Signal Transduction

of the ERK/MAPK pathway. An interesting property of RASAL is that its membrane attachment follows the progress of the Ca2 signal. A train of Ca2 spikes causes it to oscillate on and off the membrane in step with the spikes. By contrast, CAPRI binds rapidly but transiently and does not appear to sense Ca2 oscillations.41

Our knowledge of the molecules and mechanisms by which Ca2 regulates Ras signalling remains incomplete, but there are hints of a machinery that is adapted to the pulsatile nature of the Ca2 signals evoked by receptor activation. Repeated surges of Ca2 will activate the Ras GEFs and the GAPs in a periodic fashion. The resultant effect on Ras activation must depend on the relative kinetics of the two processes, but it will most likely be pulsatile if the GAPs act more rapidly than the GEFs. If the opposite is the case, the activation of Ras will build during a train of Ca2 spikes, perhaps reaching a threshold for activation of downstream pathways. Different Ras effectors may have different requirements in this respect, so that the system may decode complex Ca2 signals.

Cytoskeletal proteins

Cytoskeletal proteins are responsible for the maintenance of cell shape and for motile functions. Almost every form of cellular activation is either

preceded, accompanied, or followed by a rearrangement of at least part of the cytoskeleton. Such a change can be an essential or even defining component of the cellular response. In non-muscle cells, the cytoskeletal arrangement

of microfilaments (F-actin) is controlled by a large array of proteins, some of which bind actin.42 A number of these, e.g. -actinin and gelsolin, are sensitive to the concentration of cytosol Ca2 . These proteins have various effects on the cytoskeleton. -Actinin is a cross-linker, while gelsolin is a Ca2 -regulated actin-severing (and capping) protein. However, the cross-linking action of-actinin (which has two EF-hands) is inhibited by Ca2 and its consequent removal from F-actin allows access for gelsolin.

Paradigms of calcium signalling

Triggering neurotransmitter secretion

For many species the speedy transmission of neural signals is crucial for survival. The exchange of information between nerve cells (and between nerve and muscle) involves the release, by exocytosis, of neurotransmitter substances at synapses and neuromuscular junctions. Ca2 plays a critical role in mediating this process. Voltage-sensitive Ca2 channels in the plasma membrane of the presynaptic cell admit Ca2 directly into the region where secretory vesicles containing neurotransmitter are located, primed to undergo exocytosis. In fast exocytosis from neurons, secretion is triggered within a millisecond of channel opening. Within the presynaptic cell, the sites of exocytosis lie on average

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only 50 nm from the Ca2 channels and the concentration of Ca2 in this microdomain may rise well into the micromolar range. Cytosolic Ca2 buffers and efficient extrusion mechanisms ensure that this Ca2 signal is local and abrupt. The resting level is resumed within a few tens of milliseconds.

The mechanism by which the brief elevation of Ca2 leads to the fusion of the membrane of the synaptic vesicle with the plasma membrane, involves one or more Ca2 -sensing proteins with fast binding kinetics. For fast exocytosis, the best candidate for this task is synaptotagmin I, a transmembrane protein of synaptic vesicles. Its single cytosolic chain consists of two consecutive

C2 domains, C2A and C2B, linked by a short sequence. The C2A domain binds to acidic phospholipids in a Ca2 -dependent manner43 and this helps the protein to become tethered to the plasma membrane (see page 780). Synaptotagmin also associates with components of the SNARE complex of proteins in Ca2 -dependent secretory cells.44 This consists of the cytosolic chains of vesicle and plasma membrane proteins that, together with cytosolic proteins, form a tightly bound complex which draws the vesicle and plasma membranes together. It is only when Ca2 binds to synaptotagmin that the two membranes merge to form a fusion pore that leads to exocytosis.

Initiation of contraction in skeletal muscle

The contraction of all types of muscle depends on an increase in intracellular Ca2 . In skeletal muscle acetylcholine released at the neuromuscular junction binds to nicotinic receptors on the muscle end-plate. These are non-specific cation channels and the binding causes them to open, producing

a depolarization that propagates across the plasma membrane (sarcolemma) of the muscle cell and throughout the transverse tubular invaginations (T-tubules), which penetrate the cell in the vicinity of the contractile machinery. The voltage-sensing subunits of the sarcolemmal voltage-operated Ca2 channels are termed dihydropyridine receptors (DHPRs: dihydropyridine is a drug). These are distributed along the T-tubules and are directly coupled to ryanodine receptors (RyR1) on the membrane of sections of the sarcoplasmic reticulum that lie very close to the tubular membrane. The assemblage of T-tubules with the SR membranes forms the junctional triads. The ryanodine receptors are visible in electron micrographs of triads as‘junctional feet’(Figure 8.6).

The direct interaction between the DHPRs and the RyRs results in substantial Ca2 release from the SR (page 214) and a steep rise in concentration of Ca2 in the close vicinity of the contractile apparatus. This focusing of the Ca2 signal ensures a rapid response and has the further advantage that the resting state can be rapidly resumed, since only a small quantity of Ca2 ions have to be removed.

Within each skeletal muscle cell, the contractile machinery of actin and myosin filaments is controlled by the proteins tropomyosin and troponin. The attachment of myosin heads to actin, which causes contraction, is prevented

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Fig 8.6  Triad junctions and junctional feet.

Electron micrographs of the swimbladder muscle from the toadfish (Opsanus tau). The section is cut perpendicular to the T-tubule axis and the‘junctional feet’, which are ryanodine receptors, are indicated in the magnified image. Micrograph courtesy of Clara Franzini-Armstrong.45

by the presence of threads of tropomyosin organized on the surface of the actin filaments. Troponin, a complex of three subunits – I, T, and C – is distributed at intervals along the actin filaments and it acts to mediate the effects of Ca2 . Troponin C closely resembles calmodulin, possessing four EF-hand Ca2 binding sites. The C-terminal sites are of high affinity

(KD 10 7 mol L 1) and the two sites in the N-terminal or ‘regulatory’ lobe are of low affinity (KD 10 5 mol L 1). The C-terminal sites can bind Ca2 or Mg2 and are occupied at resting levels of Ca2 . As the concentration rises, it binds to the low-affinity N-terminal sites causing a conformational change. This is passed on to the rest of the troponin complex, resulting in the lifting of the inhibition of the myosin ATPase by tropomyosin and allowing contraction to occur. As the concentration of Ca2 returns to the resting level, inhibition by tropomyosin is re-established and contraction ceases.46

The speed with which these events occur can be very fast. For example, the muscles that surround the swim bladder of the toadfish can twitch at

frequencies in excess of 100 Hz, enabling it to emit a characteristic‘boatwhistle’ sound. Humming birds flap their wings 80 times per second, but this can rise to 200 in courtship flight. A failure to clear Ca2 between successive stimuli (depolarizations) would cause the fusion of consecutive twitches and ultimately a permanent contraction (tetany). The achievement of such high-frequency

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