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Chapter 8

Calcium effectors

Calcium-binding by proteins

In the previous chapter we have seen how transient elevations of the concentration of free Ca2 may occur in cells in response to receptor activation or membrane depolarization. We also saw how these signals may be confined at subcellular locations or may propagate and how they may be temporally encoded as spikes or oscillations. Now we ask how changes in [Ca2 ] within the cytosol are sensed and converted into downstream signals. Not surprisingly, Ca2 -binding proteins are involved. However, molecules that bind Ca2 are not necessarily mediators of signalling. For instance, cytosol proteins with very high Ca2 affinity will already be saturated and will remain unaffected by a rise in its concentration above the resting level (40–100 nmol L 1). Lower affinity proteins with dissociation constants 0.1 mol L 1 will certainly bind more Ca2 , but again it does not necessarily follow that they will take part in signalling. Instead, they may be important as buffers that stabilize Ca2 levels or help to shape Ca2 transients. On the other hand, there is a diversity of signalling proteins that bind Ca2 at regulatory sites and that are activated by increases in its level. These are the Ca2 effectors. A selection is listed in Table 8.1.

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Table 8.1  Examples of effector proteins of vertebrate origin that are activated by Ca2

Protein

Type

Ca2 -binding

Function

Main location

 

 

domains

 

 

 

 

 

 

 

Calreticulin

 

 

Ca2 buffering

ER and SR

 

 

 

 

 

Calsequestrin

 

 

Ca2 buffering

SR in muscle

 

 

 

 

 

Parvalbumin

 

EF hand

cytosolic Ca2 buffering

muscle/nerve

 

 

 

 

 

Ca2 -ATPase (PMCA)

CaM dependent

 

pumps Ca2 out of cell

plasma

 

 

 

 

membrane

 

 

 

 

 

Ca2 -ATPase (SERCA)

 

 

pumps Ca2 into stores

ER membrane

 

 

 

 

 

Calmodulin (CaM)

 

EF hand

multipurpose Ca2 sensor

 

 

 

 

 

 

Troponin C

 

EF hand

Ca2 -sensor mediating

striated muscle

 

 

 

contraction

 

 

 

 

 

 

Calmodulin kinases I, II, IV

CaM is a regulatory

 

multipurpose signalling

 

 

subunit

 

 

 

 

 

 

 

 

Myosin light chain kinase

Ca2 /CaM dependent

 

phosphorylates myosin II

smooth muscle

 

 

 

 

 

Adenylyl cyclases 1, 8

Ca2 /CaM dependent

 

makes cyclic AMP

 

 

 

 

 

 

Adenylyl cyclases 5, 6

 

 

makes cyclic AMP (Ca2 inhibits)

 

 

 

 

 

 

Transduction Signal

Cyclic nucleotide phosphodiesterase

Ca2 /CaM dependent

 

breaks down cyclic AMP

 

(1A–C)

 

 

 

 

 

 

 

 

 

Phosphorylase b kinase

CaM is a regulatory

 

phosphorylates glycogen

skeletal muscle

 

subunit

 

phosphorylase

 

 

 

 

 

 

Recoverin

Ca2+- myristoyl switch

EF hand

Ca2+- sensing mediator

photoreceptor

 

 

 

 

cells

 

 

 

 

 

Calpain

 

EF hand

protease

 

 

 

 

 

 

-Actinin

 

EF hand

cytoskeleton

 

 

 

 

 

 

Gelsolin

 

 

actin severing and capping

 

 

 

 

 

 

Synaptotagmin

Putative Ca2 sensor

C2

signalling

secretory cells

Calcineurin (protein phosphatase 2B)

Ca2 /CaM dependent

EF hand

signalling, e.g. transcription

 

 

 

 

 

 

Protein kinase C ( , 1, 2, )

 

C2

signalling

 

 

 

 

 

 

Phospholipase C (all isoforms)

 

EF hand, C2

signalling

 

 

 

 

 

 

Diacylglycerol kinase

 

EF hand

makes phosphatidate

 

 

 

 

 

 

Nitric oxide synthase

CaM is a regulatory

 

production of NO for signalling

 

 

subunit

 

 

 

CaM, calmodulin; PMCA, plasma membrane Ca2 -ATPase; SERCA, sarco/endoplasmic reticulum Ca-ATPase.

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effectors Calcium

Signal Transduction

Polypeptide modules that bind Ca2

Common Ca2 -binding sites on proteins include the EF-hand motif and the C2 domain. Their structures are described in Chapter 24. EF-hand motifs occur in pairs and a single motif binds a single Ca2 ion with KD in the range 10 7–10 5 mol L 1. However, some EF-hands have lost or never even acquired the ability to bind Ca2 , for example the EF-hands of phospholipase C (see Chapter 5). The Ca2 dissociation constants of C2 domains also vary widely (10 6–10 3 mol L 1) and again, some do not bind Ca2 at all.

Many Ca2 effectors are without either C2 domains or EF-hands, but acquire their Ca2 dependence through the Ca2 sensor protein calmodulin, which in some cases forms an integral subunit (Table 8.1). Other Ca2 -binding proteins lack C2 domains or EF-hands and function independently of calmodulin. They may be found among the wide range of channels and ATPases that conduct Ca2 ions across membranes. Further examples are the buffering proteins calreticulin and calsequestrin, present in the ER and SR, the extracellular adhesion molecule cadherin, and the actin-modifying protein gelsolin.

Decoding Ca2 signals

When Ca2 signals are transient, an effector must be able detect the change and initiate a response before the concentration subsides. When Ca2 rises transiently, sensing the change does not just depend on the stability constant of the binding, but also the rates of the‘on’and‘off’reactions. The forward reaction requires the successive displacement, one at a time, of water molecules from the solvation shell of the cation. In general, if a multidentate coordinating ligand is to associate with or dissociate from a cation rapidly, then its framework needs to be flexible. The evolution of such coordination sites in proteins has led to the emergence of Ca2 -activated regulatory enzymes that can bind and respond very rapidly to changes in Ca2 concentration.

Ca2 signals are often repetitive, taking the form of trains of spikes or pulses with periods of the order of minutes (see Figure 7.6, page 195) and it is likely that there are specific effectors that can respond to this form of temporal encoding. While a sustained rise in [Ca2 ] might be damaging because it could activate downstream effectors indiscriminately, an oscillatory signal would favour those with low effective off-rates, allowing them to retain their bound Ca2 during a downswing. It would also favour effector pathways that remain active for just long enough to ensure throughput during dips in [Ca2 ]. Evidence is accumulating in support of these ideas. For instance, in T lymphocytes a cytoplasmic Ca2 signal that oscillates can be more effective at activating transcription than one that is steady. Moreover, high-frequency oscillations can activate three different transcription factors, while at low frequencies only NF- B is activated1,2 (see page 521).

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