skip to primary navigationskip to content
 

Research Interest

Tuning ca2+ to control cell function

Introduction to Calcium Signalling

Elevations in intracellular calcium (ca2+) are key signalling events that control diverse processes throughout the lifetime of a cell. Notable examples include the wave of ca2+that spreads over the egg immediately following fertilization, and the ca2+-induced contraction of skeletal and cardiac muscle. Work in our laboratory is focused on understanding how cells generate and regulate these intracellular ca2+ signals, as well as how ca2+ elevations are interpreted by cells to determine their fate. Our work is centred on the role of ca2+ in controlling cardiac contraction and arrhythmia, cardiac hypertrophy, regulation of cell death and in neurological diseases.

Intracellular ca2+ signals are generated via several mechanisms. Specifically, binding of hormones, growth factors or neurotransmitters to their cognate receptors on the plasma membrane induce ca2+ signals by stimulating ca2+ influx from the extracellular space via plasma membrane-embedded channels or release from intracellular stores (mainly the endoplasmic reticulum, but also the Golgi, nuclear envelope etc) through channels localized there. Intracellular ca2+ returns to resting levels through the concerted action of plasma membrane and ER-localised ca2+ pumps and exchangers as well as mitochondria.

Laboratory Interests: Background

One of the principal channels responsible for ca2+ release is the inositol 1,4,5-trisphosphate receptor (InsP3R) (Berridge et al., 2003). Over the past several years, we have been testing the premise that through their specific associations with accessory proteins and their intracellular location, InsP3Rs act as coincidence detectors of intracellular signalling events. In this model, binding of accessory protein serves either to modulate the flux of ca2+ through the receptor or it acts as an immediate effector of ca2+ released through the channel. As a result, downstream targets of InsP3R activity are affected (Roderick and Bootman, 2003).

To identify novel InsP3R accessory proteins, we have been using a combination of proteomic and bioinformatics approaches. The consequnce of binding of these accessory proteins to InsP3Rs upon cell physiology has then been assessed (Figure 1).

lr_fig1.jpg
Figure 1. InsP3Rs are signal integrators and signal generators. Identification of proteins that interact with InsP3Rs will provide insight into the signals and signalling cascades that converge on the InsP3Rs as well as those cellular process that are affected by InsP3-induced ca2+ release. This strategy will provide information to place the InsP3R and InsP3-induced ca2+ release in the web of signalling pathways that control the birth, life and death of a cell.

To determine how global or local ca2+ signals arising from InsP3R can control cell function, we have been investigating how ca2+ signals impact upon excitation-contraction coupling and hypertrophy in cardiac myocytes. Cardiac myocytes are ideally suited for these studies since they have InsP3Rs with distinct sub-cellular localisations around the nucleus and plasma membrane where they may perform specific functions (Bootman et al., 2006; Roderick et al., 2007) (Figure 2).

lr_fig2.jpg
Figure 2. Signalling to cardiac hypertrophy. The figure shows the intracellular pathways that are activated downstream of diverse pro-hypertrophic factors. Many of these stimuli, activate increases in both ca2+ cycling as well as other cellular signal transduction cascades such as those mediated by PKC, MAPK and PKB. Certain stimuli also induce increases in intracellular InsP3, which subsequently promotes ca2+ release through InsP3Rs located under the plasmalemma adjacent to RyRs or located around the nucleus. ca2+ release through the plasmalemmal InsP3Rs may contribute to their inotropic function by sensitising RyRs located there whereas ca2+ release through nuclear InsP3Rs may play a role in regulating hypertrophic gene transcription. Whether these nuclear InsP3Rs release ca2+ directly into the nucleus or into the adjacent cytosol is not clear. Hypertrophic gene transcription is activated downstream of MAPK, PKC and PKB signalling pathways (purple arrow). Gene transcription is also induced by changes in ca2+ in the cytosol (orange arrow) or increases in ca2+ in the nucleus (green arrow), where it is sensed by appropriately localised ca2+ sensors/transcriptional activators/co-activators. Hypertrophic remodelling is associated with increased cardiac muscle mass as well as an increase in ca2+ signalling capacity (black dashed arrow). During heart failure ca2+ signalling capacity may be decreased (grey dashed arrow).

Regulation of inositol 1,4,5-trisphosphate receptors by accessory proteins

Figure 3. PKB phosphorylates InsP3Rs. In the presence of growth factor stimulation or during cancer, when PKB activity is high InsP3Rs are phosphorylated. As a result, in response to an apoptotic stimulus, calcium flux to the mitochondria is reduced and cell death prevented. Although mitochondria are the furnace of the cell, responsible for generating ATP, factors released from mitochondria following overload of calcium can induce cell death.

A particularly interesting InsP3R-interacting protein that we have identified and characterised is the lipid-activated kinase, protein kinase B (PKB; also known as Akt) (Szado et al., 2008). PKB has important roles in controlling cell growth, metabolism and survival, and is hyperactivated in numerous cancers. Our work has shown that PKB can phosphorylate the InsP3R and thereby diminish its ability to release ca2+ from stores. The reduction of ca2+ release caused by PKB occurs not only following stimulation by a physiological agonist, but also during apoptosis. We have determined that phosphorylation of InsP3Rs by PKB prevents the flux of ca2+ from the endoplasmic reticulum to the mitochondria, and thereby abrogates cell death. These findings also hold true for glioblastoma cancer cells that are deficient in the lipid phosphatase PTEN (phosphatase and tensin homolog), and thus have constitutively activated PKB.Together, these data place regulation of InsP3-induced ca2+ release high up the hierarchy of pro-survival targets of PKB, and could suggest InsP3Rs are target for future intervention in the treatment of cancer. These findings are encapsulated in the model presented in Figure 3.

Work from our lab has also contributed to the understanding of how the anti-apoptotic protein Bcl-2 functions. In collaboration with Clark Distelhorst (Case Western Reserve University, USA), we have demonstrated that Bcl-2 also interacts with InsP3Rs and suppresses InsP3-induced ca2+ release (Chen et al., 2004). Whilst it is well know that Bcl-2 modulates ca2+ signalling, the exact effect that Bcl-2 is somewhat controversial. We have also shown that the model system used to explore the effects of Bcl-2 on ca2+ signalling is critical to the data generated. Interfering with the interaction between InsP3Rs and Bcl-2 may provide a novel therapeutic target.

InsP3Rs are also important regulators of neuronal function. We have shown that a neuron-specific protein called 'ca2+ binding protein' can interact with InsP3Rs, thereby inhibiting InsP3 binding and ca2+ release. As a result, metabotropic signalling in neurons is suppressed (Kasri et al., 2004).

InsP3Rs in the heart

Action potential motivated ca2+ increases are resposnible for heart function. The ca2+ signals which take place in stimulated cardiac myocytes arise from the activation of ca2+ channels called ‘ryanodine receptors’ (RyRs). These channels are structurally and functionally similar to InsP3Rs, although they are not activated by InsP3, but rather by ca2+ itself. Although RyRs are the principal ca2+ release pathway in cardiac myocytes, we have demonstrated that InsP3Rs are also expressed in the heart, albeit at two orders of magnitude lower level. Recent work from our lab has uncovered that these few InsP3Rs significantly impact upon myocyte physiology and pathology.

Our current work is focussed on trying to understand the role of ca2+ in cardiac remodelling during disease. To accommodate increased homodynamic demand or as a result of disease, the heart hypertrophies. This is characterised by an increase in the size of cardiac myocytes without an increase in their number. Multiple signal transduction cascades, including those regulated by ca2+ are involved in this hypertrophic response. The ability of ca2+ to simultaneously stimulate the gene transcription required for cardiac hypertrophy however, whilst also inducing contraction during every heartbeat has fuelled great debate (Roderick et al., 2007).

Our recent data have shown that pro-hypertrophic stimulation specifically promotes InsP3-dependent ca2+ release in the nucleus, which then drives hypertrophic remodelling of cardiac myocytes.

Future studies aim to identify transcriptional activators/chromatin modifiers that are uniquely sensitive to InsP3R-mediated ca2+ release, and to determine whether epigenetics has a role to play in regulating sensitivity to hypertrophic stimuli.


ca2+ signalling in Neurological Diseases (Industrial Collaborations with Senexis, UCB Pharma, Zyentia and Stem Cell Sciences)

Using ca2+ signalling as an assay of neuronal function in primary and cultured cell models, we are carrying out projects in Parkinson’s Disease, Alzheimer’s Disease and epilepsy.

Collaborators

  • Martin Bootman, Babraham Institute, Babraham, Cambridge
  • Clark Distelhorst, Case Western Reserve University, Ohio
  • Stuart Conway, University of St Andrews, Scotland.
  • Sangeeta Chawla, Department of Pharmacology, University of Cambridge
  • Humbert De Smedt, Jan Parys, Geert Bultynck, Katholieke Universiteit Leuven, Leuven, Belgium