Potassium-Coupled Chloride Cotransport Controls Intracellular Chloride in Rat Neocortical Pyramidal Neurons

Chloride (Cl−) homeostasis is critical for many cell functions including cell signaling and volume regulation. The action of GABA at GABAAreceptors is primarily determined by the concentration of intracellular Cl−. Developmental regulation of intracellular Cl−results in a depolarizing response to GABA in immature neocortical neurons and a hyperpolarizing or shunting response in mature neocortical neurons. One protein that participates in Cl−homeostasis is the neuron-specific K+–Cl−cotransporter (KCC2). Thermodynamic considerations predict that in the physiological ranges of intracellular Cl−and extracellular K+concentrations, KCC2 can act to either extrude or accumulate Cl−. To test this hypothesis, we examined KCC2 function in pyramidal cells from rat neocortical slices in mature (18–28 d postnatal) and immature (3–6 d postnatal) rats. Intracellular Cl−concentration was estimated from the reversal potential of whole-cell currents evoked by local application of exogenous GABA. Both increasing and decreasing the extracellular K+concentration resulted in a concomitant change in intracellular Cl−concentration in neurons from mature rats. KCC2 inhibition by furosemide caused a change in the intracellular Cl−concentration that depended on the concentration of pipette Cl−; in recordings with low pipette Cl−, furosemide lowered intracellular Cl−, whereas in recordings with elevated pipette Cl−, furosemide raised intracellular Cl−. In neurons from neonatal rats, manipulation of extracellular K+had no effect on intracellular Cl−concentration, consistent with the minimal KCC2 mRNA levels observed in neocortical neurons from immature animals. These data demonstrate a physiologically relevant and developmentally regulated role for KCC2 in Cl−homeostasis via both Cl−extrusion and accumulation.