Abstract
OCTN2--the Organic Cation Transporter Novel family member 2 (SLC22A5) is known to be a xenobiotic/drug transporter. It transports as well carnitine--a compound necessary for oxidation of fatty acids and mutations of its gene cause primary carnitine deficiency. Octn2 regulation by protein kinase C (PKC) was studied in rat astrocytes--cells in which β-oxidation takes place in the brain. Activation of PKC with phorbol ester stimulated L-carnitine transport and increased cell surface presence of the transporter, although no PKC-specific phosphorylation of Octn2 could be detected. PKC activation resulted in an augmented Octn2 presence in cholesterol/sphingolipid-rich microdomains of plasma membrane (rafts) and increased co-precipitation of Octn2 with raft-proteins, caveolin-1 and flotillin-1. Deletion of potential caveolin-1 binding motifs pointed to amino acids 14-22 and 447-454 as the caveolin-1 binding sites within Octn2 sequence. A direct interaction of Octn2 with caveolin-1 in astrocytes upon PKC activation was detected by proximity ligation assay, while such an interaction was excluded in case of flotillin-1. Functioning of a multi-protein complex regulated by PKC has been postulated in rOctn2 trafficking to the cell surface, a process which could be important both under physiological conditions, when carnitine facilitates fatty acids catabolism and controls free Coenzyme A pool as well as in pathology, when transport of several drugs can induce secondary carnitine deficiency.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Astrocytes / drug effects
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Astrocytes / enzymology*
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Astrocytes / metabolism
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Astrocytes / ultrastructure
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Biological Transport / drug effects
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Carnitine / metabolism
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Caveolin 1 / chemistry
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Caveolin 1 / metabolism*
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Cell Membrane / drug effects
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Cell Membrane / metabolism
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Enzyme Activation / drug effects
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HEK293 Cells
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Humans
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Immunoprecipitation
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Membrane Microdomains / metabolism
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Membrane Proteins / metabolism
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Organic Cation Transport Proteins / metabolism*
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Phosphorylation / drug effects
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Protein Binding / drug effects
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Protein Kinase C / metabolism*
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Protein Structure, Tertiary
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Rats
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Reproducibility of Results
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Solute Carrier Family 22 Member 5
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Tetradecanoylphorbol Acetate / pharmacology
Substances
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Caveolin 1
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Membrane Proteins
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Organic Cation Transport Proteins
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Slc22a5 protein, rat
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Solute Carrier Family 22 Member 5
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flotillins
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Protein Kinase C
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Tetradecanoylphorbol Acetate
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Carnitine
Grants and funding
This work was financed by grant 4427/B/P01/2010/38 from the Polish Ministry of Science and Higher Education/National Science Centre (
http://www.ncn.gov.pl) and by the Nencki Institute (
http://www.nencki.gov.pl). KM receives a scholarship funded by the “International PhD Studies in Neurobiology” - project MPD/2009/4 of the Foundation for Polish Science (
http://www.fnp.org.pl), co-financed by the European Union Innovative Economy Operational Programme. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.