A variant of KCC2 from patients with febrile seizures impairs neuronal Cl- extrusion and dendritic spine formation

Abstract

Genetic variation in SLC12A5 which encodes KCC2, the neuron-specific cation-chloride cotransporter that is essential for hyperpolarizing GABAergic signaling and formation of cortical dendritic spines, has not been reported in human disease. Screening of SLC12A5 revealed a co-segregating variant (KCC2-R952H) in an Australian family with febrile seizures. We show that KCC2-R952H reduces neuronal Cl(-) extrusion and has a compromised ability to induce dendritic spines in vivo and in vitro. Biochemical analyses indicate a reduced surface expression of KCC2-R952H which likely contributes to the functional deficits. Our data suggest that KCC2-R952H is a bona fide susceptibility variant for febrile seizures.

Keywords: KCC2; dendritic spines; febrile seizures; genic intolerance; mutation.

© 2014 The Authors. Published under the terms of the CC BY NC ND license.

Figures

Figure 1. Segregation, conservation and location of the KCC2-R952H variant

A Pedigree of the family with the KCC2-R952H variant, showing segregation of the variant with the febrile seizures phenotype. B Amino acid sequence alignment of KCC2 shows high conservation of R952 among different species. C Putative membrane topology of KCC2. R952H is located in the distal part of the intracellular C-terminus.

Figure 2. The R952H substitution in KCC2 leads to a reduction in surface expression

A Western blot analysis of total protein shows comparable expression levels of KCC2-WT and KCC2-R952H, when heterologously expressed in C17.2 cells. B Quantification of total (n = 5) and cell surface (n = 9) protein expression of KCC2-R952H, normalized to KCC2-WT. Statistical analysis was performed using Wilcoxon matched pairs test. **P < 0.01. Error bars represent SEM. C Representative Western blot of biotinylated (s, surface) and non-biotinylated (i, internal) KCC2-WT and KCC2-R952H. Tubulin and transferrin receptor (TfR) served as loading controls. Source data are available online for this figure.

Figure 3. Cl− extrusion capacity measurements from in utero electroporated cortical neurons reveal impaired Cl− extrusion by KCC2-R952H

A IUE of EGFP and either KCC2-WT or KCC2-R952H at embryonic day 14.5 targets mouse cortical layer 2/3 pyramidal neurons (analysis at postnatal day (P) 6). Transfected neurons co-express either of the KCC2 constructs (red) with EGFP, while endogenous mKCC2 levels in non-transfected neurons at this age are low. Scale bars: 100 μm and 20 μm (insets). B Whole-cell patch clamp recordings of GABA uncaging-elicited GABAA-mediated currents (IGABA) in transfected cortical layer 2/3 pyramidal neurons with a somatically imposed Cl− load. Sample EGABA recordings and corresponding I-V curves at the soma and dendrite. Horizontal bars in the sample traces indicate the duration of the uncaging UV-flash. C Cl− extrusion capacity of P6-7 cortical pyramidal neurons expressing EGFP (n = 8), KCC2-WT (n = 15), KCC2-R952H (n = 16) or rKCC2-ΔNTD (n = 8) was quantified as the somatodendritic EGABA gradient (ΔEGABA = EGABA-soma − EGABA-dendrite). EGFP-negative neurons (n = 17) served as controls. Statistical analysis was performed using one-way ANOVA with Holm–Sidak post hoc test. *P < 0.05; **P < 0.01. Error bars represent SEM.

Figure 4. KCC2-R952H is unable to induce dendritic spines in vivo or rescue mature spine morphology of cortical mKCC2−/− neurons

A In utero electroporation of E17.5 rat embryos with KCC2-WT leads to an increase of both apical (n = 28 neurons) and basal dendrite (n = 40) spine density of cortical layer 2/3 pyramidal neurons, when compared to non-transfected neighboring neurons (apical, n = 18; basal, n = 22). In contrast, KCC2-R952H is unable to induce dendritic spines (transfected: apical, n = 37; basal = 46; non-transfected: apical, n = 22; basal, n = 28). Analysis of spine density was performed on P15. Right panel: representative confocal images of spine densities. Statistical analysis was performed using Student’s paired t-test. *P < 0.05; **P < 0.01. B Expression of KCC2-R952H is incapable of rescuing normal spine morphology in cultured mKCC2−/− cortical neurons. Aberrant spine morphology of mKCC2−/− neurons is rescued by expression of KCC2-WT (n = 19 neurons), but not KCC2-R952H (n = 12) or EGFP (n = 12). Right panel: representative confocal images of spine lengths. Statistical analysis was performed using the Kolmogorov–Smirnov test. ***P < 0.001. Data information: Scale bars, 5 μm. Error bars represent SEM.