STIM1 regulates pancreatic ß-cell behaviour: A modelling study.

Pancreatic ß-cells are equipped with the molecular machinery allowing them to respond to high glucose levels in the form of electrical activity and Ca2+ oscillations. These oscillations drive insulin secretion. Two key ionic mechanisms involved in this response are the Store-Operated Current and the current through ATP-dependent K+ channels. Both currents have been shown to be regulated by the protein STIM1, but this dual regulation by STIM1 has not been studied before. In this paper, we use mathematical modelling to gain insight into the role of STIM1 in the ß-cell response. We extended a previous ß-cell model to include the dynamics of STIM1 and described the dependence of the ATP-dependent K+ current on STIM1. Our simulations suggest that the total concentration of STIM1 modifies the bursting frequency, the burst duration and the intracellular Ca2+ levels. These results are in good agreement with experimental reports, and the contribution of the studied currents to electrical activity and Ca2+ dynamics is discussed. The model predicts that in the absence of STIM1 the excitability of the plasma membrane increases and that the glucose threshold for electrical activity is shifted to lower concentrations. These computational predictions may be related to impaired insulin secretion under conditions of reduced STIM1 in the diabetic state.

The Role of Nicotinic Receptors on Ca2+ Signaling in Bovine Chromaffin Cells.

Chromaffin cells have been used as a physiological model to understand neurosecretion in mammals for many years. Nicotinic receptors located in the cells' membrane are stimulated by acetylcholine, and they participate in the exocytosis of chromaffin granules, releasing catecholamines in response to stress. In this work, we discuss how the participation of nicotinic receptors and the localization of active zones in the borders of the cytoskeleton can generate local calcium signals leading to secretion. We use a computational model of a cytoskeleton cage to simulate Ca2+ levels in response to voltage and acetylcholine pulses. We find that nicotinic receptors are able to enhance the differences between local and average calcium values, as well as the heterogeneous distributions around the active zones, producing a non-linear, highly localized Ca2+ entry that, although consisting of a few ions, is able to improve secretion responses in chromaffin cells. Our findings emphasize the intricate interplay among nicotinic receptors, the cytoskeleton, and active zones within chromaffin cells as an example of Ca2+-dependent neurosecretion in mammals.

α3ß4 Acetylcholine Nicotinic Receptors Are Components of the Secretory Machinery Clusters in Chromaffin Cells.

The heteromeric assembly of α3 and ß4 subunits of acetylcholine nicotinic receptors (nAChRs) seems to mediate the secretory response in bovine chromaffin cells. However, there is no information about the localization of these nAChRs in relationship with the secretory active zones in this cellular model. The present work presents the first evidence that, in fact, a population of these receptors is associated through the F-actin cytoskeleton with exocytotic machinery components, as detected by SNAP-25 labeling. Furthermore, we also prove that, upon stimulation, the probability to find α3ß4 nAChRs very close to exocytotic events increases with randomized distributions, thus substantiating the clear dynamic behavior of these receptors during the secretory process. Modeling on secretory dynamics and secretory component distributions supports the idea that α3ß4 nAChR cluster mobility could help with improving the efficiency of the secretory response of chromaffin cells. Our study is limited by the use of conventional confocal microscopy; in this sense, a strengthening to our conclusions could come from the use of super-resolution microscopy techniques in the near future.

On the Electrophysiological Component of Pancreatic Alpha-Cell Models.

Glucagon, the main hormone responsible for increasing blood glucose levels, is secreted by pancreatic alphacells in a Ca2+ dependent process associated to membrane potential oscillations developed by the dynamic operation of K+, Na+ and Ca2+ channels. The mechanisms behind membrane potential and Ca2+ oscillations in alpha-cells are still under debate, and some new research works have used alpha-cell models to describe electrical activity. In this paper we studied the dynamics of electrical activity of three alpha-cell models using the Lead Potential Analysis method and Bifurcation Diagrams. Our aim is to highlight the differences in their dynamic behavior and therefore, in their response to glucose. Both issues are relevant to understand the stimulus-secretion coupling in alpha-cells and then, the mechanisms behind their dysregulation in Type 2 Diabetes.Clinical Relevance - A reliable description of the electrophysiological mechanisms in pancreatic alpha-cells is key to understand and treat the dysregulation of these cells in patients with Type 2 Diabetes.

Reduction of ER-Mitochondria Distance: a Key Feature in Alzheimer's and Parkinson's Disease, and During Cancer Treatment.

One remarkable dynamic cell structure is the region between the endoplasmic reticulum (ER) and the mitochondria, termed the mitochondria-associated membranes (MAM). MAMs carry out different cellular functions such as Ca2+ homeostasis and lipid synthesis, which depend on an adequate distance separating the ER and mitochondria. A decreased distance has been observed in Alzheimer's disease, Parkinson's disease, and during cancer treatment. It is unclear how dysregulation of the spatial characteristics of MAMs can cause abnormal Ca2+ dynamics which could end in cell death. In this work, a computational model was proposed to study the relationship between a decreased ER-mitochondria distance and mitochondria-induced cell death. Our results point towards the mitochondrial permeability transition pore (mPTP) as a key cell death signaling mechanism indirectly regulated by the spatial characteristics of MAMs.Clinical Relevance- The endoplasmic reticulum-mitochondria crosstalk plays an important role in the mPTP-induced apoptosis. This process could be behind neurodegeneration in Alzheimer's and Parkinson's diseases, as well as behind the induced cell death during cancer treatment.

Calcium Oscillations in Pancreatic α-cells Rely on Noise and ATP-Driven Changes in Membrane Electrical Activity.

In pancreatic α-cells, intracellular Ca2+ ([Ca2+]i) acts as a trigger for secretion of glucagon, a hormone that plays a key role in blood glucose homeostasis. Intracellular Ca2+ dynamics in these cells are governed by the electrical activity of voltage-gated ion channels, among which ATP-sensitive K+ (KATP) channels play a crucial role. In the majority of α-cells, the global Ca2+ response to lowering external glucose occurs in the form of oscillations that are much slower than electrical activity. These Ca2+ oscillations are highly variable as far as inter-spike intervals, shapes and amplitudes are concerned. Such observations suggest that Ca2+ dynamics in α-cells are much influenced by noise. Actually, each Ca2+ increase corresponds to multiple cycles of opening/closing of voltage gated Ca2+ channels that abruptly become silent, before the occurrence of another burst of activity a few tens of seconds later. The mechanism responsible for this intermittent activity is currently unknown. In this work, we used computational modeling to investigate the mechanism of cytosolic Ca2+ oscillations in α-cells. Given the limited population of KATP channels in this cell type, we hypothesized that the stochastic activity of these channels could play a key role in the sporadic character of the action potentials. To test this assumption, we extended a previously proposed model of the α-cells electrical activity (Diderichsen and Göpel, 2006) to take Ca2+ dynamics into account. Including molecular noise on the basis of a Langevin type description as well as realistic dynamics of opening and closing of KATP channels, we found that stochasticity at the level of the activity of this channel is on its own not able to produce Ca2+ oscillations with a time scale of a few tens of seconds. However, when taking into account the intimate relation between Ca2+ and ATP changes together with the intrinsic noise at the level of the KATP channels, simulations displayed Ca2+ oscillations that are compatible with experimental observations. We analyzed the detailed mechanism and used computational simulations to identify the factors that can affect Ca2+ oscillations in α-cells.

Electrophysiological models of the human pancreatic δ-cell: From single channels to the firing of action potentials.

Minimal mathematical models were developed to describe the electrophysiological properties of human δ-cells. Markov models of single channels were first developed based on the analysis of electrophysiological data. Monte Carlo simulations of voltage-clamp experiments were performed in an iteratively optimization procedure to estimate the number of channels required to reproduce the main characteristics of the macroscopic currents recorded experimentally. A membrane model of the firing of action potentials was then developed based on the kinetic schemes of single channels and the number of channels estimated. We showed that macroscopic currents of human δ-cells can be reproduced by minimal models of single channels when the appropriate number of channels is considered. In addition, our simulations suggest that human δ-cells are capable of generating action potentials through the interaction of the ionic currents involved. Finally, we determined the relative contribution of the currents underlying the firing of action potentials in human pancreatic δ-cells, which allowed us to propose a qualitative model of an action potential in terms of the underlying ionic currents.

Mitochondrial Ca2+ dynamics in cells and suspensions.

Mitochondria play a significant role in shaping cytosolic Ca2+ signals. Thus, transfer of Ca2+ across the mitochondrial membrane is much studied, not only in intact cells but also in artificial systems such as mitochondrial suspensions or permeabilised cells. Observed rates of Ca2+ changes vary by at least one order of magnitude. In this work, we investigate the relationship between the Ca2+ dynamics observed in various experimental conditions using a computational model calibrated on experimental data. Results confirm that mitochondrial Ca2+ exchange fluxes through the mitochondrial Ca2+ uniporter (MCU) and the Na+ /Ca2+ exchanger obey the same basic kinetics in cells and in suspensions, and emphasise the important role played by the high Ca2+ levels reached in mitochondria-associated endoplasmic reticulum membranes in intact cells. Tissue specificity can be ascribed to the different modes of regulation of the MCU by Ca2+ , probably related to the specific levels of expression of the Ca2+ sensing regulator subunit of this channel. The model emphasises the importance of mitochondrial density and buffering in controlling the rate of Ca2+ exchanges with mitochondria, as verified experimentally. Finally, we show that heterogeneity between individual mitochondria can explain the large range of amplitudes and rates of rise in mitochondrial Ca2+ concentration that have been observed experimentally.

A theoretical study of factors influencing calcium-secretion coupling in a presynaptic active zone model.

A theoretical analysis of some of the relevant factors influencing the calcium time course and the strength and timing of release probabilities of vesicles evoked by an action potential in a calyx-type active zone is presented in this paper. In particular, our study focus on the comparison of cooperative vs non-cooperative calcium binding by the release site and the effect of the number of Ca(2+) binding sites on the calcium sensitivity for release. Regarding the comparison of cooperative and non-cooperative kinetic schemes, our simulations show that quite different results are obtained when considering one or another: a reduction in the release probability of more than a 50% is obtained when considering the cooperative kinetic scheme. Also, a delay in the average time for release appears when using this model for the calcium sensor. Our study also shows that a non-cooperative kinetic binding scheme gives rise to a well defined average calcium level for release assuming that the same kinetic constants are considered for all the sites. Our results also suggest that the central value of the calcium sensitivity for release depends on the number of binding sites N and the dissociation constant KD with a scaling law depending on NKD.

Exocytotic dynamics in human chromaffin cells: experiments and modeling.

Chromaffin cells have been widely used to study neurosecretion since they exhibit similar calcium dependence of several exocytotic steps as synaptic terminals do, but having the enormous advantage of being neither as small or fast as neurons, nor as slow as endocrine cells. In the present study, secretion associated to experimental measurements of the exocytotic dynamics in human chromaffin cells of the adrenal gland was simulated by using a model that combines stochastic and deterministic approaches for short and longer depolarizing pulses, respectively. Experimental data were recorded from human chromaffin cells, obtained from healthy organ donors, using the perforated patch configuration of the patch-clamp technique. We have found that in human chromaffin cells, secretion would be mainly managed by small pools of non-equally fusion competent vesicles, slowly refilled over time. Fast secretion evoked by brief pulses can be predicted only when 75% of one of these pools (the "ready releasable pool" of vesicles, abbreviated as RRP) are co-localized to Ca²âº channels, indicating an immediately releasable pool in the range reported for isolated cells of bovine and rat (Álvarez and Marengo, J Neurochem 116:155-163, 2011). The need for spatial correlation and close proximity of vesicles to Ca²âº channels suggests that in human chromaffin cells there is a tight control of those releasable vesicles available for fast secretion.

Model for glucagon secretion by pancreatic α-cells.

Glucagon hormone is synthesized and released by pancreatic α-cells, one of the islet-cell types. This hormone, along with insulin, maintains blood glucose levels within the physiological range. Glucose stimulates glucagon release at low concentrations (hypoglycemia). However, the mechanisms involved in this secretion are still not completely clear. Here, using experimental calcium time series obtained in mouse pancreatic islets at low and high glucose conditions, we propose a glucagon secretion model for α-cells. Our model takes into account that the resupply of releasable granules is not only controlled by cytoplasmic Ca2+, as in other neuroendocrine and endocrine cells, but also by the level of extracellular glucose. We found that, although calcium oscillations are highly variable, the average secretion rates predicted by the model fall into the range of values reported in the literature, for both stimulated and non-stimulated conditions. For low glucose levels, the model predicts that there would be a well-controlled number of releasable granules refilled slowly from a large reserve pool, probably to ensure a secretion rate that could last for several minutes. Studying the α-cell response to the addition of insulin at low glucose, we observe that the presence of insulin reduces glucagon release by decreasing the islet Ca2+ level. This observation is in line with previous work reporting that Ca2+ dynamics, mainly frequency, is altered by insulin. Thus, the present results emphasize the main role played by Ca2+ and glucose in the control of glucagon secretion by α-cells. Our modeling approach also shows that calcium oscillations potentiate glucagon secretion as compared to constant levels of this cellular messenger. Altogether, the model sheds new light on the subcellular mechanisms involved in α-cell exocytosis, and provides a quantitative predictive tool for studying glucagon secretion modulators in physiological and pathological conditions.

The F-actin cortical network is a major factor influencing the organization of the secretory machinery in chromaffin cells.

We have studied how the F-actin cytoskeleton is involved in establishing the heterogeneous intracellular Ca(2+) levels ([Ca(2+)](i)) and in the organization of the exocytotic machinery in cultured bovine chromaffin cells. Simultaneous confocal visualization of [Ca(2+)](i) and transmitted light studies of the cytoskeleton showed that, following cell stimulation, the maximal signal from the Ca(2+)-sensitive fluorescent dye Fluo-3 was in the empty cytosolic spaces left by cytoskeletal cages. This was mostly due to the accumulation of the dye in spaces devoid of cytoskeletal components, as shown by the use of alternative Ca(2+)-insensitive fluorescent cytosolic markers. In addition to affecting the distribution of such compounds in the cytosol, the cytoskeleton influenced the location of L- and P-Q-type Ca(2+) channel clusters, which were associated with the borders of cytoskeletal cages in resting and stimulated cells. Indeed, syntaxin-1 and synaptotagmin-1, which are components of the secretory machinery, were present in the same location. Furthermore, granule exocytosis took place at these sites, indicating that the organization of the F-actin cytoskeletal cortex shapes the preferential sites for secretion by associating the secretory machinery with preferential sites for Ca(2+) entry. The influence of this cortical organization on the propagation of [Ca(2+)](i) can be modelled, illustrating how it serves to define rapid exocytosis.

Minimal state models for ionic channels involved in glucagon secretion.

Pancreatic alpha cells synthesize and release glucagon. This hormone along with insulin, preserves blood glucose levels within a physiological range. During low glucose levels, alpha cells exhibit electrical activity related to glucagon secretion. In this paper, we introduce minimal state models for those ionic channels involved in this electrical activity in mice alpha cells. For estimation of model parameters, we use Monte Carlo algorithms to fit steady-state channel currents. Then, we simulate dynamic ionic currents following experimental protocols. Our aims are 1) To understand the individual ionic channel functioning and modulation that could affect glucagon secretion, and 2) To simulate ionic currents actually measured in voltage-clamp alpha-cell experiments in mice. Our estimations indicate that alpha cells are highly permeable to sodium and potassium which mainly manage action potentials. We have also found that our estimated N-type calcium channel population and density in alpha cells is in good agreement to those reported for L-type calcium channels in beta cells. This finding is strongly relevant since both, L-type and N-type calcium channels, play a main role in insulin and glucagon secretion, respectively.

Association of SNAREs and calcium channels with the borders of cytoskeletal cages organizes the secretory machinery in chromaffin cells.

In chromaffin cells, SNARE proteins, forming the basic exocytotic machinery are present in membrane clusters of 500-600 nm in diameter. These microdomains containing both SNAP-25 and syntaxin-1 are dynamic and the expression of altered forms of SNAREs modifies not only their motion but also the mobility of the associated granules. It is also clear that SNARE microdomain location defines the place for individual vesicle fusion and that the alteration of cluster dynamics affects the fusion process itself. Interestingly, these SNARE patches colocalize with the borders of F-actin cages forming the cytoskeletal cortical network, and these borders also contain clusters of L- and P/Q type calcium channels. The organization of the secretory machinery in association with the borders of cytoskeletal cages seems to be an effective way to promote fast coupling between calcium entry and catecholamine release as demonstrated with the use of mathematical secretory models.

The organization of the secretory machinery in chromaffin cells as a major factor in modeling exocytosis.

The organization of cytoplasm in excitable cells was a largely ignored factor when mathematical models were developed to understand intracellular calcium and secretory behavior. Here we employed a combination of fluorescent evanescent and transmitted light microscopy to explore the F-actin cytoskeletal organization in the vicinity of secretory sites in cultured bovine chromaffin cells. This technique and confocal fluorescent microscopy show chromaffin granules associated with the borders of cortical cytoskeletal cages forming an intricate tridimensional network. Furthermore, the overexpression of SNAP-25 in these cells also reveals the association of secretory machinery clusters with the borders of these cytoskeletal cages. The importance of these F-actin cage borders is stressed when granules appear to interact and remain associated during exocytosis visualized in acridin orange loaded vesicles. These results will prompt us to propose a model of cytoskeletal cages, where the secretory machinery is associated with its borders. Both the calcium level and the secretory response are enhanced in this geometrical arrangement when compared with a random distribution of the secretory machinery that is not restricted to the borders of the cage.

Exocytotic dynamics and calcium cooperativity effects in the calyx of Held synapse: a modelling study.

A stochastic computational approach to the study of secretory processes at the calyx of Held synapse is presented in this paper. The calyx of Held is a giant synapse located in the brainstem which is widely used for experimental recording of neurotransmitter release. We focus on the study of the exocytotic dynamics for a pool of readily releasable vesicles using a Monte Carlo simulation scheme that includes models for the P-type calcium channels, the kinetic reactions of endogenous and exogenous (mobile) buffers, the kinetic reactions for the secretory vesicles, as well as the microscopic diffusion of mobile buffers and calcium ions. The simulations are performed in a 3-D orthogonal grid which approximates a cylindrical domain representing an active zone of the presynaptic terminal of the calyx. For this domain, we quantify the release rates related to calcium currents in response to depolarizing voltage pulses. The influence on simulated pulse/action potential depolarization protocols of the kinetic scheme for the calcium sensor of vesicles and the geometry of calcium channels for the kinetic cooperativity for release, is analyzed at a microdomain level. Among other aspects, our results suggest that the spatial organization of Ca2+ channels could have measurable effects in the kinetic cooperativity which could reflect developing changes in the calyx of Held synapse.

Parallel stochastic simulation of macroscopic calcium currents.

This work introduces MACACO, a macroscopic calcium currents simulator. It provides a parameter-sweep framework which computes macroscopic Ca(2+) currents from the individual aggregation of unitary currents, using a stochastic model for L-type Ca(2+) channels. MACACO uses a simplified 3-state Markov model to simulate the response of each Ca(2+) channel to different voltage inputs to the cell. In order to provide an accurate systematic view for the stochastic nature of the calcium channels, MACACO is composed of an experiment generator, a central simulation engine and a post-processing script component. Due to the computational complexity of the problem and the dimensions of the parameter space, the MACACO simulation engine employs a grid-enabled task farm. Having been designed as a computational biology tool, MACACO heavily borrows from the way cell physiologists conduct and report their experimental work.