membrane Microdomains / drug effects
Myosins / metabolism
Organic Anion Transporters, Sodium-Independent / genetics,  metabolism*
Rats
Rats, Sprague-Dawley
Up-Regulation / drug effects
beta-Cyclodextrins / pharmacology
Grant Support
ID/Acronym/Agency:
Z01 ES080031-31/ES/NIEHS NIH HHS; Z01 ES080031-32/ES/NIEHS NIH HHS
Chemical
Reg. No./Substance:
0/Actins; 0/Caveolin 1; 0/Insulin; 0/Organic Anion Transporters, Sodium-Independent; 0/beta-Cyclodextrins; 0/methyl-beta-cyclodextrin; 0/organic anion transport protein 3; 2DI9HA706A/Estrone; EC 3.6.4.1/Myosins; QTL48N278K/estrone sulfate
Comments/Corrections

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