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Related Concept Videos

Calmodulin-dependent Signaling01:16

Calmodulin-dependent Signaling

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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.
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Multicellular organisms employ a variety of ways for cells to communicate with each other. Gap junctions are specialized proteins that form pores between neighboring cells in animals, connecting the cytoplasm between the two, and allowing for the exchange of molecules and ions. They are found in a wide range of invertebrate and vertebrate species, mediate numerous functions including cell differentiation and development, and are associated with numerous human diseases, including cardiac and...
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The cytoplasm of adjacent animal cells can exchange small molecules, ions, and secondary messengers via the communication channels which form the gap junctions. These junctions comprise a few hundred to thousands of molecular channels, each made of two halves, called the connexon hemichannel. A connexon is a hexamer of six transmembrane connexin proteins, which assemble radially, thus forming a pore or channel in the center. One connexon hemichannel docks with a corresponding connexon on the...
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The extracellular matrix or ECM holds cells together to form a tissue and allows the cells within the tissue to communicate. ECM comprises proteins such as fibronectin, collagen, laminin, etc. The most abundant protein in this space is collagen. Collagen fibers are interwoven with carbohydrate-containing protein molecules called proteoglycans. ECM allows cell migration and provides a structural scaffold at cell adhesion that anchors the cell when the extracellular matrix proteins interact with...
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Related Experiment Video

Updated: Feb 22, 2026

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm
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Direct visualization of interaction between calmodulin and connexin45.

Juan Zou1, Mani Salarian1, Yanyi Chen1

  • 1Department of Chemistry, Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, GA 30303, U.S.A.

The Biochemical Journal
|October 1, 2017
PubMed
Summary

Researchers discovered the first direct interaction between calmodulin (CaM) and connexin45 (Cx45) in living cells. This Ca2+-dependent binding, crucial for Ca2+ signaling, occurs at Cx45's cytosolic loop.

Keywords:
calmodulinconnexin45gap junctionsintracellular calciumprotein–protein interactionsregulation

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Area of Science:

  • Cellular Biology
  • Biochemistry
  • Molecular Signaling

Background:

  • Calmodulin (CaM) is a key intracellular calcium (Ca2+) sensor regulating diverse cellular processes.
  • Ca2+/CaM complex involvement in gap junction regulation via connexins was hypothesized but lacked direct evidence.
  • Studying membrane protein interactions, like CaM with connexins, presents significant experimental challenges.

Purpose of the Study:

  • To provide the first direct evidence of CaM interaction with connexin45 (Cx45) in living cells.
  • To characterize the Ca2+-dependency and binding site of the CaM-Cx45 interaction.
  • To elucidate the molecular mechanism of CaM's modulation of Cx45.

Main Methods:

  • Bioluminescence resonance energy transfer (BRET) to monitor CaM-Cx45 interaction in living cells.
  • Use of CaM inhibitor W7 to confirm the interaction's Ca2+/CaM dependency.
  • Peptide model of Cx45 cytosolic loop and fluorescence-labeled CaM to study binding kinetics.
  • High-resolution nuclear magnetic resonance (NMR) spectroscopy to determine structural changes upon binding.

Main Results:

  • Direct CaM-Cx45 interaction was observed in living cells under physiological conditions.
  • The interaction is Ca2+-dependent and can be inhibited by W7.
  • A specific CaM-binding site (residues 164-186) was identified on the Cx45 cytosolic loop.
  • Ca2+-dependent binding with high affinity (Kd ~55 nM) was confirmed using a Cx45 peptide.
  • NMR revealed Ca2+-dependent global chemical shift changes in CaM upon peptide binding, indicating interaction without structural denaturation.
  • CaM's N- and C-domains interact with the Cx45 peptide, enhancing Ca2+ sensitivity.

Conclusions:

  • This study provides the first direct evidence of CaM binding to Cx45 in living cells.
  • The Ca2+-dependent interaction involves a specific site on the Cx45 cytosolic loop and modulates CaM's Ca2+ sensitivity.
  • These findings suggest that helicity and interaction mode of the Cx45 cytosolic loop contribute to CaM's regulation of connexins and Ca2+ signaling networks.