Elementary and Global Aspects of Calcium Signalling

The ubiquitous second messenger Ca²⁺ is responsible for regulating a wide range of cellular processes (Clapham, 1995). It is used at the beginning of life to mediate the process of fertilization and then is brought into play to regulate some of the cell cycle events during early development. As cells differentiate to perform specific functions, Ca²⁺ is once again called upon to regulate processes as diverse as muscle contraction, exocytosis, energy metabolism, chemotaxis and synaptic plasticity during learning and memory. Ca²⁺ is an unlikely candidate to perform this role of a universal messenger because prolonged elevations of [Ca²⁺] result in irreversible damage as occurs during cardiac or cerebral ischaemia (Trump and Berezesky, 1995). Because of its cytotoxicity, the intracellular level of Ca²⁺ in resting cells is normally held within a narrow range of 20–100 nmol l⁻¹. The signalling functions of Ca²⁺ have to be performed against this background of a tightly controlled Ca²⁺ homeostasis.

Another consequence of this rigid homeostatic control over Ca²⁺ is that this messenger has a very low diffusibility in cytoplasm. Distributed throughout the cytoplasm is an extensive array of Ca²⁺ pumps (Carafoli, 1994) which rapidly sequester Ca²⁺, thus restricting its diffusion. In order to overcome the twin problems of an inherent cytotoxicity and low diffusibility, cells have evolved an ingenious mechanism of signalling based on presenting Ca²⁺ as brief spikes often organized as regenerative waves (Cheek, 1991; Berridge, 1993; Clapham, 1995). To understand this spatiotemporal organization of Ca²⁺ signalling, it is necessary to describe the properties of the Ca²⁺ channels that regulate the entry of Ca²⁺ into the cytoplasm.

Cells have access to two sources of signal Ca²⁺. First, it can enter from the outside ⁠. Ca²⁺ enters from the outside through a variety of channels such as the voltage-operated channels (VOCs), receptor-operated channels (ROCs) or store-operated channels (SOCs). Second, it can be released from internal stores (Fig. 1). Which of these sources is used varies somewhat from cell to cell. In most cells, it is the internal stores which provide most of the signal Ca²⁺ so attention has focused on the intracellular Ca²⁺ channels, of which there are two main types (Berridge, 1993; Clapham, 1995). First, there is the ryanodine receptor (RYR) family comprising three members: RYR1 found in skeletal muscle and certain neurones (e.g. Purkinje cells), RYR2 found in cardiac muscle, brain and some other cells, and RYR3 found in smooth muscle, brain and other cells (Bennett et al. 1996; Giannini et al. 1995). Second, the inositol 1,4,5-trisphosphate receptor (InsP₃R) family has a number of members (Furuichi and Mikoshiba, 1995; Taylor and Traynor, 1995; Bezprozvanny and Ehrlich, 1995). There are four InsP₃R genes, and further diversity results from alternative splicing. These two receptor families must have evolved from a common ancestor since they display considerable sequence homology which is matched by a number of physiological similarities, particularly with regard to the control of channel opening (Taylor and Traynor, 1995). Cytosolic Ca²⁺ homeostasis in resting cells is achieved by balancing the leak of Ca²⁺ (entering from the outside or from the stores) by the constant removal of Ca²⁺ using pumps either on the plasma membrane or on the internal stores (Fig. 1). These pumps ensure that cytoplasmic [Ca²⁺] remains low and that the stores are loaded with signal Ca²⁺. The brief burst of Ca²⁺ responsible for cell activation is usually produced by the coordinated opening of either the RYRs or the InsP₃Rs. Perhaps their most important property is their sensitivity to Ca²⁺, i.e. they display the phenomenon of Ca²⁺-induced Ca²⁺ release (CICR) which is of major significance for the generation of complex signals. Ca²⁺ has a biphasic effect on the RYRs and InsP₃Rs: as its concentration is increased, it initially exerts a positive feedback effect by enhancing the opening of the channels (i.e. CICR), but as soon as the concentration reaches a certain level the feedback switches from positive to negative and Ca²⁺ then inhibits the channel (Bezprozvanny and Ehrlich, 1995). This negative feedback effect ensures that just enough Ca²⁺ is released to give a meaningful signal, thus avoiding the cytoplasm from being swamped with this potentially cytotoxic agent.

The fact that Ca²⁺ release is regenerative has important implications for signalling because it provides one of the mechanisms for coordinating the activity of individual receptors, i.e. they can communicate with each other using Ca²⁺ as a messenger (Bootman and Berridge, 1995). A specific region within the cell usually functions as an initiation site in that it is the first to release Ca²⁺ which then diffuses outwards to excite neighbouring receptors, thereby setting up a Ca²⁺ wave. A global Ca²⁺ signal is created by coordinating release from all the receptors using Ca²⁺ as the messenger. A more specialized mechanism of coordination is found in skeletal and cardiac muscle, where the opening of the RYRs is tightly coupled to the action potential sweeping over the plasma membrane (Cannell et al. 1995; Lopez-Lopez et al. 1995).

Using a regenerative process is inherently dangerous because it is liable to be triggered by the stochastic opening of a single channel. To avoid such random triggering of regenerative Ca²⁺ waves, cells have developed mechanisms for regulating the excitability of these intracellular receptors such that they are turned off in resting cells but become increasingly excitable when Ca²⁺ signals are being generated. In the case of the InsP₃Rs, excitability is regulated by the agonist-dependent generation of InsP₃ by cell surface receptors. This InsP₃ binds to the InsP₃Rs, greatly enhancing their sensitivity to the stimulatory action of Ca²⁺. In effect, the InsP₃R is under the dual regulation of two agonists – InsP₃ and Ca²⁺. The primary function of the former is to increase the Ca²⁺ sensitivity of the InsP₃R. Similarly, the RYR may also be under dual regulation, at least in some cell types (Lee, 1994; Galione and White, 1994). The putative second messenger cyclic ADP ribose (cADPR) is able to enhance the Ca²⁺ sensitivity of the RYRs.

In summary, through the ability of InsP₃ or cADPR to enhance the sensitivities of the InsP₃Rs and RYRs respectively, these messengers convert the quiescent cytoplasm into an excitable medium in which these intracellular channels can communicate with each other to generate global Ca²⁺ signals.

Recent advances in image analysis using confocal microscopy have enabled the operation of either single or small groups of these intracellular channels to be visualized (Bootman and Berridge, 1995). The brief opening of these channels gives rise to localized pulses (approximately 2 μm in diameter) such as the sparks in cardiac muscle (Cheng et al. 1993) or the blips and puffs in Xenopus oocytes (Yao et al. 1995; Parker and Yao, 1996). These elementary events of Ca²⁺ signalling have a characteristic time course: the concentration of Ca²⁺ builds up rapidly but once the channel closes, because of the negative feedback effect described earlier, the concentration falls more slowly as the Ca²⁺ gradually disperses by passive diffusion (Fig. 2). Ca²⁺ channels in the plasma membrane display similar elementary events such as the bumps in Drosophila receptors (Hardie, 1991) or the quantum emission domains (QEDs) in squid giant synapses (Sugimori et al. 1994). Attention is now focused on how these elementary events contribute to various aspects of Ca²⁺ signalling.

The spontaneous opening of Ca²⁺ channels has been observed in resting cells and this input of Ca²⁺ can contribute to the resting level of Ca²⁺. In smooth muscle cells, the RYRsdisplay elementary events referred to as sparks (Nelson et al. 1995). When these sparks occur close to the plasma membrane, they act on Ca²⁺-sensitive K⁺ channels to produce a spontaneous transient outward current (STOC). The occurrence of STOCs in coronary smooth muscle was found to induce small fluctuations in the resting level of Ca²⁺ (Ganitkevich and Isenberg, 1996). Similarly, low levels of stimulation can increase the frequency of these elementary events, resulting in an increase in the resting level of Ca²⁺ as has been described in Xenopus oocytes (Parker and Yao, 1996) and in HeLa cells (Bootman and Berridge, 1996).

Evidence is beginning to emerge that elementary events might be capable of exerting a highly localized signalling function in addition to their role in contributing to the global elevation of Ca²⁺ levels described in the next section. An interesting case concerns the smooth muscle STOCs which can cause relaxation through membrane hyperpolarization (Nelson et al. 1995). What is remarkable, therefore, is that the same messenger Ca²⁺ is able to control both contraction and relaxation. This ability of Ca²⁺ to mediate opposing responses can be explained by the spatial organization of the Ca²⁺ signalling system. Localized high-concentration pulses of Ca²⁺ near the membrane cause relaxation, whereas contraction results from the global elevation of [Ca²⁺] that occurs when a large proportion of the channels release Ca²⁺ synchronously using one of the mechanisms described below.

The primary role of elementary events is to contribute to the global Ca²⁺ signals responsible for controlling the diverse range of cellular processes described earlier (Bootman and Berridge, 1995). Such global responses depend upon the temporal coordination of a sufficient number of these elementary events so that their individual contributions will sum to give an increase in the cytosolic level of Ca²⁺. As mentioned earlier, cells have evolved two main coordinating mechanisms. First, the opening of RYRs in various muscle cells is synchronized by being tightly coupled to the action potential in the plasma membrane. Second, synchronization is achieved by the channels communicating with each other using Ca²⁺ as a coupling factor. These two mechanisms will be illustrated by describing some specific examples. The evoked release of Ca²⁺ in muscle depends upon the dihydropyridine receptor (DHPR) in the plasma membrane functioning as a voltage sensor which detects the depolarization and then transfers the information to the underlying RYRs. In skeletal muscle, the DHPRs are directly coupled to the RYR1s, and information is transferred by a process of conformational coupling (Tsugorka et al. 1995; Klein et al. 1996). The DHPR responds to membrane depolarization by undergoing a change in conformation which is transmitted to the RYR1, inducing the latter to gate Ca²⁺. Through this tight coupling, the action potential is capable of a near simultaneous recruitment of all the RYR1s to give the explosive release of Ca²⁺ responsible for the contraction of skeletal muscle.

Since the DHPRs do not associate directly with the RYR2s in cardiac cells, coupling is achieved using Ca²⁺ as an intermediary. The action potential opens the DHPRs which gate a small pulse of Ca²⁺ that is then greatly amplified as it stimulates a small group of RYR2s (Cannell et al. 1995; Lopez-Lopez et al. 1995). This coupling unit consisting of one DHPR plus approximately four RYR2s represents an autonomous elementary event of cardiac Ca²⁺ signalling. Ca²⁺ imaging has revealed that these units can fire spontaneously and independently of each other to produce sparks which fail to activate neighbouring units because of their low Ca²⁺ sensitivities (Cheng et al. 1993; Lopez-Lopez et al. 1995; Cannell et al. 1995). The fact that these elementary events can function independently of each other explains the long-standing paradox that Ca²⁺ release in cardiac cells can be graded with membrane potential. For each unit, the process of CICR operates between the DHPRs and the RYR2s, whereas the latter do not communicate with each other. However, the RYRs in the separate units can begin to communicate with each other when the cardiac cell becomes overloaded with Ca²⁺ as a consequence of an increase in the sensitivity of the RYR2s. In effect, this Ca²⁺ overload converts the cytoplasm into an excitable medium such that a Ca²⁺ spark can ignite neighbouring units, thereby setting up a regenerative Ca²⁺ wave (Cheng et al. 1993). Although these Ca²⁺ waves in overloaded cardiac cells are somewhat abnormal and can result in cardiac arrhythmias, they represent the synchronization mechanism used normally by most other cells as described below.

A characteristic feature of the Ca²⁺ signal in many non-muscle cells is that it is often organized as a wave similar to that observed in overloaded cardiac cells. There appear to be pre-determined areas which act as initiation sites where the signal first appears before spreading through the cell as a wave. This behaviour has been well-characterized in Xenopus oocytes responding to increasing concentrations of InsP₃ (Fig. 3) (Yao et al. 1995; Parker and Yao, 1996). At very low InsP₃ concentrations, there is a gradual increase in the resting level of Ca²⁺ which seems to result from the random openings of individual InsP₃ receptors to give brief pulses of Ca²⁺ called blips (Parker and Yao, 1996). As described earlier, the appearance of these elementary events represents the first indication of an increase in excitability and is associated with an increase in the resting level of Ca²⁺ (Parker and Yao, 1996; Bootman and Berridge, 1996). With further increases in stimulation, the blips in Xenopus oocytes grow into larger puffs which represent the initiation sites for the onset of Ca²⁺ waves (Fig. 3B). Such waves represent the mechanism of coordinating the release of Ca²⁺ by all the InsP₃ receptors. If the latter are sufficiently sensitized, they will respond to the Ca²⁺ diffusing away from a puff site and thereby propagate the signal through a process of CICR (Fig. 3C).

Cells maintain a rigid control over the intracellular level of Ca²⁺, thus ensuring that the level is kept low during periods of inactivity. In order to use Ca²⁺ as a messenger, cells overcome this tight homeostatic control by using sophisticated mechanisms to release Ca²⁺ in brief bursts using either InsP₃ or ryanodine receptors. These receptors display a unique autocatalytic process of Ca²⁺-induced Ca²⁺ release (CICR) which overwhelms the normal homeostatic mechanisms by producing an explosive release of Ca²⁺ to give a brief pulse of Ca²⁺ often organized as a wave that sweeps through the cytoplasm. Such waves may occur repetitively, thus establishing an oscillation whose frequency is sensitive to agonist concentration. There is reason to believe, therefore, that Ca²⁺ signalling might be frequency-modulated. By encoding information in the form of brief all-or-none spikes, the cell can use Ca²⁺ as a messenger while retaining a tight control over Ca²⁺ homeostasis.