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J Neurophysiol 81: 1939-1948, 1999;
0022-3077/99 $5.00
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The Journal of Neurophysiology Vol. 81 No. 4 April 1999, pp. 1939-1948
Copyright ©1999 by the American Physiological Society

Na+-K+-2Clminus Cotransporter in Immature Cortical Neurons: A Role in Intracellular Clminus Regulation

Dandan Sun1,2 and Sangita G. Murali1

 1Department of Neurological Surgery and  2Department of Physiology, School of Medicine, University of Wisconsin, Madison, Wisconsin 53792

Sun, Dandan and Sangita G. Murali. 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 ~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.




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