Hyperpolarizing GABAergic transmission requires the KCC2 C-terminal ISO domain

KCC2 is the neuron-specific member of the of K ⁺-Cl ^- cotransporter gene family. It is also the only member of its family that is active under physiologically normal conditions, in the absence of osmotic stress. By extruding Cl ^- from the neuron under isotonic conditions, this transporter maintains a low concentration of neuronal Cl ^-, which is essential for fast inhibitory synaptic transmission by GABA and glycine in the mature nervous system. The other members of this K ⁺-Cl ^- cotransporter gene family are exclusively swelling-activated. Here we demonstrate that a 15 aa region near the end of the C terminus, unique to KCC2 (termed the ISO domain), is required for KCC2 to cotransportK ⁺and Cl ^-out of the neuron under isotonic conditions.Wemade this discovery by overexpressing chimeric KCC2-KCC4 cDNA constructs in cultured hippocampal neurons prepared from Sprague Dawley rat embryos and assaying neuronal Cl ^- through gramicidin perforated patch-clamp recordings. We found that when neurons had been transfected with a chimeric KCC2 that lacked the unique ISO domain, hyperpolarizing responses to GABA were abolished. This finding indicates that the ISO domain is required for neuronal Cl ^- regulation. Furthermore, we discovered that when KCC2 lacks the ISO domain, it still retains swelling-activated transport, which demonstrates that there are exclusive molecular determinants of isotonic and swelling-induced K ⁺-Cl ^- cotransport in neurons.