# Biophysical mechanisms of gating and modulation in voltage-gated ion channel superfamily

> **NIH NIH R35** · WASHINGTON UNIVERSITY · 2022 · $985,622

## Abstract

Project Summary
Members of the voltage-gated ion channels (VGICs) are critical for electrical and chemical signaling throughout
the three kingdoms of life. Dysfunction of ion channels underlie a wide range of pathophysiology and they are
one of the primary targets for new drug development. Although they share a common membrane architecture,
the channels in this superfamily exhibit surprising diversity of function. Most open in response to a membrane
depolarization but some open on hyperpolarization. Many of them are also polymodal- their activity is regulated
by second messengers such as cyclic nucleotide or a physical stimulus such as temperature. The main objective
of this proposal is to probe the molecular driving forces in order to understand the fundamental mechanisms of
voltage-gating and its modulation by temperature and ligand. Current mechanistic approach tends to be structure
focused to the extent that protein dynamics is either ignored or treated as secondary. Although the structures of
many highly temperature-sensitive ion channels are now available, our understanding of the mechanism of tem-
perature-sensitivity remains limited, in large part, due to our inability to directly probe the molecular forces. To
address this issue, we are using a multi-pronged approach that combines new and existing tools to systematically
characterize the molecular interactions that determine polarity of voltage-gating, exquisite temperature-sensitiv-
ity and unusual allostery in VGICs. We are using the HCN channel as a model system to study gating polarity
and ligand activation. Using zero model waveguides and newly developed high-throughput analysis algorithms
we were able to probe the cooperativity of ligand binding in a model system. We are now poised to extend these
studies to full-length channels and receptors. With regards to mechanisms of gating polarity, we have made a
surprising discovery that a bipartite switch regulates gating polarity in HCN channels. Microsecond scale simu-
lations in Anton supercomputer suggest a gating model which we will be tested further. We will carry out structural
studies and combine it with voltage clamp fluorometry in order to annotate these structures. Next, we will also
use ancestral protein reconstruction approach, to identify the deep allosteric networks that regulate gating po-
larity in these channels. Our studies on temperature-dependent gating is based on two model systems: a) Tem-
perature-sensitive Shaker mutant and, b) archaeal MthK channel. In order to determine the essential elements
that are responsible for “sensing” temperature, we have to measure the thermodynamic properties such as heat
capacity. We propose to develop a new approach involving single molecule force spectroscopy to extract these
energetic parameters. Overall, our “molecular forces” focused approach has the potential to provide unparalleled
insights into the mechanisms of voltage gating and its regulation by temperature in VGICs.

## Key facts

- **NIH application ID:** 10400913
- **Project number:** 5R35NS116850-04
- **Recipient organization:** WASHINGTON UNIVERSITY
- **Principal Investigator:** Baron Chanda
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $985,622
- **Award type:** 5
- **Project period:** 2020-05-03 → 2028-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10400913

## Citation

> US National Institutes of Health, RePORTER application 10400913, Biophysical mechanisms of gating and modulation in voltage-gated ion channel superfamily (5R35NS116850-04). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10400913. Licensed CC0.

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