# Neurosteroid and Cholesterol Binding to Integral Membrane Proteins

> **NIH NIH R35** · WASHINGTON UNIVERSITY · 2024 · $552,219

## Abstract

Project Summary
The Evers laboratory studies the binding interactions of neurosteroids and cholesterol with integral membrane
proteins, with the aim of identifying the specific binding events underlying sterol modulation of protein function.
Our major focus is on neurosteroid (NS) modulation of -aminobutyric acid type A receptors (GABAA-
R). Neurosteroids are important modulators of neuronal excitability and nervous system development with
enormous therapeutic potential as anesthetics, anti-depressants and neuro-protectants. We have shown
that there are multiple, subunit-specific binding sites for neurosteroids on GABAA receptors, each of which
contributes to the functional effects of neurosteroids. In the proposed research, we will use photolabeling
techniques to define the precise sites at which the major classes of neurosteroids bind on the most abundant
forms of synaptic and extra-synaptic GABAA receptors and determine the functional significance of each
identified binding site by assessing the effect of targeted amino acid substitutions on NS modulation of GABAA-
R currents. To identify photo-labeled residues we will utilize state-of-the-art protein chemistry and expression
techniques in conjunction with cutting edge mass spectrometry (MS) methods, including middle-down and
intact protein MS. High-resolution cryogenic-electron microscopy structures will be obtained to identify the
atomic details of novel NS binding sites and to investigate binding interactions that appear to stabilize
conformations not captured in current structures. Fluorescence-based binding assays will then be used to
measure the site-specific affinity of various NS for the identified binding sites. These assays will be
adapted to stopped-flow fluorimetry to determine the state-dependence of binding and to a plate reader
format to screen for site-specific agonists and antagonists. The long term goal of our NS program is to
develop and use site-specific NS ligands to probe the role of specific NS binding sites and GABAA-R subtypes
in the behavioral effects of endogenous NS and the mechanisms of action of NS sedatives and anesthetics.
 We have also used cholesterol-analogue photolabeling to identify specific binding sites that
mediate cholesterol inhibition of the lipid scramblase, nhTMEM16, and cholesterol modulation of
mTOR1 by the lysosomal membrane protein GPR155. Both nhTMEM16 and GPR155 have two specific
cholesterol binding sites per protein monomer and we are using targeted amino acid substitution to
understand the functional role of each site. We are also developing fluorescence-based binding assays to
measure cholesterol affinity and sterol specificity for these sites. Novel cholesterol binding sites present new
targets for small molecule allosteric modulators of membrane protein function and the tools we have
developed are widely applicable to identifying binding sites on other cholesterol-modulated proteins and
screening for site-specific ligands.

## Key facts

- **NIH application ID:** 10840472
- **Project number:** 5R35GM149287-02
- **Recipient organization:** WASHINGTON UNIVERSITY
- **Principal Investigator:** ALEX S. EVERS
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $552,219
- **Award type:** 5
- **Project period:** 2023-06-01 → 2028-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10840472

## Citation

> US National Institutes of Health, RePORTER application 10840472, Neurosteroid and Cholesterol Binding to Integral Membrane Proteins (5R35GM149287-02). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/10840472. Licensed CC0.

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