Why does fever trigger febrile seizures? GABAA receptor gamma2 subunit mutations associated with idiopathic generalized epilepsies have temperature-dependent trafficking deficiencies - PubMed (original) (raw)

Comparative Study

Why does fever trigger febrile seizures? GABAA receptor gamma2 subunit mutations associated with idiopathic generalized epilepsies have temperature-dependent trafficking deficiencies

Jing-Qiong Kang et al. J Neurosci. 2006.

Abstract

With a worldwide incidence as high as 6.7% of children, febrile seizures are one of the most common reasons for seeking pediatric care, but the mechanisms underlying generation of febrile seizures are poorly understood. Febrile seizures have been suspected to have a genetic basis, and recently, mutations in GABAA receptor and sodium channel genes have been identified that are associated with febrile seizures and generalized seizures with febrile seizures plus pedigrees. Pentameric GABAA receptors mediate the majority of fast synaptic inhibition in the brain and are composed of combinations of alpha(1-6), beta(1-3), and gamma(1-3) subunits. In alphabetagamma2 GABAA receptors, the gamma2 subunit is critical for receptor trafficking, clustering, and synaptic maintenance, and mutations in the gamma2 subunit have been monogenically associated with autosomal dominant transmission of febrile seizures. Here, we report that whereas trafficking of wild-type alpha1beta2gamma2 receptors was slightly temperature dependent, trafficking of mutant alpha1beta2gamma2 receptors containing gamma2 subunit mutations [gamma2(R43Q), gamma2(K289M), and gamma2(Q351X)] associated with febrile seizures was highly temperature dependent. In contrast, trafficking of mutant alpha1beta2gamma2 receptors containing an alpha1 subunit mutation [alpha1(A322D)] not associated with febrile seizures was not highly temperature dependent. Brief increases in temperature from 37 to 40 degrees C rapidly (<10 min) impaired trafficking and/or accelerated endocytosis of heterozygous mutant alpha1beta2gamma2 receptors containing gamma2 subunit mutations associated with febrile seizures but not of wild-type alpha1beta2gamma2 receptors or heterozygous mutant alpha1(A322D)beta2gamma2 receptors, suggesting that febrile seizures may be produced by a temperature-induced dynamic reduction of susceptible mutant surface GABAA receptors in response to fever.

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Figures

Figure 1.

Figure 1.

Elevated temperature further reduced surface protein expression of mutant γ2S subunit-containing GABAA receptors. A, Western blot analysis of biotinylated γ2S and α1 subunit surface proteins from HEK293T cells expressing wild-type and heterozygous mutant α1β2γ2S-EYFP receptors incubated at 37 or 40°C for 1 h. The cell membranes were biotinylated, and equal amounts of protein were conjugated with beads and loaded, and the membranes were immunoblotted with mouse monoclonal anti-GFP and anti-α1 antibodies. B, Quantification of Western blots of biotinylated EYFP-tagged γ2S subunit surface protein from HEK293T cells expressing wild-type and mutant α1β2γ2S-EYFP receptors incubated at different temperatures.C, Quantification of Western blots of biotinylated α1 subunit surface protein from HEK293T cells expressing wild-type and mutant GABAA α1β2γ2S-EYFP receptors incubated at different temperatures. B, C, In all groups, data represent the mean ± SEM (n = 4; *p < 0.05, **p < 0.01 vs wild type at 37°C; ††p < 0.01 vs wild type at 40°C; §p < 0.05 vs the same mutation at 37°C; two-tailed unpaired Student’s t test).

Figure 2.

Figure 2.

Elevated temperature rapidly increased intracellular retention of mutant γ2S subunit-containing GABAA receptors. A, Confocal microscopy images of fluorescence-tagged wild-type and mutant γ2S subunit-containing α1β2γ2S receptors and α1(A322D) subunit-containing α1β2γ2S receptors in COS-7 cells after a 30 min incubation at 40°C. wt, ECFP-tagged wild-type subunits; mut, EYFP-tagged mutant subunits; mem, membrane marker FM4-64; co, colocalization of all ECFP, EYFP, and FM4-64 channels. With heterozygous expression, mutant and wild-type γ2S(R43Q), γ2S(K289M), and γ2S(Q351X) subunits and mutant α1(A322D), but not wild-type α1, subunits were localized intracellularly in compartments that had the morphology of the ER and colocalized with the ECFP-ER marker (data not shown). The loss of surface receptor illustrated here was an extreme example to illustrate the point. Other cells showed less extensive loss of surface receptor.B, Total membrane surface receptor fluorescence pixel intensity values of heterozygous α1β2γ2S(R43Q), α1β2γ2S(K289M), and α1β2γ2S(Q351X) receptors were reduced after incubation at 37°C compared with wild-type receptors (filled bars) and were further reduced after a 30 min incubation at 40°C (open bars). In all groups, data represent the mean ± SEM (n = 19–23 cells from 5 transfections; *p < 0.05 vs wild type at 37°C;†p < 0.05,††p < 0.01 vs wild type at 40°C;§p < 0.05,§§p < 0.01 vs the same mutation at 37°C; two-tailed unpaired Student’s t test).

Figure 3.

Figure 3.

Reduction of surface receptor and increased intracellular retention with elevated temperature in mutant γ2(K289M) subunit-containing α1β2γ2 receptors was rapid and dynamic in heterologous cells. The representative images illustrate the rapid time course of the reduction of heterozygous α1β2γ2S-ECFP/γ2S(K289M)-EYFP surface receptors in COS-7 cells. Cells were cotransfected with heterozygous α1β2γ2S/γ2S(K289M) receptors with the wild-type γ2S tagged with ECFP and the mutant γ2S subunit tagged with EYFP. The receptors displayed a merged aqua color both on the surface and intracellularly after incubation at 40°C for 5 min (boxed area). With incubation at 40°C over a rapid time course, the fluorescence intensity of the surface receptors progressively diminished (7 and 11 min, bottom panel, red boxes) with loss of cyan color on the cell surface (changes of cyan color in the red box, red arrow), and the intracellular fluorescence intensity progressively increased with the accumulation of intracellular receptors (changes of cyan color of the coregistered image in the red box; red double arrow). wt, ECFP-tagged wild-type subunits; mut, EYFP-tagged mutant subunits; co, colocalization of all ECFP and EYFP channels.

Figure 4.

Figure 4.

Rapid reduction of heterozygous α1β2γ2/α1β2γ2(Q351X) receptors with elevated temperature on the membrane surface of hippocampal neurons.A, Heterozygous α1β2γ2L-pHluorin/α1β2γ2L(Q351X)-pHluorin receptor on the surface of rat hippocampal neurons was reduced rapidly by temperature elevation to 40°C. As illustrated in the left panels, neurons were cotransfected with heterozygous α1β2γ2L-pHluorin/α1β2γ2L(Q351X)-pHluorin receptors, and receptors were imaged as puncta on the surface of neurons. With incubation at 40°C, the fluorescent puncta were reduced, with loss or fading of fluorescence on the cell surface (red arrows). TI, Transmitted image; wt, wild-type α1β2γ2L-pHluorin receptors; mut, heterozygous mutant α1β2γ2L-pHluorin/α1β2γ2L(Q351X)-pHluorin receptors. B, Membrane surface fluorescence clusters of heterozygous mutant α1β2γ2L-pHluorin/α1β2γ2L(Q351X)-pHluorin receptors were reduced after incubation at 40°C at different times compared with wild-type receptors measured over equivalent areas and were further reduced after a 20 min incubation at 40°C compared with the receptor fluorescence puncta in the same regions. In all groups, data represent the mean ± SEM (n = 10–14 neurons from 4 transfections; *p < 0.05 vs wild type at the same time points;†p < 0.05 vs the same mutation after incubation at 40°C for 3 min; two-tailed unpaired Student’s_t_ test).

Figure 5.

Figure 5.

Both wild-type and mutant γ2S subunit-containing GABAA receptors demonstrated a trafficking/recycling deficiency with elevated temperature.A, Representative currents are presented from a single cell expressing wild-type α1β2γ2S receptors recovering at 25°C from 2.5 h of incubation at 40°C. B, Wild-type receptor peak current amplitudes increased over a 45 min time course at 25°C and then stabilized after incubation at 40°C for 2.5 h. The ordinate denotes current amplitudes at each time point over the current amplitude obtained at 120 min after incubation at 25°C. Data were averaged from four cells.C, Representative currents are presented that were recorded at room temperature from cells that were incubated at 40°C for 1 h.D, Mutant γ2S subunit-containing receptors challenged with 40°C had reduced peak currents compared with wild-type and with the same mutant γ2S subunit-containing receptors at 37°C. In each group, data represent the mean ± SEM (n = 12–17; *p < 0.05 vs wild type at 37°C;†p < 0.05 and††p < 0.01 vs wild type at 40°C;§p < 0.05 and§§§p < 0.001 vs the same mutations at 37°C; two-tailed unpaired Student’s t test). All currents were recorded in HEK293T cells under lifted whole-cell configurations and were voltage clamped at −50 mV.

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