The GABAA receptor gamma2 subunit R43Q mutation linked to childhood absence epilepsy and febrile seizures causes retention of alpha1beta2gamma2S receptors in the endoplasmic reticulum - PubMed (original) (raw)

The GABAA receptor gamma2 subunit R43Q mutation linked to childhood absence epilepsy and febrile seizures causes retention of alpha1beta2gamma2S receptors in the endoplasmic reticulum

Jing-Qiong Kang et al. J Neurosci. 2004.

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Abstract

The GABA(A) receptor gamma2 subunit mutation R43Q is an autosomal dominant mutation associated with childhood absence epilepsy and febrile seizures. Previously, we demonstrated that homozygous alpha1beta3gamma2L(R43Q) receptor whole-cell currents had reduced amplitude with unaltered time course, suggesting reduced cell surface expression of functional receptors. In human embryonic kidney 293-T cells, we demonstrate that both heterozygous and homozygous alpha1beta2gamma2S(R43Q) GABA(A) receptor current amplitudes were reduced when receptors were assembled from coexpressed alpha1, beta2, and gamma2S subunits and from beta2-alpha1 tandem subunits coexpressed with the gamma2L subunit. Using fluorescence confocal microscopy, we demonstrated that mutant receptors containing enhanced yellow fluorescent protein-tagged gamma2S subunits had reduced surface expression and were retained in the endoplasmic reticulum. In addition, using biotinylation of surface receptors and immunoblotting, we confirmed that alpha1beta2gamma2S(R43Q) receptors had reduced surface expression. These results provide evidence that the gamma2S(R43Q) mutation impaired GABA(A) receptor function by compromising receptor trafficking and reducing surface expression.

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Figures

Figure 1.

Figure 1.

Heterozygous and homozygous α1β2γ2S(R43Q) receptors had unaltered diazepam sensitivity. A, Whole-cell recordings were made from lifted HEK293-T cells expressing wild-type (wt) hα1β2γ2S or heterozygous (het) or homozygous (hom) hα1β2γ2S(R43Q) receptors. An approximate EC20 value of GABA concentration (2 μ

m

) was applied for 6 sec to cells voltage clamped at -50 mV, and 1 μ

m

diazepam was coapplied with GABA. B, Diazepam enhancement is shown as a percentage of control current (average peak current before and after diazepam coapplication). The γ2S(R43Q) mutation did not alter the magnitude of diazepam enhancement for either heterozygous or homozygous expression with both free hα1β2γ2S assembly and forced rβ2-α1/γ2L assembly (n = 6 for each group).

Figure 2.

Figure 2.

Heterozygous and homozygous α1β2γ2S(R43Q) receptors had reduced current amplitudes. A, Whole-cell currents were obtained from either hα1β2γ2S (wt), hα1β2γ2S(R43Q) (het), or hα1β2γ2S (R43Q) (hom) receptors in free assembly or forced rβ2-α1γ2L assembly. GABA (1 m

m

) was applied for 28 sec. Currents are shown to scale (dark traces) and normalized to wild-type currents (gray traces). The time scale for the first trace applies to all traces. B, C, Peak amplitudes (B) and relative current intensities (C) of heterozygous and homozygous hα1β2γ2S(R43Q) and rβ2-α1γ2L(R43Q) receptor currents were significantly reduced with both free and forced assembly (*p < 0.01 vs wild type; †p < 0.01 vs heterozygous; data are from 9-17 patches from 4 batches of cells).

Figure 3.

Figure 3.

Heterozygous and homozygous hα1β2γ2S(R43Q) receptors were trapped in the ER. A, Representative confocal fluorescence images of Cos-7 cells transfected with hα1β2γ2S-EYFP or hα1β2γ2S(R43Q)-EYFP receptors are presented. R, Receptor; Co, colocalized image. Wt α1β2γ2S-EYFP receptors were primarily in the cell membrane. Heterozygous (het) α1β2γ2S(R43Q)-EYFP receptors were found in both membrane and intracellular compartments. Homozygous (hom)α1β2γ2S(R43Q)-EYFP receptors were found primarily in intracellular compartments with minimal cell surface localization. Both heterozygous and homozygous receptors had a fluorescence pattern that was similar in distribution to the pECFP-ER fluorescence pattern (

supplemental material

, available at

www.jneurosci.org

). In B, the relative membrane/ER fluorescence intensity ratios for heterozygous and homozygous receptors were significantly reduced compared with that for the wild-type receptors (see Materials and Methods) (for each group, 9-11 randomly chosen cells were measured from 5 batches; *p < 0.01 vs wild type).

Figure 4.

Figure 4.

Heterozygous and homozygous hα1β2γ2S(R43Q) receptors had reduced surface expression on HEK293-T cells. A, B, HEK293-T cells transfected with wild-type (wt) or heterozygous (het) or homozygous (hom) hα1β2γ2S(R43Q) receptors were biotinylated and immunoblotted with antibodies against the α1 subunit (A) and GFP variant EYFP (B). Expression of heterozygous or homozygous α1β2γ2S(R43Q) receptors resulted in similar levels of whole-cell protein expression (total) but reduced cell surface protein expression (surface) compared with wild-type receptors. C, HEK293-T cells transfected with wild-type or heterozygous or homozygous α1β2S(R43Q) receptors and pECFP-Mem were biotinylated, and whole-cell protein was precipitated with antibody against the human α1 subunit and detected by antibody against the γ2 subunit. Heterozygous and homozygous α1β2γ2S(R43Q) receptors revealed a similar protein expression in whole-cell level (total) but a reduced protein expression in the cell surface (surface). β-Actin was used as a control to demonstrate that equal amounts of protein were loaded (β-actin). Similarly, pECFP-Mem protein expression was also similar in each group (pECFP-Mem). D, The optical absorbency of Western blots was quantified with Bio-Rad QuantifyOne. In each group, heterozygous protein intensities were lower than wild type but higher than homozygous receptors (*p < 0.05 vs wild type; †p < 0.05 vs heterozygous; data are from 5 experiments).

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References

    1. Angelotti TP, Macdonald RL (1993) Assembly of GABAA receptor subunits: α1β1 and α1β1γ2S subunits produce unique ion channels with dissimilar single-channel properties. J Neurosci 13: 1429-1440. - PMC - PubMed
    1. Baumann SW, Baur R, Sigel E (2001) Subunit arrangement of γ-aminobutyric acid type A receptors. J Biol Chem 276: 36275-36280. - PubMed
    1. Bianchi MT, Song L, Zhang H, Macdonald RL (2002) Two different mechanisms of disinhibition produced by GABAA receptor mutations linked to epilepsy in humans. J Neurosci 22: 5321-5327. - PMC - PubMed
    1. Boileau AJ, Li T, Benkwitz C, Czajkowski C, Pearce RA (2003) Effects of γ2S subunit incorporation on GABAA receptor macroscopic kinetics. Neuropharmacology 44: 1003-1012. - PubMed
    1. Bolte S, Talbot C, Boutte Y, Catrice O, Read ND, Satiat-Jeunemaitre B (2004) FM-dyes as experimental probes for dissecting vesicle trafficking in living plant cells. J Microsc 214: 159-173. - PubMed

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