# Small-molecule probes for study of CLC-2 chloride-channel function in the central nervous system

> **NIH NIH R01** · STANFORD UNIVERSITY · 2021 · $545,163

## Abstract

The CLC chloride channel family is a class of membrane proteins that controls the flux of
chloride ions across cell membranes. Nine unique CLC homologs are differentially expressed in
mammalian tissue and function in diverse physiological roles, ranging from electrical excitation
of muscles and neurons to regulation of electrolyte balance. One subtype, CLC-2, is a voltage-dependent channel expressed broadly in the brain. Although the presence of CLC-2 in the brain
has been known for decades, the role of this CLC homolog in neuronal signaling and proper
brain function remains poorly understood, in part due to the absence of potent and selective
small-molecule tools that enable studies of the molecular physiology of this channel. A recent
breakthrough in our laboratories now opens the door to developing small molecule tools specific
to CLC-2. Through a compound-library screen, we identified 'hit' compounds that inhibit CLC-2
activity. We developed one of these into a potent and selective CLC-2 inhibitor, FA44, which
has an IC50 of 18 nM for CLC-2 and no off-target effects on the closest CLC homolog or on a
panel of 65 CNS channels, receptors, and transporters. The efficacy and selectivity of FA44 for
CLC-2 is further supported by our electrophysiological recordings of brain slices from wild-type
versus CLC-2 knock-out mice. In this project, we will continue our collaborative efforts to
develop, characterize, and use chemical tool compounds for studying CLC-2. In Aim 1, we will
identify the mechanism of action and molecular determinants for inhibition of CLC-2. In Aim 2,
we will develop novel probes, including small-molecule activators and fluorescent imaging
probes for localizing channel expression. In Aim 3, we will leverage our tool compounds to
query the role of CLC-2 in excitatory synaptic transmission and network excitability in the
thalamus and to evaluate the potential causative link between CLC-2 malfunction and epilepsy.
Our team's combined expertise in synthetic chemistry (Du Bois), ion-channel structure-function
(Maduke), computation (Dror), and cellular neuroscience/epilepsy (Huguenard) ideally positions
us to advance this research program.

## Key facts

- **NIH application ID:** 10119335
- **Project number:** 5R01NS113611-02
- **Recipient organization:** STANFORD UNIVERSITY
- **Principal Investigator:** Justin Du Bois
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $545,163
- **Award type:** 5
- **Project period:** 2020-03-15 → 2025-02-28

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10119335

## Citation

> US National Institutes of Health, RePORTER application 10119335, Small-molecule probes for study of CLC-2 chloride-channel function in the central nervous system (5R01NS113611-02). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10119335. Licensed CC0.

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