Hyperpolarizing GABAergic transmission requires the KCC2 C-terminal ISO domain

Abstract

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 cotransport K(+) and Cl(-) out of the neuron under isotonic conditions. We made 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.

Figure 1.

KCC2ΔISO does not transport K⁺ or Cl⁻ under isotonic conditions. A, Fluorescent images of cells overexpressing KCC2ΔISO; transfected neurons were chosen for electrophysiological experiments based on their expression of GFP. B, Examples of gramicidin perforated patch-clamp recordings obtained from neurons overexpressing KCC2 (i), KCC2ΔISO (ii), and vector alone (iii). The IPSC amplitude was plotted against the holding potential, and the intercept of this curve with the x-axis was taken as E_Cl (arrows). Insets, Traces of IPSCs for the examples shown. Scale bars: 60 pA, 150 ms. C, Summary of all experiments similar to B shown as a distribution plot. ***p < 0.001.

Figure 2.

Expression of KCC2-KCC4 chimeric proteins in hippocampal neurons. A, Confocal microscopic immunofluorescent images from single optical sections of anti-HA and GFP in cultured hippocampal neurons. Neurons were stained for anti-HA (Cy3, red). i, ii, KCC2ΔISO-HA. iii, iv, KCC2-HA. Scale bar, 20 μm. B, Summary of anti-HA normalized cluster intensity for all GFP-positive neurons (KCC2ΔISO-HA: n = 13; KCC2-HA: n = 12; p = 0.5). Neurons expressing the KCC2-HA construct show hyperpolarized values of E_cl while KCC2ΔISO-HA is devoid of chloride transport (Bii). *p < 0.05.

Figure 3.

Hypotonic shock recovers K⁺-Cl⁻ transport function in KCC2ΔISO. A, Examples of gramicidin perforated patch-clamp recordings obtained from neurons in hippocampal culture overexpressing KCC2ΔISO in isotonic (300 mOsM) (i) and hypotonic (180 mOsM) (ii) extracellular solution. The IPSC amplitude was plotted against the holding potential, and the intercept of this curve with the x-axis was taken as E_Cl (arrows). Insets, Traces of IPSCs for the examples shown. Scale bars: 100 pA, 150 ms. B, Summary of the change in E_Cl from isotonic to hypotonic conditions for both untransfected and transfected cultured hippocampal neurons. Transfected neurons overexpressed KCC2, KCC2ΔISO, KCC4, or an empty vector. Data were not paired, but were obtained from separate populations of neurons (in either isotonic or hypotonic conditions). ***p < 0.001.

Figure 4.

The KCC4-ISO rescue of isotonic transport depends on neuronal Cl⁻. A, Summary figure plotting the distribution of E_Cl in neurons overexpressing KCC2-KCC4 chimeric proteins under isotonic extracellular conditions. B, Summary figure plotting the distribution of E_Cl in neurons overexpressing KCC2-KCC4 chimeric proteins and loaded with 50 mm intracellular Cl⁻ under isotonic extracellular conditions. *p < 0.05, ***p < 0.001.