# Defining architecture of EC coupling machinery in situ

> **NIH NIH R21** · UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON · 2024 · $166,452

## Abstract

Project Summary/Abstract
The focus of this proposal is on excitation-contraction coupling (ECC) in skeletal muscle. The ECC consists of a
series of physiological events linking the depolarization of muscle cell’s plasma membrane to the release of Ca2+
from the sarcoplasmic reticulum (SR) into cytoplasm, resulting in muscle contraction. ECC is restricted spatially
to a subcompartment of muscle cells (‘triad junction’) and regulated precisely via a physical interaction between
the voltage-gated Ca2+ channel (dihydropyridine receptor, DHPR) on the plasma membrane and the Ca2+-release
channel (type 1 ryanodine receptor, RyR1) in the SR. Many drugs currently in use to treat muscle disorders
target these two Ca2+ channels. Despite recent remarkable advances in the structural characterization of these
two channels, the molecular mechanisms underlying their interactions remain elusive due to the lack of detailed
3D architecture of the ECC machinery comprising both channels and associated regulatory proteins. Determining
architecture of such multiprotein complexes is a formidable challenge given their native location in lipid
membranes and the lack a general means to preserve the complex integrity upon extraction with detergents from
their lipid bilayer environment. In this project, we will address this challenge by utilizing advanced cryogenic
electron tomography (cryoET) to study frozen-hydrated triad junctions isolated from skeletal muscle (aim 1) as
well as within myotubes cultured on EM grids (aim 2). To accomplish these studies, we endeavor to develop the
experimental workflow for in situ cryoET analysis of the ECC complex. This workflow will consist of the following
major steps: preparation of the membrane-embedded ECC complexes suitable for cryoET analysis; cryoET data
collection, image analysis, tomographic reconstruction and subtomogram averaging; visualization and
annotation of densities in cryo-tomograms. The determined structures will reveal mechanistically informative
features underlying protein-protein interactions in the ECC Ca2+ release complex that will allow important
functional insights into the ECC process. In the future, we will apply the workflow developed here to structure-
functional characterization of ECC in different types of muscle and under pathological conditions. Overall, the
proposed studies are highly significant, as they will provide mechanistic structural insights into the ECC
machinery illuminating the pathological consequences of deregulated Ca2+ signaling, that will ultimately aid in
search for novel therapies targeting neuromuscular diseases. The workflow developed, as part of this research
will have broad applicability to studies of other integral membrane protein complexes.

## Key facts

- **NIH application ID:** 10872299
- **Project number:** 5R21AR082833-02
- **Recipient organization:** UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
- **Principal Investigator:** Irina I Serysheva
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $166,452
- **Award type:** 5
- **Project period:** 2023-07-01 → 2025-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10872299

## Citation

> US National Institutes of Health, RePORTER application 10872299, Defining architecture of EC coupling machinery in situ (5R21AR082833-02). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10872299. Licensed CC0.

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