Mutations in SLC12A5 in epilepsy of infancy with migrating focal seizures

Abstract

The potassium-chloride co-transporter KCC2, encoded by SLC12A5, plays a fundamental role in fast synaptic inhibition by maintaining a hyperpolarizing gradient for chloride ions. KCC2 dysfunction has been implicated in human epilepsy, but to date, no monogenic KCC2-related epilepsy disorders have been described. Here we show recessive loss-of-function SLC12A5 mutations in patients with a severe infantile-onset pharmacoresistant epilepsy syndrome, epilepsy of infancy with migrating focal seizures (EIMFS). Decreased KCC2 surface expression, reduced protein glycosylation and impaired chloride extrusion contribute to loss of KCC2 activity, thereby impairing normal synaptic inhibition and promoting neuronal excitability in this early-onset epileptic encephalopathy.

Figure 1. Clinical and molecular genetic findings in families with EIMFS.

(a) Family A (Caucasian origin) and Family B (Pakistani origin). Squares represent males. Circles represent females. Affected individuals are represented by black shading. Double parallel horizontal bars indicate consanguinity in Family B. (b) The left upper and lower EEG recording are two consecutive EEG segments, each 1 min long, from the ictal EEG of proband A-II:1. Seizure activity is boxed in black with rhythmic spike-waves evident initially on the left hemisphere (red), then fading and seizure activity starting up in the right (blue) temporal area. Expanded segments below (10 s) show the spike-wave activity in more detail. Montage: Longitudinal bipolar according to the 10–20 system. Only lateral channels shown. The right upper (compressed) and lower EEG recording from proband B-II:4 reveal central/vertex and right parietal epileptiform activity (purple/red and boxed in red, upper right figure) that wanes with a return to the interictal state before onset of left centro-temporal ictal activity associated with facial twitching (blue and boxed in red, lower right figure). (c) SLC12A5 consists of 26 exons (purple rectangles). Both wild-type (WT) and mutant DNA sequence is displayed. Three point mutations were identified in four patients with EIMFS (WT sequence indicated by yellow arrow, mutated base by orange arrow). The children from Family A harboured missense mutations in exon 9 (c.1277T>C, L426P) and 13 [c.1652G>A, G551D], while the two affected Family B patients were homozygous for missense variant c.932T>A (L311H) in exon 8. (d) The KCC2 protein structure consists of 12 transmembrane (TM) helices (numbered blue cylinders) interconnected by a series of extracellular and intracellular loops. Sites of phosphorylation at the C-terminus are depicted as light green hexagons. N-linked glycosylated sites are indicated as dark green Y-shaped structures present at the extracellular loop between TM5 and TM6. The EIMFS mutations are indicated in red circles, located in the extracellular loop between TM5 and TM6 (L311H), within TM6 (L426P) and in the intracellular loop between TM8 and TM9 (G551D). Variants associated with idiopathic generalized epilepsy and febrile convulsions are represented as purple circles at the C-terminus.

Figure 2. Structural modeling of KCC2 mutants.

(a) A homology model of KCC2 showing a transmembrane (TM) protein consisting of 12 TM helices interconnected by intracellular and extracellular loops. (b) In our homology model, L426 (shown in a surface representation) is located in the unwound TM6 (TM6a). This helix, together with TM1, is pivotal for the transporter function. In the L426P mutant, the substitution of a leucine with a proline (not shown) is likely to introduce a kink in TM6, which will result in alternation of the interaction with TM1b. A comparative analysis of different structures of transporters (LeuT, vSGLT and ApcT structures) shows that the conformation of TM1b plays a key role in allowing access to the substrate binding pocket (maintaining a conformation that completely occludes access to the post-synaptic space but only partially occludes access to the cytosolic side). Thus, the presence of a proline in KCC2 TM6 could lead to an alteration of the extracellular occlusion formed by TM1b and TM6. (c) Our KCC2 model predicts that G551 (shown in a surface representation) is located in the intracellular loop between TM8 and TM9 (residues 539–557). In the G551D mutant, the substitution of the glycine with an aspartic acid—a larger and negatively charged amino acid—is likely to introduce a distortion in the orientation of TM8. The latter helix contains the putative substrate binding residues A521 and S525, based on homology to ApcT (A287 and T291, respectively, PDB: 3GIA). Therefore, introducing an aspartic acid in position 551 could lead to disruption of the binding site thereby affecting substrate binding.

Figure 3. Electrophysiology of wild-type and mutant KCC2.

(a–c) KCC2 mutants exhibit a depolarized E_Cl. (a) Examples of I–V relationships in HEK293 cells transfected either with GlyR α₂ alone (no KCC2), or GlyR α₂ with a wild-type KCC2 construct. I_Gly amplitude was plotted against the holding potential and the intercept of the line of fit of this relation with the abscissa was taken as E_Cl. Superimposed traces show examples of glycine-evoked currents recorded from individual cells at different holding potentials (–80 to +20 mV). (b) Examples of I–V relationships in HEK293 cells co-transfected with GlyR α₂ and mutant KCC2, as indicated. E_Cl in mutant KCC2 is depolarized in comparison to wild-type (fit of I–V relation indicated by dashed-line). Calibration bars for 3a and 3b: 1nA, 1sec. (c) Summary chart plotting the distribution of E_Cl for each cell group: no KCC2: –14.7±0.8 mV (n=5), GlyR-KCC2: –29±3.5 mV (n=9), G551D: –17.4±1.2 mV (n=5), L426P: –17.9±2.9 mV (n=5), L311H: –15±3.1 mV (n=9); *P=0.006, one-way analysis of variance (ANOVA). Grey symbols represent measurements from individual cells. Boxes represent the mean±standard error of the mean. GlyR α₂: glycine receptor α₂, Wt-KCC2: wild-type K⁺-Cl⁻ co-transporter. (d–f) The rate of Cl⁻ extrusion is impaired in KCC2 mutants. (d) I_Gly amplitude plotted against time in a HEK293 cell co-transfected with GlyRα₂ and wild-type KCC2 and held at different membrane potentials as indicated in the top image. Vertical bars indicate the times of successive glycine applications. Example traces show glycine-evoked currents at times indicated by letters in italic. Calibration bars: 1 nA, 300 ms. (e) Rate of recovery of I_Gly amplitude after switching back the holding potential from +40 to −80 mV. Fine lines replace symbols and error bars are omitted for legibility. An exponential decay function was used to fit I_Gly amplitude ratio in each cell group. (f) Summary bar graph of the time-constant of I_Gly recovery in each group (no KCC2: 224±56.7 s, n=6; WT-KCC2: 59.5±7.7 s, n=13; L426P: 162.5±42.1 s, n=4; G551D: 340.1±137 s, n=4; L311H: 88.1±11.5 s, n=7). *Different from WT-KCC2 (P<0.01), ANOVA.

Figure 4. Immunoblotting studies for KCC2 mutants.

Immunoblotting studies including cell surface biotinylation and total cell lysate studies. Two bands are observed for WT and the three mutant KCC2 proteins closely located to each other at ∼130 kDa and corresponding to glycosylated (upper band) and unglycosylated (lower band) states.

Figure 5. Immunofluorescence microscopy for KCC2 mutants.

L311H, L426P and G551D substitutions impair cell-surface expression of KCC2. HEK293 cells were co-transfected with KCC2-FLAG and HsRed-NLS and immunostained using anti-FLAG and AlexaFluor 488 antibodies. KCC2 WT is detected at the cell surface of intact cells (a) whereas KCC2-L311H, -L426P and -G551D display very little cell surface expression (b–d) Expression of KCC2-WT,-L311H,-L426P and -G551D is detected in permeabilised cells (e–h) Scale bar is 20 μm.

Figure 6. Schematic representation of postulated disease mechanisms in KCC2-EIMFS.

A post-synaptic neuron is schematically represented for (a) wild-type and (b) mutant KCC2 in EIMFS. Neurotransmitters and ions are depicted as shapes, namely GABA (red circle), potassium (K⁺, magenta diamond) and chloride (Cl⁻, blue circle). The mature KCC2 transporter (orange) with N-linked glycosylated extracellular domains (black branched structure) is located at the synaptic membrane. The GABA_A receptor (GABA_AR) is a purple oval. In normal mature neuronal cells (a) KCC2 transporter (bold orange) maintains low intraneuronal chloride (represented by blue fluid level) through chloride extrusion (solid orange lines). With GABA binding, the resultant gradient allows an influx of chloride via the GABA_AR resulting in a hyperpolarizing IPSP (inhibitory post-synaptic potential), which contributes to neuronal inhibition. In KCC2-EIMFS (b) a number of mechanisms contribute to loss of KCC2 function (faded orange KCC2 transporter) and impaired transporter ability to extrude chloride (dashed orange lines), including reduced cell surface transporter expression and abnormal protein glycosylation. This results in a depolarizing inhibitory postsynaptic potential, thereby leading to impaired neuronal inhibition.