Cross Talk Between the GABAAReceptor and the Na-K-Cl Cotransporter Is Mediated by Intracellular Cl−

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Abstract
It has been suggested that the GABAAreceptor-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 GABAAreceptor led to stimulation of NKCC1. This stimulation could be a result of GABAAreceptor-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 GABAAreceptor activation. The basal levels of intracellular36Cl were 0.70 ± 0.04 μmol/mg protein. The intracellular36Cl content decreased to 0.53 ± 0.03 μmol/mg protein in response to 30 μM muscimol ( P < 0.05). The loss of intracellular36Cl was blocked by 10 μM bicuculline. Muscimol triggered a rise in [Ca2+]i, but did not cause cell shrinkage. In contrast, 10–50 mM [Cl−]oor 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−]oand muscimol conditions. These results suggest that a GABA-mediated loss of intracellular Cl−, but not a subsequent rise in [Ca2+]ior 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.