Enhanced Membrane Incorporation of H289Y Mutant GluK1 Receptors from the Audiogenic Seizure-Prone GASH/Sal Model: Functional and Morphological Impacts on Xenopus Oocytes

Abstract

Epilepsy is a neurological disorder characterized by abnormal neuronal excitability, with glutamate playing a key role as the predominant excitatory neurotransmitter involved in seizures. Animal models of epilepsy are crucial in advancing epilepsy research by faithfully replicating the diverse symptoms of this disorder. In particular, the GASH/Sal (genetically audiogenic seizure-prone hamster from Salamanca) model exhibits seizures resembling human generalized tonic-clonic convulsions. A single nucleotide polymorphism (SNP; C9586732T, p.His289Tyr) in the Grik1 gene (which encodes the kainate receptor GluK1) has been previously identified in this strain. The H289Y mutation affects the amino-terminal domain of GluK1, which is related to the subunit assembly and trafficking. We used confocal microscopy in Xenopus oocytes to investigate how the H289Y mutation, compared to the wild type (WT), affects the expression and cell-surface trafficking of GluK1 receptors. Additionally, we employed the two-electrode voltage-clamp technique to examine the functional effects of the H289Y mutation. Our results indicate that this mutation increases the expression and incorporation of GluK1 receptors into an oocyte's membrane, enhancing kainate-evoked currents, without affecting their functional properties. Although further research is needed to fully understand the molecular mechanisms responsible for this epilepsy, the H289Y mutation in GluK1 may be part of the molecular basis underlying the seizure-prone circuitry in the GASH/Sal model.

Keywords: GluK1; Xenopus oocytes; epilepsy; genetic variant; kainate currents; membrane incorporation.

Figure 1

(A) Schematic representation of the protein sequences of GluK1, WT and mutated (H289Y). Protein domains are represented with colored bars in GluK1_WT: ATD (amino-terminal domain), S1 and S2 are two segments of the polypeptide chain that together form the ligand-binding domain (LBD), M1–M4 represent transmembrane domains, and CTD is the C-terminal domain. Color arrows show protein regions corresponding to the SNP (H289Y) in the ATD, immunogen peptide region (380–430 aa), and the ER retention sequence (amino acids 841–905). The asterisk (*) in yellow indicates the location of single-point mutation in the protein sequences. (B) Schematic representation of the plasmid for in vitro transcription of the distinct variants of Grik1 gene. (C) WebLogo representation of the consensus alignment of the SNP containing region using 50 GluK1 ortholog proteins from different species (78% of conservation), including hamster and humans. It is important to note that the residue at position 289 in WT GluK1 receptor is a histidine (H) in Mesocricetus auratus, Mus musculus, Rattus norvegicus, and Homo sapiens. Color code: polar amino acids (G, S, T, Y, C, Q, N) shown in green, basic (K, R, H) in blue, acidic (D, E) in red, and hydrophobic amino acids (A, V, L, I, P, W, F, M) in black. Bits, in the y-axis, indicate the frequency of the corresponding amino acid with the overall height of each stack proportional to the sequence conservation.

Figure 2

Distribution of GluK1-2a-immunolabeling in Xenopus oocytes containing WT GluK1-2a and H289Y GluK1-2a receptors. Representative confocal microscopy images showing GluK1-2a-immunolabeling in cross-sections of oocytes expressing WT GluK1-2a (A) and H289Y GluK1-2a (B) receptors. Scale bar = 200 μm. MATLAB maps of the oocyte cross-sections corresponding to panels A and B showing comparison of GluK1-2a-immunolabeling between WT GluK1-2a (C) and H289Y GluK1-2a (D) receptors. Inset in the right of the maps show the optical density-to-color calibration bars. NaN stands for not-a-number values. (E) Comparative levels of the sum of the values of the pixels (RawIntDen) corresponding to images (C,D). Each bar represents the mean number of particles ± hemistandard deviation (SEM). Statistical significance: ** p < 0.01.

Figure 3

Confocal immunofluorescence micrographs showing representative cross-sections of animal and vegetal oocyte hemispheres. Immunolabeling for WT GluK1-2a (A–F) and H289Y GluK1-2a (G–L) receptors. Scale bar = 50 μm in (A,D,G,J) and 10 μm in (B,C,E,F,H,I,K,L). (M,N) Relative emission intensity levels (a.u.—arbitrary units—) of immunofluorescence for WT GluK1-2a and H289Y GluK1-2a receptors in the animal (M) and vegetal (N) oocyte hemispheres. Each bar represents the relative intensity ± SEM. (O,P) Expression levels vs. distribution of WT GluK1-2a (O) and H289Y GluK1-2a (P) receptors across 150 μm of the oocyte membrane (as indicated in the oocyte drawing in the left part of the panel). Each bar represents the relative intensity ± SEM. Statistical significance: * p < 0.05, ** p < 0.01.

Figure 4

(A) Representative I_Kas obtained through application of 100 µM kainate (Ka) for 5 s in two oocytes from the same donor, one injected with WT GluK1-2a-coding mRNA (left, black recording) and the other with H289Y GluK1-2a-coding mRNA (right, red recording). Henceforth, bars above the traces show the timing of agonist application, downward deflections represent inward currents, and, unless otherwise stated, the holding potential was −60 mV. (B) Bar diagram showing the average percentage of the normalized I_Kas for cells incorporating either WT or H289Y GluK1-2a receptors. The asterisk (*) indicates significant differences between the two values (p < 0.05 in Mann–Whitney rank-sum test). “n” and “N” specify the number of oocytes and donors (frogs), respectively.

Figure 5

(A) Recordings obtained by applying 1, 5, 10, 50, 100, and 500 μM Ka to an oocyte bearing either WT (black recordings) or H289Y (red recordings) GluK1-2aRs. (B) Averaged Ka concentration–I_Ka amplitude curves for WT (black solid symbols; n = 4, N = 3) and H289Y (red solid symbols; n = 7, N = 3) GluK1-2aRs. Data were normalized to the maximal I_Ka elicited and the Hill equation was fitted to them (continuous lines).

Figure 6

(A) I_Kas evoked using 100 µM Ka when applying voltage pulses from −120 to +60 mV, as shown underneath, in oocytes bearing WT (black recording) or H289Y (red recording) GluK1-2aRs. (B) Net I–V relationship of I_Kas elicited through the protocol shown in A. Black and red symbols are for I_Kas elicited in oocytes that expressed WT (n = 7; N = 3) or H289Y (n = 6; N = 3) GluK1-2aRs, respectively. Net I_Kas were normalized as the percentage of the I_Ka obtained at −60 mV.