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.

Public Health Relevance

The malfunction of chloride ion channels and transporter (CLCs) ? proteins critical for human physiology ? has been linked to musculoskeletal, cardiovascular, and neurological disease. Although the functions of some CLCs are well understood, others remain poorly described, in large part due to the lack of selective 'tools' for analyzing the mechanisms of these proteins in regulating cellular processes. We are advancing high-precision chemical agents to study CLC-2, a chloride-selective ion channel expressed broadly throughout the brain and implicated in epilepsy as well as other disorders of the central nervous system.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS113611-02
Application #
10119335
Study Section
Neurotransporters, Receptors, and Calcium Signaling Study Section (NTRC)
Program Officer
Umanah, George Kwabena Essien
Project Start
2020-03-15
Project End
2025-02-28
Budget Start
2021-03-01
Budget End
2022-02-28
Support Year
2
Fiscal Year
2021
Total Cost
Indirect Cost
Name
Stanford University
Department
Biophysics
Type
Schools of Medicine
DUNS #
009214214
City
Stanford
State
CA
Country
United States
Zip Code
94305