# Molecular Sites of Neurosteroid Binding

> **NIH NIH R01** · WASHINGTON UNIVERSITY · 2021 · $589,789

## Abstract

Neurosteroids are important modulators of neuronal excitability and nervous system development with
enormous therapeutic potential as anti-depressants, anesthetics and neuro-protectants. The principal
molecular target of neurosteroids is the GABAA receptor. We have shown that there are multiple, subunit-
specific binding sites for neurosteroids on GABAA receptors, each of which contributes to the effects of
neurosteroids on receptor expression and function. This project will use photolabeling techniques to define the
precise sites of neurosteroid binding on the most abundant forms of synaptic and extrasynaptic GABAA
receptors. Novel neurosteroid analogue photolabeling reagents will be developed and used to determine the
number of neurosteroid binding sites on each subunit, to identify the binding sites and to determine the
orientation of the neurosteroids in these sites. To achieve these goals we will utilize state-of-the-art protein
chemistry and expression techniques in conjunction with cutting edge mass spectrometry (MS) methods,
including middle-down, intact protein and native MS. We will then mutate amino acids in each of the identified
binding sites and use functional readouts to determine which binding sites mediate the various effects of
neurosteroids on GABAA receptor expression and function. The Project has two specific aims:
In Aim 1, a viral expression system (BacMam) will be used to express large quantities of synaptic (α1β2γ2)
and extrasynaptic (α4β3δ) GABAA receptors and a suite of neurosteroid analogue photolabeling reagents will
be synthesized in which the photolabeling moieties are placed at various positions around the neurosteroid
backbone. The expressed receptors will be used in conjunction with the photolabeling reagents to determine
the number of labeling sites on each subunit using intact protein MS and on each pentameric receptor using
native MS. The specific amino acids modified by photolabeling will be identified using middle-down MS. The
photolabeling data will then be used in conjunction with molecular modeling and docking to determine the
preferred orientation of neurosteroids in each of the binding pockets and to identify critical residues which may
be necessary for neurosteroid binding or effect. In Aim 2 receptors will be expressed in which these critical
residues are mutagenized. The mutant receptors will be used in functional assays (channel gating, trafficking
of receptors to the surface and modulation of orthosteric ligand binding) to determine the contribution of each
binding site to neurosteroid action(s). Neurosteroid labeling and orientation will be then be assessed in the
mutated receptors (as in Aim 1) to determine how the mutations alter neurosteroid binding and/or effect. The
data from these studies will provide insight into the specific molecular interactions underlying neurosteroid
actions at each of its binding sites on GABAA receptors. These data will provide a structural template for
development o...

## Key facts

- **NIH application ID:** 10105332
- **Project number:** 5R01GM108799-07
- **Recipient organization:** WASHINGTON UNIVERSITY
- **Principal Investigator:** ALEX S. EVERS
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $589,789
- **Award type:** 5
- **Project period:** 2014-05-01 → 2023-02-28

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10105332

## Citation

> US National Institutes of Health, RePORTER application 10105332, Molecular Sites of Neurosteroid Binding (5R01GM108799-07). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10105332. Licensed CC0.

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