Novel Functional Properties of Missense Mutations in the Glycine Receptor β Subunit in Startle Disease

Abstract

Startle disease is a rare disorder associated with mutations in GLRA1 and GLRB, encoding glycine receptor (GlyR) α1 and β subunits, which enable fast synaptic inhibitory transmission in the spinal cord and brainstem. The GlyR β subunit is important for synaptic localization via interactions with gephyrin and contributes to agonist binding and ion channel conductance. Here, we have studied three GLRB missense mutations, Y252S, S321F, and A455P, identified in startle disease patients. For Y252S in M1 a disrupted stacking interaction with surrounding aromatic residues in M3 and M4 is suggested which is accompanied by an increased EC₅₀ value. By contrast, S321F in M3 might stabilize stacking interactions with aromatic residues in M1 and M4. No significant differences in glycine potency or efficacy were observed for S321F. The A455P variant was not predicted to impact on subunit folding but surprisingly displayed increased maximal currents which were not accompanied by enhanced surface expression, suggesting that A455P is a gain-of-function mutation. All three GlyR β variants are trafficked effectively with the α1 subunit through intracellular compartments and inserted into the cellular membrane. In vivo, the GlyR β subunit is transported together with α1 and the scaffolding protein gephyrin to synaptic sites. The interaction of these proteins was studied using eGFP-gephyrin, forming cytosolic aggregates in non-neuronal cells. eGFP-gephyrin and β subunit co-expression resulted in the recruitment of both wild-type and mutant GlyR β subunits to gephyrin aggregates. However, a significantly lower number of GlyR β aggregates was observed for Y252S, while for mutants S321F and A455P, the area and the perimeter of GlyR β subunit aggregates was increased in comparison to wild-type β. Transfection of hippocampal neurons confirmed differences in GlyR-gephyrin clustering with Y252S and A455P, leading to a significant reduction in GlyR β-positive synapses. Although none of the mutations studied is directly located within the gephyrin-binding motif in the GlyR β M3-M4 loop, we suggest that structural changes within the GlyR β subunit result in differences in GlyR β-gephyrin interactions. Hence, we conclude that loss- or gain-of-function, or alterations in synaptic GlyR clustering may underlie disease pathology in startle disease patients carrying GLRB mutations.

Keywords: GLRB; gephyrin; glycine receptor; hyperekplexia; startle disease.

FIGURE 1

Molecular modeling of novel GlyR β subunit mutants. (A) Alignment of GlyR subunits α1, α2, α3, β subunits from human and the β subunit variants concentrating on transmembrane segments. Numbers of amino acid residues in mutant variants refer to non-mature protein, (SP) signal peptide. (B–D) Cartoon representation of the 3α:2β glycine receptor heteropentamer homology model viewed from extracellular domain (B), from the intracellular side (C) and the membrane plane (D). GlyR α-subunits are colored orange and yellow and β-subunits are colored in green. (E) Close-up views of the interaction of residues in WT (left panels) and the mutant (right panels) model. All critical residues are shown as ball and stick, whereas the backbone is shown in cartoon representation, numbering refers to mature protein.

FIGURE 2

Subcellular expression and trafficking of GlyR β mutants. (A) Immunocytochemical staining of HEK-293 (upper two lanes) or COS-7 cells (lower lanes) transfected with GlyR α1 and β WT or β subunit variants (1:10 = α1 WT:β WT or β^x variant with x = either Y252S, S321F, or A455P). GlyR β or β^x variants were stained with an anti-myc antibody (cyan), cellular compartment marker (GAP-43: cell membrane, calreticulin (Cal): endoplasmic reticulum, ERGIC: ER-Golgi intermediate compartment, GM130: Golgi) are always shown in magenta. Enlargements (marked by white box) are provided on the right next to each image, arrow heads point to co-localization or accumulation of both labeled proteins. Scale bars refer to 10 μm. (B) Representative Western blot of whole cell protein lysates from HEK-293 cells transfected with α1 WT and β WT or β^x variants in a ratio of 1:2. GlyR β is detected at 58 kDa. Pan-cadherin (Pan-Cad) served as loading control (130 kDa). (C) Quantitative analysis of GlyR β and β^x variants from whole cell lysates (n = 3, three independent experiments) of co-expressed GlyR α1 together with the GlyR β subunit (red bar) or GlyR β^x variants (black bars).

FIGURE 3

Glycine receptor β subunit mutants alter functional properties of the chloride channel. Electrophysiological measurements of transfected HEK-293 cells. GlyR α1 alone (green bar), co-transfection of α1 and β WT (red bar) or β variants (Y252S, S321F, and A455P; black bars). (A) Block of the glycine-gated response using 100 μM glycine and 100 μM of picrotoxinin. Bars show residual currents after picrotoxinin block normalized to currents evoked by 100 μM glycine application. Dotted line shows cut-off for determination of heteromeric receptor configuration. Only cells above the cut-off were used for analysis. (B,C) I_max and EC₅₀ mean values are depicted. (D–F) Dose-response curves for α1β (red line) or α1β^x variants [Y252S (D), S321F (E), and A455P (F); black lines] using increasing glycine concentrations (10, 30, 60, 100, 300, 600, and 1,000 μM). Values were normalized to the I_max mean value of the according α1β variant following application of 1,000 μM glycine. (G–I) Dose-response curves of α1β (red line) or α1β^x variants [(G) Y252S, (H) S321F, and (I) A455P black lines] normalized to WT α1β I_max mean value following application of 1,000 μM glycine. Representative traces at 1,000 μM glycine application of α1β (red traces) and α1β^x variants are depicted in the upper left corner of each diagram. Significance values are *p < 0.05, **p < 0.01, and ***p < 0.001.

FIGURE 4

GlyR β variants for cytoplasmic aggregates in the presence of gephyrin. (A) Immunocytochemical stainings of HEK-293 cells transfected with eGFP-gephyrin alone or GlyR α1, eGFP-gephyrin and β WT or β variants (Y252S, S321F and A455P; ratio 1:5:10). Myc-tagged GlyR β WT or missense variants were stained with a specific anti-myc antibody (cyan, lower lane), eGFP-gephyrin is shown in yellow (middle lane), merge images are depicted in the upper lane. Nucleus is marked in blue (DAPI). Scale bar refers to 10 μm in all images. (B–G) Bar graphs of gephyrin aggregate analysis of HEK-293 cells expressing eGFP-gephyrin (blue bars), eGFP-gephyrin and α1β WT (red bars) or eGFP-gephyrin and α1β variants (black bars): (B) number of gephyrin aggregates, (C) area of gephyrin aggregates, (D) perimeter of gephyrin aggregates, (E) number of β (c-myc) aggregates, (F) area of β (c-myc) aggregates, (G) perimeter of β (c-myc) aggregates, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 5

Membrane expression of GlyR β variants is altered in the presence of gephyrin. (A) HEK-293 transfected with GlyR α1, eGFP-gephyrin and β WT or β^x variants (Y252S, S321F or A455P). Cellular compartment markers (GAP-43: cell membrane, calreticulin (Cal): endoplasmic reticulum) are always co-transfected (magenta). GlyR β WT or variants are stained with an anti-myc antibody (cyan), eGFP-gephyrin is shown in yellow. White boxes mark the areas shown in the enlarged images. Arrow heads point to co-localization or accumulation of eGFP-gephyrin and β WT or β variants. Scale bars refer to 10 μm. (B,C) Quantitative analysis of eGFP-gephyrin in whole cell fraction (B) and surface (C) fractions (n = 4, four independent experiments) of co-expressed GlyR α1 together with β WT (red bar) or GlyR β variants (black bars) and eGFP-gephyrin. (D) Representative Western blots of whole cell and surface fractions from transfected HEK-293 cells. eGFP-gephyrin (geph) is detected at the appropriate molecular weight of 93 kDa, Pan-cadherin (Pan-Cad) served as loading control for surface fraction (130 kDa) and GAPDH served as loading control for whole cell fraction (30 kDa). Level of significance *p < 0.05.

FIGURE 6

GlyR functional properties do not change in the presence of gephyrin. (A) Verification of heteromeric receptor configuration using picrotoxinin block. GlyR α1β WT and α1β^x variants were co-expressed with eGFP-gephyrin in HEK-293 cells. The remaining current (%) upon co-application of 100 μM glycine and 100 μM picrotoxinin compared to 100 μM glycine alone is shown. Dotted blue line at 50% picrotoxinin block divides between homomeric (below blue line) and heteromeric (above blue line) receptor configuration. (B) Stack plot of the ratio between homomeric (green) and heteromeric (black) receptor configurations of α1β WT and α1β^x variants in the absence and presence of eGFP-gephyrin. Significance value *p < 0.05. (C) Glycine-activated currents at saturating glycine concentration (1 mM). Note, the GlyR β^A455P variant again exhibited significantly increased I_max values. (D) EC₅₀ values for α1β WT and α1β^x variants in the presence of eGFP-gephyrin were determined following application of a concentration series of glycine (10, 30, 60, 100, 300, 600, and 1,000 μM). (E) Dose response curves of α1β^Y252S shows a rightward shift to higher glycine concentration compared to α1β WT in the presence of eGFP-gephyrin. *p < 0.05, ***p < 0.001.

FIGURE 7

GlyR β variants form clusters with endogenous gephyrin but exhibit less synaptic localization. (A) Hippocampal neurons transfected with α1β WT and α1β^x variants. The GlyR β subunit was detected with a myc-antibody (cyan), as well as endogenous gephyrin (yellow), and synapsin (magenta). White boxes indicate dendritic areas shown in the enlarged images on the right. White open arrowheads show co-localization of GlyR β in gephyrin-positive and synapsin-positive clusters. Scale bar refers to 25 μm. (B) Quantification of GlyR β-positive synapses. Calculation was performed from 40 to 92 dendrites (n = 40–92) from transfected neurons obtained from two independent experiments. A ratio of myc-GlyR β-positive synapses versus gephyrin-positive synapses was estimated and is given as β-positive synapses in %. Level of significance *p < 0.05.