# Mechanisms of Permeation and Gating of Voltage-Sensing Domains

> **NIH NIH R01** · UNIVERSITY OF CALIFORNIA-IRVINE · 2024 · $498,393

## Abstract

Project Summary
The voltage-gated proton channel Hv1 plays important roles in numerous biological processes, including
pH homeostasis and the immune response. Its activity has been found to worsen brain damage after
ischemic stroke, to exacerbate the effect of traumatic brain injury and spinal cord injury, and to increase
the metastatic potential of different types of cancer. The development of small-molecule modulators of
Hv1 activity could lead to new anti-inflammatory agents and anticancer drugs. In addition, Hv1
modulators can provide useful pharmacological tools for studying the function of the channel in health
and disease. Hv1 belongs to the large family of voltage-gated ion channels (VGICs). The majority of
these proteins consist of four voltage-sensing domains (VSDs) surrounding a central pore domain. While
many types of drugs bind the pore domain of VGICs, the number of organic molecules known to bind
VSDs is limited. The Hv1 channel is made of only two VSDs and does not contain a pore domain,
providing a simplified model for studying how ligands interact with VSDs. We have previously discovered
small molecules that inhibit Hv1 activity by binding within the intracellular vestibule of the channel VSD
in the open state (class I.1 ligands). Using a rational design approach that combines experimental and
computational methods, we identified related compounds that are able to bind the channel also in the
closed state (class I.2 ligands). Some of the new ligands display inhibitory properties that are superior
to those of class I.1 compounds and provide a promising scaffold for further development of high-affinity
Hv1 antagonists. However, little is known about how effective class I.2 ligands are at inhibiting Hv1-
regulated cellular processes, such as ROS production by NOX enzymes, or how specifically they target
the Hv1 VSD versus VSDs of other VGICs. In aim 1 of this project, we will apply our rational design
approach to develop I.2 ligands with improved potency and corresponding negative controls. We will
also use electrophysiological methods to investigate potential effects of Hv1 ligands on other members
of the VGIC family. In aim 2, we will utilize a variety of live cell imaging assays on wild type and Hv1
knockout cells to examine how I.2 ligands inhibit NOX-mediated ROS production in phagocytes and
how they affect proliferation and migration of cancer cells in a Hv1-dependent manner. The Hv1 channel
contains a VSD-VSD interface unique among VGICs. As a result, ligands that bind such interface are
expected to be more specific channel modulators than ligands that bind other transmembrane regions.
The structure of the Hv1 dimer has yet to be determined, and alternative dimer models have been
proposed by different groups with different VSD-VSD interfaces. In aim 3, we will use molecular
dynamics simulations combined with multichemistry cross-linking mass spectrometry to probe the
different models and derive a consensus dimer interface.

## Key facts

- **NIH application ID:** 10843136
- **Project number:** 5R01GM098973-12
- **Recipient organization:** UNIVERSITY OF CALIFORNIA-IRVINE
- **Principal Investigator:** Francesco Tombola
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $498,393
- **Award type:** 5
- **Project period:** 2011-07-01 → 2026-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10843136

## Citation

> US National Institutes of Health, RePORTER application 10843136, Mechanisms of Permeation and Gating of Voltage-Sensing Domains (5R01GM098973-12). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10843136. Licensed CC0.

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