Protein kinase C-dependent trafficking of glutamate transporters excitatory amino acid carrier 1 and glutamate transporter 1b in cultured cerebellar granule cells.
Previous data showed that cell surface expression of the glutamate transporters GLT1a and excitatory amino acid carrier 1 (EAAC1), localized in glia and neurons of the CNS, can be regulated by protein kinase C (PKC). Regulation and physiological importance of GLT1b, a splice variant of GLT1a, is not understood. In the present study we used cultured cerebellar granule cells (CGCs) from mice to investigate PKC dependent trafficking of GLT1b in comparison to GLT1a and EAAC1 using immunohistochemistry and subcellular fractionation followed by Western blotting. In neurites of CGCs, GLT1b and EAAC1 were localized to different aggregates of vesicles that were different from vesicle aggregates containing vesicular glutamate transporters. In CGCs cultured with low-potassium medium, stimulation of PKC by phorbol ester enhanced the formation of large varicosities in neurites that exhibited immunoreactivity for GLT1a, GLT1b, and EAAC1. Stimulation of PKC leads to a significant increase of GLT1b and EAAC1 in the plasma membrane whereas GLT1a in the plasma membrane was decreased. Following PKC stimulation, also a significant increase of transporter-mediated glutamate uptake representing sodium dependent glutamate uptake, was observed. Similarly, the fraction of glutamate uptake, that was sensitive to the inhibitor WAY-213613 and represents uptake by GLT1a and GLT1b, was increased after stimulation by PKC. The findings suggest that PKC is similarly involved in regulation of surface trafficking of GLT1b and EAAC1 and that PKC stimulated increase in surface location of GLT1b and EAAC1 in glutamatergic CGCs.
University of Würzburg, Institute of Anatomy and Cell Biology, Würzburg, Germany.
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U
Karatas-Wulf
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H
Koepsell
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M
Bergert
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S
Sönnekes
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P
Kugler
D000818:Animals; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D002531:Cerebellum / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D020536:Enzyme Activators / Q000008:administration & dosage; D027342:Excitatory Amino Acid Transporter 2 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D050580:Excitatory Amino Acid Transporter 3 / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D009474:Neurons / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D010703:Phorbol Esters / Q000008:administration & dosage; D011188:Potassium / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism*; D021381:Protein Transport; D013347:Subcellular Fractions / Q000378:metabolism; D013572:Synaptic Vesicles / Q000378:metabolism
D020536:Enzyme Activators; D027342:Excitatory Amino Acid Transporter 2; D050580:Excitatory Amino Acid Transporter 3; D010703:Phorbol Esters; C493399:Slc1a1 protein, mouse; D018698:Glutamic Acid; D011493:Protein Kinase C; D011188:Potassium