Differential protein structural disturbances and suppression of assembly partners produced by nonsense GABRG2 epilepsy mutations: implications for disease phenotypic heterogeneity.

Juexin Wang,Dingding Shen,Jing-Qiong Kang,Robert L Macdonald,Geqing Xia,Dong Xu,Wangzhen Shen

doi:10.1038/srep35294

Juexin Wang, Dingding Shen + Show 5 more

Open Access

https://doi.org/10.1038/srep35294

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Abstract

Mutations in GABAA receptor subunit genes are frequently associated with epilepsy, and nonsense mutations in GABRG2 are associated with several epilepsy syndromes including childhood absence epilepsy, generalized tonic clonic seizures and the epileptic encephalopathy, Dravet syndrome. The molecular basis for the phenotypic heterogeneity of mutations is unclear. Here we focused on three nonsense mutations in GABRG2 (GABRG2(R136*), GABRG2(Q390*) and GABRG2(W429*)) associated with epilepsies of different severities. Structural modeling and structure-based analysis indicated that the surface of the wild-type γ2 subunit was naturally hydrophobic, which is suitable to be buried in the cell membrane. Different mutant γ2 subunits had different stabilities and different interactions with their wild-type subunit binding partners because they adopted different conformations and had different surface hydrophobicities and different tendency to dimerize. We utilized flow cytometry and biochemical approaches in combination with lifted whole cell patch-clamp recordings. We demonstrated that the truncated subunits had no to minimal surface expression and unchanged or reduced surface expression of wild-type partnering subunits. The amplitudes of GABA-evoked currents from the mutant α1β2γ2(R136*), α1β2γ2(Q390*) and α1β2γ2(W429*) receptors were reduced compared to the currents from α1β2γ2 receptors but with differentially reduced levels. This thus suggests differential protein structure disturbances are correlated with disease severity.

Highlights

Basis for the mutant protein’s stability, and the correlating biochemistry and function of the mutant subunits has not been reported
The GABRG2(Q390*) mutation resulted in the loss of the downstream 78 amino acids in the middle of the intracellular TM3-TM4 loop towards the C-terminus while the GABRG2(W429*) mutation resulted in loss of the downstream 39 amino acids in the middle of the TM3-TM4 intracellular loop towards the C-terminus
It is of note that all the mutant γ2 subunits we presented here are whole proteins including sequences of N-terminus, transmembrane domain to intracellular loop while only part of the γ2(Q390*) protein model was reported in our previous study[7]

Summary

Introduction

Basis for the mutant protein’s stability, and the correlating biochemistry and function of the mutant subunits has not been reported. With structural modeling and protein docking, there are already several successes in predicting function-related structural conformational differences between mutant/truncated and wild-type structures[7,8,9,10,11]. We characterized the properties of the three FS and epilepsy associated truncated mutant γ2 subunits based on structural modeling. Based on the predicted GABAA receptor subunit structural models and a series of computational analyses, we quantitatively inferred the protein-protein interaction stabilities among these subunits in the complexes. With various protein docking processes, we have determined that different mutant subunits have different interactions with the remaining wild-type partnering subunits, like α1 subunits, and the stabilities of the dimers of different mutant subunits are different. With whole-cell patch clamp recordings, we have identified different extents of preservation of wild-type channel function

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