Related Experiment Video

Updated: Jul 17, 2026

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading (TED)
09:32

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading (TED)

Published on: May 7, 2013

Phosphorylation sites on calcium channel alpha1 and beta subunits regulate ERK-dependent modulation of neuronal

S W Martin1, A J Butcher, N S Berrow

  • 1Department of Pharmacology, University College London, Gower Street, London WC1E 6BT, UK.

Cell Calcium
|January 13, 2006
PubMed

Insights

Voltage-dependent calcium channels (VDCCs) in sensory neurons are regulated by extracellular signal-regulated kinase (ERK) signaling. This study identifies specific phosphorylation sites on Ca(v)2.2 and Ca(v)beta subunits critical for ERK-dependent channel modulation.

Area of Science:

  • Neuroscience
  • Molecular Biology
  • Ion Channel Physiology

Background:

  • Voltage-dependent calcium channels (VDCCs), specifically N-type (Ca(v)2.2), are tonically upregulated in sensory neurons through Ras/extracellular signal-regulated kinase (ERK) signaling pathways.
  • The presence of potential ERK phosphorylation sites on the intracellular loop of Ca(v)2.2 and calcium channel beta (Ca(v)beta) subunits suggests these proteins may be direct substrates of ERK.

Purpose of the Study:

  • To investigate whether Ca(v)2.2 and Ca(v)beta subunits are substrates for ERK1/2 phosphorylation.
  • To identify specific serine residues on Ca(v)2.2 and Ca(v)beta subunits involved in ERK-mediated channel modulation.
  • To elucidate the role of these phosphorylation sites in the regulation of N-type VDCC activity by ERK.

Main Methods:

  • In vitro phosphorylation assays using GST-Ca(v)2.2 I-II loop and Ca(v)beta1b-His(6) with ERK1/2.
  • Site-directed mutagenesis of putative serine phosphorylation sites (Ser-409, Ser-447 on Ca(v)2.2; Ser-161, Ser-348 on Ca(v)beta1b).
  • Electrophysiological recordings (patch-clamp) of wild-type and mutant channels expressed with auxiliary subunits in response to MEK inhibitor UO126.

Main Results:

  • GST-Ca(v)2.2 I-II loop and Ca(v)beta1b were identified as substrates for ERK1/2 phosphorylation.
  • Mutation of Ser-447 on Ca(v)2.2 significantly reduced ERK phosphorylation and UO126-induced current inhibition, with Ser-409 also playing a role.
  • Co-expression of Ca(v)2.2(S447A) with Ca(v)beta1b(S161,348A) abolished UO126 effects, indicating Ser-447 on Ca(v)2.2 and Ser-161/348 on Ca(v)beta1b are crucial for ERK modulation.

Conclusions:

  • Ser-447 on the Ca(v)2.2 alpha1 subunit and Ser-161/348 on the Ca(v)beta1b subunit are necessary and sufficient for ERK-dependent modulation of N-type VDCCs.
  • ERK-mediated modulation of neuronal N-type VDCCs involves direct phosphorylation of the Ca(v)2.2 alpha1 subunit and, to a lesser extent, Ca(v)beta subunits.

More Related Videos

Assaying Protein Kinase Activity with Radiolabeled ATP08:05

Assaying Protein Kinase Activity with Radiolabeled ATP

Published on: May 26, 2017

Study of the Functions and Activities of Neuronal K-Cl Co-Transporter KCC2 Using Western Blotting10:08

Study of the Functions and Activities of Neuronal K-Cl Co-Transporter KCC2 Using Western Blotting

Published on: December 9, 2022

Related Concept Videos

Protein Kinases and Phosphatases02:54

Protein Kinases and Phosphatases

Proteins undergo chemical modifications that trigger changes in the charge, structure, and conformation of the proteins. Phosphorylation, acetylation, glycosylation, nitrosylation, ubiquitination, lipidation, methylation, and proteolysis are various protein modifications that regulate protein activity. Such modifications are usually enzyme-driven.
Protein kinases
Many proteins in the cell are regulated by phosphorylation, the addition of a phosphate group. A family of enzymes called kinases...
Amplifying Signals via Enzymatic Cascade01:22

Amplifying Signals via Enzymatic Cascade

When a ligand binds to a cell-surface receptor, the receptor's intracellular domain changes shape, which may either activate its enzyme function or allow its binding to other molecules. The initial signal is amplified by most signal transduction pathways. This means that a single ligand molecule can activate multiple molecules of a downstream target. Proteins that relay a signal are most commonly phosphorylated at one or more sites, activating or inactivating the protein. Kinases catalyze the...
G-Protein Gated Ion Channels01:21

G-Protein Gated Ion Channels

GPCRs are primarily responsible for our sense of smell, taste, and vision.  The binding of a sensory stimulus activates GPCR to stimulate effector proteins, many of which are ion channels in the sensory organs. GPCRs modulate the opening and closing of the target ion channels either directly by binding them, or by releasing second messengers that activate these channels. As ions move across the membrane, the membrane potential is altered, which induces an appropriate response.
Sensory organs,...
Feedback Regulation of Calcium Concentration01:27

Feedback Regulation of Calcium Concentration

Calcium is an essential signaling molecule required for various cellular functions. Calcium pumps and ion channels on cell and organellar membranes, such as those on the endoplasmic reticulum (ER), regulate calcium concentrations inside the cell. They remain closed, keeping the cytosolic calcium levels low at a resting state.
Various transmembrane receptors, such as G protein-coupled receptors (GPCRs), elicit a response to extracellular signals by increasing cytosolic calcium. Activated GPCRs...
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.
The Ca2+-CaM complex does not have enzymatic activity by itself. Instead, the complex binds downstream target proteins, including membrane proteins or enzymes,...
Ligand-Gated Ion Channel Receptor: Gating Mechanism01:30

Ligand-Gated Ion Channel Receptor: Gating Mechanism

Ligand-gated ion channels are transmembrane proteins that play a vital role in intercellular communication and functions of the nervous system. They allow the influx of ions across the membrane once the neurotransmitter binds, allowing the subsequent transmission of electrical excitation across the neurons. Other ligand-gated ion channels, like the γ-aminobutyric acid (GABA) receptor, permit anions like chloride into the cells on the binding of the GABA molecule. Their entry into the cell...