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Calcium signalling

Introduction

Welcome to the world of calcium signalling. This webpage will break down many facets of calcium signalling within cells including entry, compartmentalisation and release of calcium ions, into short digestible chunks. Images are included, these can be enlarged by clicking if there is any difficulty during viewing.

Brief overview of signal transduction

As humans we have to adapt to changing seasons, by wearing a coat in the winter for example. Cells behave in a similar manner in that they also have to adapt to changes in their environment. They do this by relaying environmental changes as 'signals' to their interior - termed signal transduction. So where do calcium ions come into this? In cells, calcium ions can act as second messengers. Second messengers help 'transduce' the signal inside the cell, in order to facilitate a change in cell behaviour.

SIGNAL (ENVIRONMENTAL CHANGE) ---->  CELL ----> SIGNAL TRANSDUCTION ----> CHANGE IN CELL BEHAVIOUR

Uses of calcium

Calcium ions regulate many cellular activities, examples include:

  • Fertilisation
  • Muscle contraction
  • Neuron excitability
  • Metabolism

Gradients - setting the stage for calcium signalling

Calcium signalling is driven by gradients across membranes. How so? Well there are less calcium ions in the cytosol (~100nM) than in the extracellular space (mM). This means that there are more positive calcium ions on the extracellular side of the plasma membrane (NB - Ca2+). As a result of this electrochemical gradient - calcium ions are pulled into the cell to even things out....

 

However - too much calcium in the cytosol has a toxic effect and spells bad news for the cell. Therefore cells strive to maintain a low cytosolic ca2+ concentration using three main methods.

  1. Actively pump calcium out of the cell.
  2. Chelate calcium using Ca2+ binding proteins.
  3. Compartmentalise Ca2+ into intracellular stores.

 

There is a higher concentration of calcium ions outside the cell than inside. When channels are open

1) Actively pumping calcium ions out of the cell

There are two main methods of calcium extrusion via the plasma membrane

1) Plasma membrane Ca2+/ATPase (PMCA) pumps

Out: 1 Ca  In: 2H+  Energy used: 1 ATP

Rate: 30 ions per second (high affinity for calcium)

 

2) Exchangers - 2000 Ca per second

Na/Ca K (NCKX) -> Out: Ca and K  In: 4Na

Na/Ca (NCX) -> Out: Ca   In: 3Na

 

Exchangers work at faster rates than ATPases. They facilitate rapid changes in calcium concentration - as required in neurons during generation of action potentials.

Pumps - maintain low cytosolic concentrations of calcium over longer durations of time than exchangers.



2) Sequestering calcium into intracellular stores

Calcium needs to be shifted from the cytosol into intracellular stores (if not extruded or chelated). Calcium can be stored in the endoplasmic reticulum/sarcoplasmic reticulum. This requires opening of sarco(endo)plasmic reticulum calcium ATPase (SERCA) pumps. Alternatively, calcium ions can traverse the outer mitochondrial membrane via simple diffusion down a proton gradient. Beyond this they require opening of highly selective mitochondrial calcium (MiCa) uniporter ion channels in order to be sequestered in the mitochondrial lumen.

 

Remember:

1) SERCA Pump (to ER/SR)    2) MiCa (to mitochondria)

3) Chelating calcium: Calcium binding proteins

The third method of removing free calcium ions from the cytosol is to bind them to proteins. These proteins fall under two groups:

Buffers

  • Function to control the amount of free calcium ions floating in the cytosol.
  • eg - calbindin (in the brain), and calsequestrin (in the ER/SR).

 

Sensors

  • Use C2 protein domains - allow the protein to bind calcium directly.
  • eg phospholipase C, PI3K, protein kinase C.
  • OR - use calcium-binding proteins to interact with calcium eg calmodulin.
  • examples of these include - IP3 receptor and the ryanodine receptor (both below).

 

Calmodulin (CaM 1-4)

  • CALcium MODULated proteIN.
  • an ubiquitous adaptor protein.
  • conserved over evolution - its expression levels were a determinant for the beak shapes of Darwin's finches.

 

Structure

  • Calmodulin is a dumbbell shaped protein
  • Contains two globular domains joined by a flexible linker.
  • Two Ca2+ binding sites (EF hands) are on each globular domain (Two per end = four Ca2+ total)  (see image).

 

Calcium binding sites (EF Hands)

  • Named after E and F regions of parvalbumin.
  • Present in many proteins.
  • Two per globular end of calmodulin.
  • helix-loop-helix motif -> negative oxygen atoms in two orthogonal helices cradle positive Ca2+ ions into a twelve amino acid loop.

When Calcium is bound to Calmodulin:

  • A conformational change ensues, exposing hydrophobic surfaces on the protein.
  • Binding/target proteins can then attack these surfaces using ampipathic regions of their protein structure.

 Effect of protein-protein interactions

1) Conformational change causing removal of intrinsic autoinhibition eg activation of Ca/Calmodulin dependent kinase and calcineurin.

2) Remodelling of active sites.

3) Dimerisation of proteins.

 

Calcineurin

  • Calcineurin is a serine/threonine phosphatase.
  • It is important in activation of T cell immune responses.
  • It is normally inactive - via a C-terminal autoinhibitory domain.
  • Ca2+ or calmodulin + Ca2+ can bind at the C-terminus - causing a conformational change that liberates the autoinhibition - allowing phosphatase activity of calcineurin.

 

How does calcineurin work?

  • Calcineurin dephosphorylates nuclear factor of activated cells (NF-ATc 1-4).
  • This allows NFAT to translocate into the nucleus.
  • Here it affects transcription allowing upregulation of interleukin-2 - allowing an immune response to be elicited.

 

How calcium enters the cytosol: Opening of plasma membrane channels

Before reviewing calcium signalling pathways, it is important to understand how calcium enters the cytosol from outside the cell. Each entry method has a specific 'gating' mechanism: voltage gated calcium channels, receptor operated channels, cyclic nucleotide gated channels and store-operated channels.

 

Voltage gated channels

  • Found in excitable cells such as nerve and muscle.
  • Paddle shaped helix-turn-helix mostly containing positive amino acid residues.
  • Voltage changes cause the paddle to turn, opening the 'gate' and allowing Ca2+ to enter the cytosol.

 

Receptor operated channels

  • Open in response to binding of specific extracellular agonists (usually neurotransmitters).
  • eg NDMA receptors, nicotinic receptors).
  • Once the specific ligand binds - the channel opens allowing Ca2+ to enter the cell.

 

Store operated channels (SOCs)

  • In non-excitable cells - calcium entry via the plasma membrane is limited (eg in red blood cells).
  • PMCA pumps out Ca2+ faster than it can be repleted via the PMCA.
  • This depletion quickly activates store operated calcium entry (specifically low ER Ca2+).
  • This allows a small conductance of calcium called calcium release activated current (CRAC).
  • Details of this conductance is still under investigation, but STIM1 and Orai1 have been implicated.

 

Stim1 and Orai1

  • Stim1 is a single spanning transmembrane protein found on the ER thought to be key for activation of CRAC.
  • Orai1 is a protein found that spans the plasma membrane four times (4TM) - important for CRAC. calcium release. It is thought to be the channel-forming subunit used in CRAC.

 

 

Calcium release from intracellular stores - receptors

Here is a brief overview of the receptors used in releasing calcium from intracellular stores. This will make the next section a little clearer. The two receptors important for calcium release from the ER are: IP3 receptors and ryanodine receptors (RyR).

 

IP3 receptors

  • Large, non-selective cation channels found on the ER.
  • Conduct Ca2+ from the ER to the cytosol.
  • Vary in tissues/have tissue specific isoforms.
  • Regulated by an intrinsic suppressor domain that opens in response to - cytosolic IP3 but also low calcium levels and phopshorylation events.

 

Ryanodine receptors

  • Large tetrameric channels found on the ER/SR.
  • Allow Ca2+ to traverse from the ER/SR to the cytosol in response to the plant alkaloid ryanodine.
  • Involved in CICR - calcium-induced calcium release.

 

Calcium-induced calcium release (CICR)

Voltage gated dihydropyridine receptors (DHPR) respond to voltage changes on the plasma membrane. This response allows calcium to enter the cytosol, however the cytosolic concentration is still very low (uM). Low calcium concentrations cause a change in conformation of the ryanodine receptor, opening its pore to allow calcium from the ER/SR to enter the cytosol. Conversely, if the cytosolic Ca2+ concentration is too high, the RyR remains shut.

 

Calcium signalling - phospholipase C pathway

Normally, cytosolic calcium concentrations are kept low. However, this can be increased via release from intracellular stores as we have seen above. The main mechanism inducing this release is via the phospholipase C (PLC) pathway. A ligand, for example a hormone, binds a G protein coupled receptor on the membrane (mainly Gq/11 subtype). The alpha subunit is liberated and activates phospholipase C(B) (alternatively receptor tyrosine kinases can activate PLC(y) ).

 

Phospholipase C pathway

  • PLC cleaves phosphatidylinositol 4,5 bisphosphate (PIP2), producing second messengers 1,4,5-inositol triphosphate (IP3) and diaglycerol (DAG). 
  • DAG is membrane bound, IP3 enters the cytosol.
  • IP3 diffuses to the IP3 receptor on the ER - causing it to open.
  • Ca2+ is released from the ER to the cytosol.
  • Depleted ER stores allow opening of store operated channels in the plasma membrane, allowing more calcium to enter the cytosol.

 

Free cytosolic calcium in this pathway

  • Binds C2 domains on Protein Kinase C. Together they translocate to the plasma membrane, where DAG is waiting to activate protein kinase C.
  • Calcium is also involved in recycling of DAG.
  • DAG kinase is sensitive to calcium - it phosphorylates DAG to create phosphatidic acid.
  • DAG lipase - breaks DAG down to arachidonic acid.
  • These products along with protein kinase C are involved in further signalling events.

 

This may be too complex to understand by text only, therefore I have attached a link: http://bcs.whfreeman.com/lodish5e/content/cat_010/13010-01.htm?v=chapter&i=13010.01&s=13000&n=00010&o this is an animation of what has been described above and may be helpful if there is any difficulty understanding what has been reviewed.

 

Summary

Calcium is a small ion, yet here it has been demonstrated the vast importance it has during signalling in cells. The activities it partakes in is largely controlled by gradients between membranes (especially embedded receptors/channels) in the cell, allowing it to cooperate with proteins it is able to link to. The overall effect of these binding events is to induce a range of potential changes in cell behaviour. The fact it governs so many of the cells activities provides sound reasoning for the extensive research that goes into breaking the locks we have still yet to understand about calcium signalling. 

References

Clapham, D.E. (2007) Calcium signaling. Cell, 131, 1047–1058

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