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Calmodulin-dependent Signaling01:16

Calmodulin-dependent Signaling

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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Serotonin, a crucial neurotransmitter synthesized by enterochromaffin cells, plays a cardinal role in regulating gastrointestinal (GI) motility. With over 90% of the body's total serotonin in the GI tract, its influence on digestive processes is profound. Serotonin is swiftly released upon various stimuli, such as food boluses or certain drugs, triggering intrinsic sensory neurons in the myenteric plexus and extrinsic vagal and spinal sensory neurons. This leads to the activation of the...
Feedback Regulation of Calcium Concentration01:27

Feedback Regulation of Calcium Concentration

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Cell-based Calcium Assay for Medium to High Throughput Screening of TRP Channel Functions using FlexStation 3
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Calcium modulation of 5-HT3 receptor binding and function.

Andrew J Thompson1, Sarah C R Lummis

  • 1Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QW, UK.

Neuropharmacology
|August 5, 2008
PubMed
Summary

Calcium ions modulate serotonin 5-HT3 receptor function by affecting current amplitude and kinetics. Specific residues in the extracellular domain are crucial for receptor assembly and ligand binding, though the primary modulation site is intracellular.

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Pull-down of Calmodulin-binding Proteins
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Pull-down of Calmodulin-binding Proteins
07:51

Pull-down of Calmodulin-binding Proteins

Published on: January 23, 2012

Area of Science:

  • Neuroscience
  • Molecular Biology
  • Biochemistry

Background:

  • Calcium ions (Ca2+) are known to modulate the function of serotonin 5-HT3 receptors.
  • The precise binding sites and mechanisms underlying calcium's modulatory effects on 5-HT3 receptors remain largely unknown.
  • Previous studies suggest potential involvement of extracellular residues in calcium binding.

Purpose of the Study:

  • To investigate the role of specific extracellular residues (E213-E215-E218 and D204-E218-V219) in calcium modulation of 5-HT3 receptors.
  • To elucidate the binding sites and mechanisms by which calcium affects 5-HT3 receptor function, including ligand binding and ion channel activity.

Main Methods:

  • Radioligand binding assays using [3H]granisetron to assess ligand binding affinity.
  • Whole-cell patch-clamp electrophysiology to measure receptor currents and kinetics.
  • Site-directed mutagenesis to substitute key amino acid residues.
  • Immunofluorescence to confirm receptor localization at the cell surface.

Main Results:

  • Mutations E213Q, E215Q, D204N, and V219L abolished the calcium-induced increase in the dissociation constant (Kd) for [3H]granisetron.
  • The E218Q mutation resulted in non-functional receptors that did not traffic to the cell surface.
  • Mutations D204N and V219L rendered the receptors non-functional.
  • While E213Q altered desensitization rates, calcium's relative effects remained similar to wild-type receptors.
  • A calcium-impermeable mutant (E277A/S297R) showed no changes in peak amplitude or kinetics with increased calcium.
  • The primary site for calcium-dependent modulation of 5-HT3 receptor current appears to be within the ion channel or cell interior.

Conclusions:

  • Residues D204, E218, and V219 are critical for 5-HT3 receptor assembly, structure, and/or trafficking.
  • Calcium may stabilize receptor structure and facilitate trafficking via these residues.
  • Residues E213, E215, D204, and V219 may contribute to a calcium binding site influencing ligand binding.
  • The major site for calcium's modulatory effects on 5-HT3 receptor current is located intracellularly or within the ion channel pore.