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 intr acellular Cl- accumulation in neurons at both early developmental and adult stages. In this report, we extensively characterized the Na+-K+-2Cl(-) cot ransporter in primary culture of cortical neurons that were dissected from cerebral cortex of rat fetus at embryonic day 17. The Na+-K+-2Cl(-) cotrans porter was expressed abundantly in soma and dendritic processes of cortical neurons evaluated by immunocytochemical staining. Western blot analysis re vealed that an similar to 145-kDa cotransporter protein was present in cere bral cortex at the early postnatal (P0-P9) and adult stages. There was a ti me-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-sensitiv e K+ influx was abolished completely in the absence of either extracellular Na+ or Cl-. Opening of gamma-aminobutyric acid (GABA)-activated Cl- channe l or depletion of intracellular Cl- significantly stimulated the cotranspor ter activity. Moreover, the cotransporter activity was elevated significant ly 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 intracel lular Cl- and may be responsible for GABA-mediated excitatory effect in imm ature cortical neurons.