Correlations of receptor desensitization of gain-of-function GABRB3 variants with clinical severity

Abstract

Genetic variants associated with developmental and epileptic encephalopathies have been identified in the GABRB3 gene that encodes the β3 subunit of GABAA receptors. Typically, variants alter receptor sensitivity to GABA resulting in either gain- or loss-of-function, which correlates with patient phenotypes. However, it is unclear how another important receptor property, desensitization, contributes to the greater clinical severity of gain-of-function variants. Desensitization properties of 20 gain-of-function GABRB3 variant receptors were evaluated using two-electrode voltage-clamp electrophysiology. The parameters measured included current decay rates and steady-state currents. Selected variants with increased or reduced desensitization were also evaluated using whole-cell electrophysiology in transfected mammalian cell lines. Of the 20 gain-of-function variants assessed, 13 were found to alter receptor desensitization properties. Seven variants reduced desensitization at equilibrium, which acts to worsen gain-of-function traits. Six variants accelerated current decay kinetics, which limits gain-of-function traits. All affected patients displayed severe clinical phenotypes with intellectual disability and difficult-to-treat epilepsy. Nevertheless, variants that reduced desensitization at equilibrium were associated with more severe clinical outcomes. This included younger age of first seizure onset (median 0.5 months), movement disorders (dystonia and dyskinesia), epilepsy of infancy with migrating focal seizures (EIMFS) and risk of early mortality. Variants that accelerated current decay kinetics were associated with slightly milder phenotypes with later seizure onset (median 4 months), unclassifiable developmental and epileptic encephalopathies or Lennox-Gastaut syndrome and no movement disorders. Our study reveals that gain-of-function GABRB3 variants can increase or decrease receptor desensitization properties and that there is a correlation with the degree of disease severity. Variants that reduced the desensitization at equilibrium were clustered in the transmembrane regions that constitute the channel pore and correlated with greater disease severity, while variants that accelerated current decay were clustered in the coupling loops responsible for receptor activation and correlated with lesser severity.

Distinct neurodevelopmental and epileptic phenotypes associated with gain- and loss-of-function GABRB2 variants

“…Recently, we reported that more severe phenotypes observed in individuals with GABRB3 GOF variants in M1 could be attributed to decreased receptor desensitisation. 36 In the GABRB2 Low-shift GOF group, one individual (#18) harboured the L255V variant, which is a paralogue of the GABRB3 L256Q variant previously shown to cause decreased desensitisation. This individual suffered from treatment-resistant epilepsy with severe movement disorders and moderate developmental delay, ultimately succumbing to SUDEP at the age of 4 years.…”

“…For desensitisation experiments, another setup with ultra-low bath volumes was used to ensure rapid liquid exchange. 36 3 M KCl-filled borosilicate glass microelectrodes with resistance of 0.2–1.6 MΩ were inserted into cells then clamped at −60 mV with constant perfusion of ND96 solution (96 mM NaCl, 2 mM KCl, 1 mM MgCl 2 , 5 mM HEPES, 1.8 mM CaCl 2 , pH 7.4) through a gravity-driven semi-automatic system at 1 mL/min. A Warner OC-725C amplifier (Warner Instruments) was used for amplifying GABA-evoked currents that were then filtered and digitised at 10 Hz using the Powerlab 8/35 with LabChart reader version 8.1 (AD Instruments).…”

“…The peak current amplitude induced by the second 3 mM GABA application was normalised to that induced by 10 mM GABA and 10 μM etomidate to calculate the maximum GABA-evoked receptor open probability (Est P O(max) ). 36 For desensitisation experiments, non-linear regression was performed with GraphPad Prism 8. The following equation was used to fit traces to one-phase exponential decay: represents current amplitude and the time.…”