Molecular Biochemistry I

Ca++ Signals

Contents of this page:
cytosolic Ca++
Ca++-release channels
calmodulin

Note: Only selected aspects of the complex subject of calcium signaling will be covered here. See also sections on phosphatidylinositol signals and calcium pumps.

Modulation of Cytosolic [Ca++]

  • Cytosolic [Ca++] is usually less than one micromolar, except during a Ca++ signal event.
    Ca++-ATPase pumps
    in the plasma membranes and in endoplasmic reticulum (ER) membranes maintain this low concentration by transporting Ca++ away from the cytosol, either out of the cell or into the ER. 
  • Extracellular [Ca++] in mammalian organisms is in the millimolar range. Opening of plasma membrane Ca++ channels may initiate or sustain a Ca++ signal.
  • [Ca++] is also relatively high in the lumen of the ER, which serves as the major internal reservoir from which Ca++ is released to the cytosol during Ca++ signaling.
  • Mitochondria and lysosomes also serve as reservoirs for Ca++ subject to release under certain conditions.

Ca++-binding proteins within the ER lumen "buffer" the free Ca++ concentration, and increase the capacity for Ca++ storage. ER Ca++-binding proteins have 20-50 low-affinity Ca++-binding sites per molecule, consisting of acidic residues. Examples:

Ca++ concentration, within the cytosol or other cell compartments, may be monitored using indicator dyes or proteins that are either luminescent or change their fluorescence when they bind Ca++. Fluorescent indicators used with confocal fluorescence microscopy can provide high-resolution imaging and quantitation of Ca++ fluctuations within cells.

A transient increase in cytosolic Ca++ may be localized to the vicinity of one or a few activated Ca++-release or Ca++-entry channels. Such a localized Ca++ "puff" or "spark" may activate activate effectors that induce additional Ca++ release, leading to a more widespread increase in cytosolic Ca++. A “wave” of higher cytosolic Ca++ may  spread to neighboring cells.

For example, see a website maintained by E. Niggli showing recordings of Ca++ sparks and waves, using fluorescent Ca++ indicators.

Ryanodine Receptor: A Ca++ Release Channel

A large Ca++-release channel in the membrane of muscle sarcoplasmic reticulum (SR) is called the ryanodine receptor, because of its sensitivity to inhibition by a plant alkaloid ryanodine. Skeletal and cardiac muscle contraction is activated when Ca++ is released from the SR lumen to the cytosol via the ryanodine receptor. 

T tubules are invaginations of muscle cell plasma membrane. Voltage-gated Ca++ channels in the T tubule membrane interact with ryanodine receptors in the closely apposed SR membrane. 

Activation of the voltage-gated Ca++ channels, by an action potential in the T tubule, leads to opening of ryanodine-sensitive Ca++ release channels. Ca++ moves from the SR lumen to the cytosol, passing first through the transmembrane portion of the ryanodine receptor, and then through the ryanodine receptor's cytoplasmic assembly. 

  • The ryanodine receptor is itself activated by cytosolic Ca++ at micromolar concentrations. Thus entry of a small amount of Ca++ into the cytosol causes further Ca++ release.
  • High (e.g., mM) cytosolic Ca++ inactivates the ryanodine receptor channel, contributing to signal turn-off.
At right are three views of a 3D reconstruction of the structure of the ryanodine-sensitive Ca++ channel, at 30 resolution, based on micrographs obtained by cryo-electron microscopy at varied tilt angles.
See also an animation of conformational changes during channel opening & closing.

A somewhat higher resolution structure now available indicates the presence of bent a-helices adjacent to the lumen in the transmembrane pore domain, but an atomic resolution structure of the whole channel has not yet been achieved. For diagrams see article by Ludtke et al. (journal subscription required).
 


The images above were provided by Terrence Wagenknecht of the Wadsworth Center, NY State Dept. of Health. (For references & more information see a Wadsworth Center Web page.)  

 

IP3 receptor Ca++ Release Channel

In many mammalian cells, IP3 (inositol-1,4,5-trisphosphate) triggers Ca++ release from the endoplasmic reticulum. The "second messenger" IP3 is produced, e.g., in response to hormonal signals, from the membrane lipid phosphatidylinositol.  (Phosphatidylinositol signals are discussed in more detail elsewhere.)

Explore at right the structure of IP3. The receptor-bound IP3, from PDB 1N4K, structure solved by I. Bosanac, J. R. Alattia, T. K. Mal, J. Chan, S. Talarico, F. K. Tong, K. I. Tong, F. Yoshikawa, T. Furuichi, M. Iwai, T. Michikawa, K. Mikoshiba & M. Ikura in 2002.

Recommended display options:

View with different display settings, e.g., ball & stick, sticks, spacefill.

Note the stereospecific orientation of substituted and un-substituted hydroxyl groups relative to the two sides of the ring.
Compare to the structure above.


   C   O   P   
  • The IP3 receptor is a ligand-gated Ca++-release channel embedded in endoplasmic reticulum membranes. It is distinct from but partly homologous to the ryanodine receptor channel.
  • IP3 binds to a cytosolic domain of the receptor, promoting channel opening.
    IP3 may displace a regulatory phospho-protein IRBIT, which binds at the same site.
    See a diagram in a website of the RIKEN Institute, Japan.
  • Ca++ also binds to the ligand-binding domain of the IP3 receptor, and promotes channel opening.
    However, high cytosolic Ca++, which develops after channel opening, promotes channel closure.
    Thus both the IP3-activated & ryanodine-sensitive channels are activated by low cytosolic Ca++ and inhibited by high cytosolic Ca++.
    The feedback inhibition of Ca++ release by high cytosolic Ca++, along with activity of Ca++-ATPase pumps, contributes to signal turnoff and makes possible observed oscillations in Ca++ concentration.

Structures of cytosolic domains of the IP3 receptor, including the IP3 binding site, have been solved, but the pore structure of the IP3 receptor has not yet been determined at atomic resolution. 

See image in a website for a low-resolution structure of the IP3 receptor (AIST, Japan, findings of C. Sato et al.).


of Ca++ release &
accumulation by ER 

Calmodulin

Calmodulin, a Ca++-activated switch protein, mediates many of the signal functions of Ca++. Calmodulin cooperatively binds 4 Ca++

At each binding site, Ca++ interacts with oxygen atoms, mainly of glutamate and aspartate side-chain carboxyl groups, and of the protein backbone, in a loop domain between two a-helices at right angles. This helix-loop-helix motif is called an EF hand (diagram p. 642). 


There are four helix-loop-helix motifs in calmodulin, two at each end of the molecule, which has a dumbbell shape.

Ca++ binding to the 4 helix-loop-helix motifs promotes a conformational change that exposes hydrophobic residues along a concave patch on each of the 2 lobes. These residues are involved in protein-protein interactions. Ca++-calmodulin then changes conformation again as it wraps around the target domain of a protein.

A typical calmodulin-binding target domain is a positively charged, amphipathic a-helix, with polar and non-polar surfaces. Terminal methyl groups of methionine side-chains of calmodulin participate in binding to hydrophobic residues in target domains of some enzymes that are regulated by calmodulin. However the interaction of Ca++-calmodulin with some target proteins is different from what is described here.

Some proteins have bound calmodulin as part of their quaternary structure, even in the absence of Ca++. In either case, Ca++ binding to calmodulin may induce a conformational change that alters activity of the target protein.

Many enzymes are regulated by Ca++-calmodulin. For example:

  • Some protein kinases that transfer phosphate from ATP to hydroxyl residues on other enzymes to be regulated are activated by Ca++-calmodulin. These are referred to as CaM Kinases.
  • The plasma membrane Ca++-ATPase that pumps Ca++ out of the cell, is one of the target proteins activated by Ca++-calmodulin. Thus cytosolic Ca++ itself contributes further to turning off Ca++ signals.

 

View at right:

  • an animation of binding of calmodulin to a target peptide

  • the structure of calmodulin complexed with a target peptide.

 
Calmodulin conformation change


Calmodulin

Defects in genes coding for Ca++ channel proteins, Ca++-ATPases, and intracellular Ca++ sensors are associated with disease or death.

Copyright 1998-2007 by Joyce J. Diwan. All rights reserved.

Additional material on Ca++ Signals:
Readings, Test Questions & Tutorials

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