Na+-K+-2Cl- cotransporter in immature cortical neurons: A role in intracellular Cl- regulation.
Na+-K+-2Cl- cotransporter has been suggested to contribute to active intracellular Cl- accumulation in neurons at both early developmental and adult stages. In this report, we extensively characterized the Na+-K+-2Cl- cotransporter in primary culture of cortical neurons that were dissected from cerebral cortex of rat fetus at embryonic day 17. The Na+-K+-2Cl- cotransporter was expressed abundantly in soma and dendritic processes of cortical neurons evaluated by immunocytochemical staining. Western blot analysis revealed that an approximately 145-kDa cotransporter protein was present in cerebral cortex at the early postnatal (P0-P9) and adult stages. There was a time-dependent upregulation of the cotransporter activity in cortical neurons during the early postnatal development. A substantial level of bumetanide-sensitive K+ influx was detected in neurons cultured for 4-8 days in vitro (DIV 4-8). The cotransporter activity was increased significantly at DIV 12 and maintained at a steady level throughout DIV 12-14. Bumetanide-sensitive K+ influx was abolished completely in the absence of either extracellular Na+ or Cl-. Opening of gamma-aminobutyric acid (GABA)-activated Cl- channel or depletion of intracellular Cl- significantly stimulated the cotransporter activity. Moreover, the cotransporter activity was elevated significantly by activation of N-methyl-D-aspartate ionotropic glutamate receptor via a Ca2+-dependent mechanism. These results imply that the inwardly directed Na+-K+-2Cl- cotransporter is important in active accumulation of intracellular Cl- and may be responsible for GABA-mediated excitatory effect in immature cortical neurons.
Sun
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Department of Neurological Surgery, School of Medicine, University of Wisconsin, Madison, Wisconsin 53792, USA.
Murali
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SG
D000818:Animals; D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D015153:Blotting, Western; D002352:Carrier Proteins / Q000032:analysis / Q000502:physiology*; D002540:Cerebral Cortex / Q000166:cytology / Q000254:growth & development; D002614:Chelating Agents / Q000494:pharmacology; D002712:Chlorides / Q000378:metabolism*; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D018690:Excitatory Amino Acid Agonists / Q000494:pharmacology; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D005260:Female; D007150:Immunohistochemistry; D016202:N-Methylaspartate / Q000494:pharmacology; D009474:Neurons / Q000737:chemistry* / Q000378:metabolism*; D010880:Piperidines / Q000494:pharmacology; D011247:Pregnancy; D051381:Rats; D017207:Rats, Sprague-Dawley; D011963:Receptors, GABA-A / Q000502:physiology; D016194:Receptors, N-Methyl-D-Aspartate / Q000502:physiology; D028021:Sodium-Potassium-Chloride Symporters; D013194:Staining and Labeling; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology
D002352:Carrier Proteins; D002614:Chelating Agents; D002712:Chlorides; D018690:Excitatory Amino Acid Agonists; D018691:Excitatory Amino Acid Antagonists; D010880:Piperidines; D011963:Receptors, GABA-A; D016194:Receptors, N-Methyl-D-Aspartate; D028021:Sodium-Potassium-Chloride Symporters; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D004533:Egtazic Acid; D005680:gamma-Aminobutyric Acid; D016202:N-Methylaspartate; C010739:ifenprodil