Understanding Cellular Calcium Signals
4 April 2008, University of Nottingham

Invited speakers

Abstracts

Dr Martin Falcke

Hierarchic modelling of intracellular Ca²⁺ oscillations

We have shown experimentally that molecular fluctuations drive intracellular Ca²⁺ dynamics. Modelling of these dynamics requires to take concentration gradients into account and to carry molecular fluctuations from molecule subunit level to cell level. We will present a modelling concept meeting these requirements. It is based on hybrid deterministic-stochastic simulations using 3-component Green's functions for linearized bulk dynamics and local quasi-static approximations.

Dr Llewelyn Roderick

Nuclear inositol 1,4,5-trisphosphate-induced Ca²⁺ signals control cardiac hypertrophy

Cardiac hypertrophy is a major predictor of heart failure, and is caused by a range of cardiac stressors. Ca²⁺ controls both contraction (excitation-contraction coupling, ECC) and hypertrophic remodelling in cardiac myocytes. How Ca²⁺ can perform both of these functions simultaneously and specifically is a matter of debate. Here we dissected the specific contributions of Ca²⁺ transients associated with ECC and InsP3-induced Ca²⁺ release (IICR) to the hypertrophic action of the vasoactive peptide endothelin (ET-1). We found ET-1 elicited its effect through activation of IICR but not enhancement of ECC. Moreover, ET-1 promoted Ca²⁺ increases in the nuclear region of cardiac myocytes, possibly isolating its effects from the bulk changes in cytosolic Ca²⁺ that occurred during ECC. Significantly, although the β-adrenergic agonist, isoproterenol, and the L-type channel agonist, BayK8644, which both increased ECC, also induced hypertrophy, their pro-hypertrophic action was also dependent upon IICR. The InsP3-dependence of BayK8644- and isoproterenol-induced hypertrophy was found to be through a mechanism involving autocrine/paracrine ET-1. In summary, we have shown that IICR is a key component of hypertrophic signalling and we have identified IICR as a link between myocyte activity, ET-1-secretion and hypertrophic remodelling.

Dr Krasimira Tsaneva

Calcium regulation of spontaneous and receptor-controlled electrical activity in pituitary somatotrophs

Cultured pituitary somatotrophs release growth hormone in response to spontaneous Ca²⁺ entry through voltage-gated calcium channels (VGCC), which is governed by plateau-bursting electrical activity and is regulated by several neurohormones, including GH-releasing hormone (GHRH) and somatostatin. Here we combine experiments and theory to clarify the mechanisms underlying spontaneous and receptor-controlled electrical activity. Experiments support a role of a Na⁺-conducting and cAMP-dependent cation channel in controlling spontaneous and GHRH-stimulated pacemaking; an opposing role of spontaneously active inwardly rectifying K⁺ (K_ir) channels and G-protein-regulated Kir channels in somatostatin-mediated inhibition of pacemaking, as well as a role of VGCC in spiking and BK-type Ca²⁺-activated K⁺ channels in plateau-bursting. In the mathematical model, the plateau-bursting is controlled by two functional populations of BK channels, characterized by distance from the VGCC. The rapid activation of the proximal BK channels is critical for the establishment of the plateau, whereas slow recruitment of the distal BK channels terminates the plateau. The model is compatible with a wide variety of experimental data involving pharmacology and ion substitution and supports the importance of the cAMP-dependent cation channels in maintaining spontaneous pacemaking in pituitary somatotrophs.

Dr Helen Kennedy

Calcium signals at the efferent synapse of developing inner hair cells

Inner hair cells are responsible for detecting and encoding sound information by translating mechanical vibrations into neuronal signals. Early in development inner hair cells fire spontaneous calcium based action potentials that release bursts of neurotransmitter onto the auditory nerve. At a similar time during development inner hair cells are transiently innervated by medial olivocochlear efferent fibres that release ACh triggering a depolarising calcium influx through the α9/α10 nicotinic acetylcholine receptors. The resulting calcium signal subsequently activates SK currents that serve to hyperpolarise the cell, preventing action potential firing. These signaling events occur early in development, before the onset of hearing, and are thought to be crucial to the developmental of the auditory pathway.

Both action potential firing and efferent inhibition rely on rises in intracellular calcium to activate small conductance calcium activated potassium channels (SK2). The aim of this study was to determine whether calcium induced calcium release (CICR) formed an important component of this calcium signal, and thus determine the role for CICR in developing IHCs. In our experiments we used intracellular ryanodine to modulate CICR, at either high concentrations (100µM) that reduces CICR, or low concentrations (1µM) that should enhance calcium release.

Action potential repolarisation was dramatically affected by blocking CICR. During trains of action potentials this effect was cumulative with reploarisation becoming gradually less efficient and eventually failing. Efferent inhibition was only inhibited by 12% when CICR was blocked but enhanced by 74% in low ryanodine.

Our results demonstrate that release of calcium from intracellular stores is involved in both action potential firing and efferent inhibition but that the degree of contribution of CICR in these processes is different. Such differences likely reflect the spatial localisation of membrane channels and intracellular calcium release channels.

Dr Yulia Timofeeva

Calcium and electrical signalling in neural cells

Calcium is known to be actively involved in regulating a large variety of neuronal processes including excitability, associativity, neurotransmitter release and synaptic plasticity. The entry of Ca²⁺ from the extracellular medium into the cell is primarily regulated by voltage-gated calcium channels (VGCCs). Another important contribution to Ca²⁺ concentration is provided by the Ca²⁺ release from the endoplasmic reticulum (ER) through the inositol (1,4,5)-triphosphate IP₃ receptors (IP₃Rs) and the ryanodine receptors (RyRs). The ER in neural cells appears to be a continuous membrane system and may be considered as a neuron-within-a-neuron concept by forming a binary membrane system together with the plasma membrane. The Ca²⁺ signal triggered by the VGCCs can be quite localised whereas the sensitivity of IP₃Rs and RyRs to calcium can set up global calcium waves via Ca²⁺-induced Ca²⁺ release mechanism. Here I will present a biophysically realistic and computationally inexpensive model of a nerve cell that incorporates the notions of discrete distributions of VGCCs and Ca²⁺-sensitive receptors. Mimicking the nonlinear properties of Ca²⁺ channels by a threshold process allows us to solve the model using a mixture of analysis and numerical simulations. The proposed model can be expected to provide a better understanding of the interaction between membrane voltage and action potential-evoked Ca²⁺ signals.

Dr John Love

Non-invasive calcium imaging in plant cells, tissues and organs

We aim to define the role of circadian oscillations in cytoplasmic [Ca²⁺] and dark-induced bursts in chloroplastic [Ca²⁺] in photoperiodic signalling in Arabidopsis thaliana . We use a combination of pharmacological and transgenic approaches to manipulate sub-cellular Ca²⁺ in plants grown in various photoperiods. To quantify and image in vivo [Ca²⁺] we use non-invasive biosensors. [Ca²⁺]-dependent luminescence of cytoplasmic and chloroplast-targeted aequorin enables measurement of [Ca²⁺] dynamics in whole plants and seedlings over an extended period of time. We have also recently developed and expressed in A. thaliana various “cameleons” that permit subcellular [Ca²⁺] imaging, and enable us to address questions of cell type specificity and individual cell involvement in establishing and propagating [Ca²⁺] oscillations in plants. Our talk will focus on different ways in which these empirical approaches may inform models of Ca²⁺ signalling, and vice versa.

Meeting report

Dr Martin Falcke (Berlin) opened the meeting by introducing the toolbox of intracellular Calcium signalling. He pointed out that intracellular Calcium dynamics is a multiscale phenomenon in both space and time. To go from the fastest processes on the nanometre scale to global cellular responses he familiarised the audience with ideas of hierarchical modelling and demonstrated this approach by three dimensional simulations of Calcium release. Dr Llewelyn Roderick (Cambridge) moved the focus to cardiac dynamics and illustrated how the interplay between Calcium dynamics and gene transcription can give rise to diseases like hypertrophy. In addition to elucidating major molecular components such as IP3 and ET-1, he emphasised the distinctive roles of the nucleus and the cytosol as two different locations for Calcium signalling. This underpinned the notion of cells as a spatially heterogeneous environment. Later on Dr Helen Kennedy (Bristol) took up this idea when she presented results for Calcium induced Calcium release in the development of inner hair cells. Although this process is involved in both action potential firing and efferent inhibition, its strength varies. Dr Kennedy argued that this could result from the spatially distinctive recruitment of signalling components on either the plasma membrane or the membrane of internal Calcium stores. Dr Krasimira Tsaneva (Bristol) reported another example for the spatial arrangement of signalling cascades. She demonstrated that the best model to explain the initiation and termination of bursting in pituitary somatotrophs required two populations of Calcium activated Potassium channels which surround a voltage gated Calcium channel. Those closest to the VGCC were responsible for the initiation whereas those further apart controlled the final phase of bursting. VGCCs are crucial in neuronal dynamics, and Dr Yulia Timofeeva (Warwick) proposed a model to couple the spreading of membrane potential to a cytosolic Calcium wave. Her approach was based on threshold dynamics and hence allowed for a computationally efficient model that took the spatially discrete arrangements of both VGCCs and Calcium gated ion channels on the membrane of internal storage compartments into account. Dr John Love (Exeter) concluded the workshop by presenting recent experiments on the circadian rhythm in plants. He showed results for different cell types obtained by non-invasive imaging and highlighted how to control intracellular Calcium dynamics on the subcellular level in plants. Lively discussions continued after the last talk with food and beer (unfunded) at the Victoria Hotel in Beeston.