Related Experiment Video
Updated: May 29, 2025
12:26
Author Spotlight: Functional Site-Directed Fluorometry in Native Cells to Study Skeletal Muscle Excitability
Published on: June 2, 2023
898
Comprehensive mutational characterization of the calcium-sensing STIM1 EF-hand reveals residues essential for
Nisha D Kamath1, Kenneth A Matreyek1
1Department of Pathology, Case Western Reserve University School of Medicine, Cleveland, Ohio, USA.
Biorxiv : the Preprint Server for Biology
|February 3, 2025
Summary
We functionally characterized 706 STIM1 EF-hand variants using cell-based fitness assays. This reveals how STIM1 sequence variations impact calcium signaling and disease, aiding clinical interpretation.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Calcium signaling regulates fundamental cellular processes.
- Store-operated calcium entry (SOCE) involves STIM1 sensing ER calcium and triggering influx.
- STIM1 variants can cause diseases like myopathy and immunodeficiency, but few are characterized.
Purpose of the Study:
- To functionally characterize the STIM1 canonical EF-hand domain by assessing 706 single amino acid variants.
- To understand how STIM1 sequence variations impact its function in calcium signaling.
- To aid in the clinical interpretation of STIM1 variants found in patients.
Main Methods:
- Utilized a high-throughput fitness assay based on STIM1 variant overexpression in cultured human cells.
- Analyzed mutational patterns in calcium-coordinating residues and STIM1 helices.
- Employed orthogonal measurements including STIM1 oligomerization, cytoplasmic calcium influx, and cellular stress.
Main Results:
- Identified varying mutational patterns in EF-hand calcium-coordinating residues.
- Discovered a core of immutable residues in the trailing helix, potentially restraining STIM1 aggregation.
- Found that the leading helix variants caused toxicity only in the presence of endogenous STIM1, suggesting a multimerization-dependent regulation.
Conclusions:
- The STIM1 canonical EF-hand sequence contains complex roles critical for SOCE.
- Cytotoxicity assays, combined with oligomerization and calcium influx measurements, enhance variant functional understanding.
- Most pathogenic STIM1 variants were not overtly cytotoxic in this assay, highlighting the need for complementary analyses.
Related Concept Videos
01:16Calmodulin-dependent Signaling
5.1K
Calmodulin (CaM) is a calcium-binding protein in eukaryotes that controls various calcium-regulated cellular processes. It has four calcium-binding sites that bind calcium to form the calcium-calmodulin ( Ca2+-CaM) complex. GPCR stimulation increases the calcium levels in the cells that bind to CaM and induces a conformational change.
The Ca2+-CaM complex does not have enzymatic activity by itself. Instead, the complex binds downstream target proteins, including membrane proteins or enzymes,...
The Ca2+-CaM complex does not have enzymatic activity by itself. Instead, the complex binds downstream target proteins, including membrane proteins or enzymes,...
5.1K
01:49Conserved Binding Sites
4.2K
Many proteins’ biological role depends on their interactions with their ligands, small molecules that bind to specific locations on the protein known as ligand-binding sites. Ligand-binding sites are often conserved among homologous proteins as these sites are critical for protein function.
Binding sites are often located in large pockets, and if their location on a protein’s surface is unknown, it can be predicted using various approaches. The energetic method computationally...
Binding sites are often located in large pockets, and if their location on a protein’s surface is unknown, it can be predicted using various approaches. The energetic method computationally...
4.2K
01:25Structure of Cadherins
3.3K
The cadherins were one of the first cell adhesion molecules discovered; the term “cadherins” is based on their calcium-dependent adhering properties. The first cadherins discovered on the epithelial, neuronal, and placental cells were named E-cadherin, P-cadherin, and N-cadherin, respectively. These classical cadherins share sequence and structural similarities. Other cadherins, including those involved in cell signaling, are grouped into non-classical cadherins. This...
3.3K
01:33Overview of Secretory Vesicles
8.3K
Secretory vesicles, also known as dense core vesicles (DCVs), are membrane-bound vesicles that transport secretory proteins, such as hormones or neurotransmitters. Regulated secretory vesicles transport proteins from the trans-Golgi network to the exterior of the cell. Proteins present in regulated secretory vesicles are required to be rapidly exocytosed in large amounts upon a specific stimulus.
Various proteins regulate the aggregation of molecules inside the secretory vesicles. Chromogranins...
Various proteins regulate the aggregation of molecules inside the secretory vesicles. Chromogranins...
8.3K