Thermodynamic regulation of NKCC1-mediated Cl- cotransport underlies plasticity of GABAA signaling in neonatal neurons

In the adult brain, chloride (Cl-) influx through GABA(A) receptors is an important mechanism of synaptic inhibition. However, under a variety of circumstances, including acquired epilepsy, neuropathic pain, after trains of action potentials or trauma, and during normal early brain development, GABAA receptor activation excites neurons by gating Cl- efflux because the intracellular Cl- concentration (Cl-i) is elevated. These findings require an inducible, active mechanism of chloride accumulation. We used gramicidin-perforated patch recordings to characterize Cl- transport via NKCC1, the principal neuronal Cl- accumulator, in neonatal CA1 pyramidal neurons. NKCC1 activity was required to maintain elevated Cli such that GABAA receptor activation was depolarizing. Kinetic analysis of NKCC1 revealed reversible transmembrane Cl- transport characterized by a large maximum velocity (v(max)) and high affinity (K-m), so that NKCC1 transport was limited only by the net electrochemical driving force for Na+, K+, and Cl+. At the steady-state Cl-i, NKCC1 was at thermodynamic equilibrium, and there was no evidence of net Cl- transport. Trains of action potentials that have been previously shown to induce persistent changes in neuronal E-Cl (reversal potential for Cl-) did not alter vmax or Km of NKCC1. Rather, action potentials shifted the thermodynamic set point, the steady-state Cl-i at which there was no net NKCC1-mediated Cl- transport. The persistent increase in Cl-i required intact alpha 2/alpha 3 Na+ -K+ -ATPase activity, indicating that trains of action potentials reset the thermodynamic equilibrium for NKCC1 transport by lowering Na-i. Activity-induced changes in Na+- K+ -ATPase activity comprise a novel mechanism for persistent alterations in synaptic signaling mediated by GABA.