Potassium-coupled chloride cotransport controls intracellular chloride in rat neocortical pyramidal neurons

Chloride (Cl-) homeostasis is critical for many cell functions including ce ll signaling and volume regulation. The action of GABA at GABA(A) receptors is primarily determined by the concentration of intracellular Cl-. Develop mental regulation of intracellular Cl- results in a depolarizing response t o GABA in immature neocortical neurons and a hyperpolarizing or shunting re sponse in mature neocortical neurons. One protein that participates in Cl- homeostasis is the neuron-specific K+-Cl+ cotransporter (KCC2). Thermodynam ic 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 pyra midal cells from rat neocortical slices in mature (18-28 d postnatal) and i mmature (3-6 d postnatal) rats. Intracellular Cl- concentration was estimat ed from the reversal potential of whole-cell currents evoked by local appli cation of exogenous GABA. Both increasing and decreasing the extracellular K+ concentration resulted in a concomitant change in intracellular Cl- conc entration in neurons from mature rats. KCC2 inhibition by furosemide caused a change in the intracellular Cl- concentration that depended on the conce ntration of pipette Cl-; in recordings with low pipette Cl-, furosemide low ered intracellular Cl-, whereas in recordings with elevated pipette Cl-, fu rosemide raised intracellular Cl-. In neurons from neonatal rats, manipulat ion of extracellular K+ had no effect on intracellular Cl- concentration, c onsistent with the minimal KCC2 mRNA levels observed in neocortical neurons from immature animals. These data demonstrate a physiologically relevant a nd developmentally regulated role for KCC2 in Cl- homeostasis via both Cl- extrusion and accumulation.