# Structural dynamics in cyclic nucleotide-modulated channels

> **NIH NIH R01** · WEILL MEDICAL COLL OF CORNELL UNIV · 2022 · $399,686

## Abstract

ABSTRACT
Cyclic nucleotide-modulated channels play major roles in pacemaking activity in heart and brain as well as in
olfactory and visual signal transduction in the nervous system. Defects in the functioning of these channels lead
to diseases such as epilepsy, cardiac arrhythmia, and color blindness. The overall objective of this grant is to
understand how binding of cyclic nucleotides gates (opens/closes) the channels and how other factors such as
lipids and proline isomerization modulate this gating. We will accomplish this by combining state-of-the-art
techniques: single-particle cryo electron microscopy (cryo-EM) with atomic force microscopy force spectroscopy
(AFM-FS), native mass spectrometry (MS), and functional assays like single-channel electrophysiology and
stopped flow fluorescence assays of channels incorporated in liposomes. We will employ SthK, a model
prokaryotic cyclic nucleotide-modulated channel, and also eukaryotic HCN1 and HCN2 for select sub-aims. Our
first aim is to determine the molecular mechanisms for partial agonism and ligand selectivity in SthK. We will
determine the structures of specific voltage-sensor SthK mutants that display increased open probability and
correlate class averages with the single-channel electrophysiology. To determine the molecular mechanism for
ligand selectivity we will use AFM-FS to determine at the single-molecule level the binding kinetics of cAMP and
cGMP to either the SthK cyclic nucleotide binding domain alone or in the context of the full-length channel. This
will yield the energetics of binding of both cyclic nucleotides and will isolate the contribution of the pore to the
binding. This aim will shed light on why cAMP binding does not fully open the SthK channel and why cGMP is
an antagonist, although its binding modality to the binding pocket is similar to that of cAMP. Our second aim is
to understand how lipids modulate channel activity. We will systematically test the effect of lipids on SthK activity
using stopped-flow fluorescence assays and single-channel electrophysiology where channels are in liposomes
of controlled composition. We will determine the lipids tightly bound to the channels (both SthK and HCN1) using
native MS and determine the mechanism of how they increase activity by perturbing the residues that appear to
coordinate these lipid-protein interactions with functional assays. The third aim is to characterize functionally
and structurally the regulation of SthK as well as potentially HCN channels by a newly discovered modality: prolyl
isomerization of a conserved proline in the cyclic nucleotide binding domain, which appears to be responsible
for SthK’s biphasic activation with cAMP. This can be highly impactful, as proline isomerization may turn out to
be yet another means to regulate pacemaking activity in the heart and brain. All aims are geared towards
unravelling the molecular mechanisms of cyclic nucleotide-modulated channels’ synergistic regulation by
ligands,...

## Key facts

- **NIH application ID:** 10458032
- **Project number:** 5R01GM124451-06
- **Recipient organization:** WEILL MEDICAL COLL OF CORNELL UNIV
- **Principal Investigator:** Crina M Nimigean
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $399,686
- **Award type:** 5
- **Project period:** 2017-09-01 → 2025-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10458032

## Citation

> US National Institutes of Health, RePORTER application 10458032, Structural dynamics in cyclic nucleotide-modulated channels (5R01GM124451-06). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10458032. Licensed CC0.

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