Martin Bootman
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Functional characterisation of the calcium signalling toolkit

Calcium regulates a diverse array of cellular processes. For example, elevation of intracellular calcium is one of the first events to occur following sperm/egg fusion at fertilisation. Pulsatile calcium increases trigger contraction of the beating heart. Dysregulation of calcium homeostasis is known to underlie a variety of pathological conditions and can trigger cell death.

The Calcium Group within the Laboratory of Molecular Signalling has a long-standing interest in how cells generate and interpret calcium signals. Cellular calcium signals are highly versatile- they can occur as brief events localised to specific regions within cells or traverse whole tissues in a repetitive manner. The enormous diversity of calcium signals arises from the complex interplay of different mechanisms that serve to increase or decrease calcium within cells. The types of responses that a cell can display are a function of its ‘calcium signalling proteome'. Such proteomes are cell specific: no two cell types utilise exactly the same combinations of calcium signalling systems and they therefore have unique responses.

The goal of our research is to characterise the elements that make up calcium signalling proteomes, to understand the factors controlling the expression of different elements of such proteomes, and also to elucidate how cells detect and respond to their specific calcium patterns. Our work therefore has the intertwined themes of characterising calcium signalling machinery and understanding physiological/pathological responses to calcium.

Of particular interest in the laboratory are the mechanisms regulating calcium release from intracellular stores. Mobilisation of calcium stores is achieved through the activation of channels expressed on the surface of endomembrane compartments, such as the endoplasmic reticulum or sarcoplasmic reticulum. A principal type of calcium release channel is the inositol 1,4,5-trisphosphate receptor (InsP3Rs). These are large (~1200 kDa) tetrameric proteins, with an amino-terminal domain projecting into the cytoplasm, and an integral calcium channel formed by six membrane-spanning regions in the carboxy-terminal portion of each subunit. InsP3 binding within residues 226-576 of the amino terminus causes a conformational change that promotes channel opening. Between the InsP3 binding site and the transmembrane regions is a large stretch of amino acids where a significant proportion of regulatory interactions occur. InsP3Rs are expressed and participate in calcium release within almost all mammalian tissue, although their function in some cell types is unclear. Three InsP3R isoforms have been cloned and splice variants have been described, leading to the possibility of heteromultimeric channels with distinctive properties based on their subunit content.

We are currently using mass spectrometry and bioinformatics approaches to examine proteins that interact with InsP3Rs. So far, a group of >15 proteins with significant roles in cell biology have emerged as binding partners and regulators of InsP3Rs. One example is the anti-apoptotic protein Bcl-2, which is a key regulator of cell death. Our work has demonstrated that this protein binds to InsP3Rs, decreases the amount of calcium released during cell stimulation and thereby reduces apoptosis. The precise mechanism by which Bcl-2 exerts this effect and its binding site are currently under investigation. Other InsP3R binding partners include cytochrome C, calmodulin and members of the neuronal calcium sensor family. In addition to these proteins with known cellular functions, we are finding InsP3R binding partners with, as yet, unknown function. Furthermore, InsP3Rs also dock kinases, phosphatases and cytoskeletal components, suggesting that they posses the machinery to receive and convey messages inside a cell. In addition to revealing the complexity of InsP3R regulation, these studies will provide a basis for an integrative view of the role of InsP3Rs and calcium release in cell behaviour.

We are also interested in the modulation of cardiac myocytes by InsP3Rs. In particular, we have focussed on the regulation of excitation contraction-coupling in cardiomyocytes from the atrial chambers of the heart. We have demonstrated that perfusion of atrial cardiac myocytes with cardioactive hormones, such as endothelin-1 (ET-1), leads to an increase in the amplitude of calcium transients during excitation contraction-coupling. This is a beneficial ‘positive inotropic' effect that causes greater contractility and can increase blood flow. However, concomitant with the stimulation of contractility, ET-1 triggers spontaneous calcium transients during otherwise quiet diastolic periods. Such spontaneous calcium transients can lead to unsynchronised activity in the heart, known as arrhythmia. A further consequence of stimulating cardiac myocytes with ET-1 is the development of hypertrophy. That is, an increase in cell size without concomitant increase in cell number. Since cardiac myocytes are terminally differentiated and do not divide, hypertrophy is an important mechanism for the heart to respond to demands for greater pumping capacity. However, hypertrophy can also lead to deleterious remodelling of the heart, expression of inappropriate genes and eventual heart failure.

Although it is established that ET-1 activates the pathway that leads to InsP3 production, a role for InsP3-induced calcium signalling in the heart is not generally established. We have shown that cardiac myocytes express functional InsP3Rs that can augment the contractility of cardiac cells, cause arrhythmic calcium release events and also promote cardiac hypertrophy.