Cross talk between the GABA(A) receptor and the Na-K-Cl cotransporter is mediated by intracellular Cl-

It has been suggested that the GABA(A) receptor-mediated depolarization in immature neurons depends on a high intracellular Cl- concentration maintained by Na-K-Cl cotransporter isoform1 (NKCC1). We previously found that activation of the GABA(A) receptor led to stimulation of NKCC1. This stimulation could be a result of GABA(A) receptor-mediated Cl- efflux. However, a loss of intracellular Cl- is associated with cell shrinkage, membrane depolarization, as well as a rise of intracellular Ca2+ concentration ([Ca2+](i)). To determine which cellular mechanism is underlying NKCC1 stimulation, we investigated changes of intracellular Cl- content, [Ca2+](i), cell volume, and NKCC1 activity following GABA(A) receptor activation. The basal levels of intracellular Cl-36 were 0.70 +/- 0.04 mumol/mg protein. The intracellular Cl-36 content decreased to 0.53 +/- 0.03 mumol/mg protein in response to 30 muM muscimol (P < 0.05). The loss of intracellular Cl-36 was blocked by 10 mu M bicuculline. Muscimol triggered a rise in [Ca2+](i), but did not cause cell shrinkage. In contrast, 10-50 mM [Cl-](o) or hypertonic HEPES-MEM resulted in reversible cell shrinkage (P < 0.05). Moreover, the GABA-mediated stimulation of NKCC1 activity was not abolished either by removal of extracellular Ca2+ or BAPTA-AM. An increase in phosphorylation of NKCC1 was detected under both 10 mM [Cl-](o) and muscimol conditions. These results suggest that a GABA-mediated loss of intracellular Cl-, but not a subsequent rise in [Ca2+](i) or shrinkage, leads to stimulation of NKCC1. This stimulation may be an important positive feedback mechanism to maintain intracellular Cl- level and GABA function in immature neurons.