Components of neuronal chloride transport in rat and human neocortex

Non-technical summary The inhibitory neurotransmitter GABA activates two distinct receptors of which the GABA(A) receptor is mainly a Cl- conducting ion channel. The proper functioning of inhibition at the GABA(A) receptor depends on the ionic gradient prevailing during receptor activation, and in epilepsy there is an aberrant Cl- gradient. Using rat and human cortical neurones and pharmacological inhibitors, we calculated the contributions of neuronal Cl- extrusion by the Na+-K+-2Cl- transporter NKCC1, the K+-coupled Cl- transporter KCC2 and the voltage-gated Cl- channel ClC2. We found that KCC2 is the major route of Cl- extrusion and that reduced KCC2 Cl- extrusion is likely to be the initial step of disturbed Cl- regulation. The results contribute to our understanding of epilepsy.Considerable evidence indicates disturbances in the ionic gradient of GABA(A) receptor-mediated inhibition of neurones in human epileptogenic tissues. Two contending mechanisms have been proposed, reduced outward and increased inward Cl- transporters. We investigated the properties of Cl- transport in human and rat neocortical neurones (layer II/III) using intracellular recordings in slices of cortical tissue. We measured the alterations in reversal potential of the pharmacologically isolated inhibitory postsynaptic potential mediated by GABA(A) receptors (IPSP(A)) to estimate the ionic gradient and kinetics of Cl- efflux after Cl- injections before and during application of selected blockers of Cl- routes (furosemide, bumetanide, 9-anthracene carboxylic acid and Cs+). Neurones from human epileptogenic cortex exhibited a fairly depolarized reversal potential of GABA(A) receptor-mediated inhibition (E(IPSP-A)) of -61.9 +/- 8.3 mV. In about half of the neurones, the E(IPSP-A) averaged -55.2 +/- 5.7 mV, in the other half, 68.6 +/- 2.3 mV, similar to rat neurones (-68.9 +/- 2.6 mV). After injections of Cl-, IPSP(A) recovered in human neurones with an average time constant (tau) of 19.0 +/- 9.6 s (rat neurones: 7.2 +/- 2.4 s). We calculated Cl- extrusion rates (1/tau) via individual routes from the tau values obtained in different experimental conditions, revealing that, for example, the K+-coupled Cl- transporter KCC2 comprises 45.3% of the total rate in rat neurones. In human neurones, the total rate of Cl- extrusion was 63.9% smaller, and rates via KCC2, the Na+-K+-2Cl- transporter NKCC1 and the voltage-gated Cl- channel ClC were smaller than in rat neurones by 80.0%, 61.7% and 79.9%, respectively. The rate via anion exchangers conversely was 14.4% larger in human than in rat neurones. We propose that (i) KCC2 is the major route of Cl- extrusion in cortical neurones, (ii) reduced KCC2 is the initial step of disturbed Cl- regulation and (iii) reductions in KCC2 contribute to depolarizing E(IPSP-A) of neurones in human epileptogenic neocortex.