Loss, gain and altered function of GABA-A receptors in genetic epilepsies – Effects of Shisa7 modulation

Project description

Genetic epilepsy affects millions of people worldwide and over 30% of patients do not respond to currently available therapies. Accompanied by other neurodevelopmental disorders, this disease is one of the most significant unmet challenges in health care. Genetic mutations to GABA-A receptors (GABAAR) are one of the major causes of genetic epilepsy. This project will focus on how these mutations affect receptor function and how this may lead to development of epilepsy.
GABAARs are protein complexes that generate inhibitory postsynaptic currents (IPSCs) when they are activated by neurotransmitter, GABA. For decades, GABAARs were treated as neuroreceptors without accessory proteins. However, a recent study showed that Shisa7 forms complexes with GABAARs and regulates the strength of GABAergic transmission. We and others have measured IPSCs and the change in their properties has been described for several mutations. However, Shisa7 was not included in any of the studies and therefore we do not know the properties of GABAergic currents in epilepsy patients.
Aims:
1: To determine the effect of epilepsy-causing mutations on the electrophysiological properties of GABAergic synaptic currents when GABAARs are in complex with Shisa7.
2. To determine surface expression of mutant GABAARs with and without Shisa7.
3. Molecular dynamics simulations of mutant GABAARs when they are in complex with Shisa7. Simulations will focus on characterising the permeation of ions through the GABAAR pore in the resting, pre-open and desensitised states.
Methodology:
Aim 1: We will use patch-clamp electrophysiology recordings to record IPSCs in neurons and in hetero-synapses formed between neuronal pre-synaptic terminals and HEK293 cells.
Aim 2: We will use fluorescent microscopy to measure GABAAR surface expression.
Aim 3: Molecular modelling and advanced simulation techniques will be carried out to model Shisa7 bound GABAAR structures and study mutations-induced conformational transitions modulating the ion-permeation process.

Outcomes

This project will uncover a spectrum of pathomechanisms associated with GABAAR mutations found in epilepsy patients, via structure-function analysis in a synaptic context. It will also test novel drug-based therapies for either inhibiting or restoring GABAAR function, building the foundation for future personalised pharmacotherapies.
A prospective PhD student will learn patch-clamp electrophysiology, confocal fluorescent microscopy, and molecular dynamics simulation methods.
Expected outcomes per aim:
1. Aim 1 will answer the vital question about how mutant-containing GABAARs shape IPSCs that give rise to impaired neurotransmission. The data obtained will also offer insights into how to therapeutically target mutant AMPARs.
2. Many of the GABAAR variants implicated in epilepsy have reduced surface expression. Shisa7 increases expression of healthy GABAARs, but we do not know if it has the same effect on the mutants. Aim 2 will answer this question.
3. Artificial intelligence-based modelling in combination with enhanced sampling molecular dynamics simulations will provide Shisa7 bound GABAAR structures, unveil conformational transitions, and characterise permeation of ions through the channel pore and conformational changes induced by epilepsy-causing mutations. Together with cellular/functional data from aims 1 and 2, this will provide predictive models for understanding the effects of mutations on GABAAR function.

Essential capabilities

Excellent oral and written communication skills. Ability to work independently and to efficiently collaborate with colleagues

Desireable capabilities

Some experience in microscopy, molecular biology, laboratory management and training, and/or animal work

Expected qualifications (Course/Degrees etc.)

Undergraduate or Masters degree in Biology, Chemistry, Computational Biology, Biophysics, Neuroscience, or related disciplines.