Thermodynamic regulation of NKCC1-mediated Cl- cotransport underlies plasticity of GABA(A) 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, GABA(A) 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 Cl(i) such that GABA(A) receptor activation was depolarizing. Kinetic analysis of NKCC1 revealed reversible transmembrane Cl- transport characterized by a large maximum velocity (vmax) and high affinity (Km), 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 alpha2/alpha3 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.