Local Calcium Signals Research Articles

IQ Motif-Containing G (Iqcg) Is Required for Mouse Spermiogenesis

Spermiogenesis in mammals is the process by which the newly formed products of meiosis, haploid spermatids, undergo a dramatic morphological transformation from round cells into flagellated spermatozoa. The underlying genetic control of spermiogenesis is complicated and not well-characterized. We have used forward genetic screens in mice to illuminate the mechanisms of spermatozoon development. Here, we report that the oligoasthenoteratospermia in a male-specific infertility mutant (esgd12d) is attributable to disruption of a gene called Iqcg (IQ motif-containing G). The causality of the mutation was confirmed with a targeted null allele. Loss of Iqcg disrupts spermiogenesis such that tail formation either occurs incompletely or breaks apart from the sperm heads. Orthologs are present in diverse species as distant as hemichordates, mollusks, and green algae. Consistent with a conserved role in flagellar formation and/or function, the orthologous Chlamydomonas protein is present in that organism’s flagella. Because IQ motif-containing genes typically regulate calmodulin (CaM), which in turn can impact the actin cytoskeleton, these findings suggest a potential role for localized calcium signaling in sperm flagellum morphogenesis.

Potassium conductances mediate bidirectional state-dependent modulation of action potential evoked dendritic calcium signals in dentate gyrus granule cells

Event Abstract Back to Event Potassium conductances mediate bidirectional state-dependent modulation of action potential evoked dendritic calcium signals in dentate gyrus granule cells János Brunner1* and János Szabadics1 1 Institute of Experimental Medicine Hungarian Academy of Sciences, Department of Pharmacology, Hungary Backpropagating action potentials (bAPs) and local calcium signals that they trigger are fundamental for dendritic functions. Here we addressed the question what extent the changes of local dendritic membrane properties can contribute to the shaping of the coupling between dendritic action potentials and the local calcium responses. Using a combination of in vitro electrophysiological and confocal imaging techniques we found that activation of dendritic GIRK channels via mGlu2 or GABAB receptors enhanced the bAP¬-triggered calcium signals in the dendrites of dentate gyrus granule cells (GCs). The enhancement of calcium signals was significant only in those dendritic regions, where these receptors are predominantly expressed. Similarly to GIRK channel activation, somatic hyperpolarization by DC current injection (from -64 mV to -77 mV), significantly increased bAP-associated calcium signals in the proximal dendrites. The hyperpolarization was associated with a decrease in the input resistance due to the rectification of the membrane potential of GCs. The effect of hyperpolarization on the calcium signals was maintained when T-type calcium currents were blocked but it decreased when GIRK channels were inhibited. Simultaneous dual somato-dendritic recordings from GCs showed that somatic hyperpolarization accelerated the repolarization phase of dendritic bAP in the proximal region whereas the rising phase and peak amplitude was not affected. We hypothesize that the larger driving force for calcium ions during the faster repolarization can contribute to the increasing in calcium signals. Employment of previously recorded dendritic bAP waveforms from hyperpolarized membrane potential as voltage command evoked larger calcium currents in nucleated patches compared to bAP waveform from the same recording at depolarized membrane potential. Furthermore, addition of native, high-voltage activated, inactivating potassium conductance by somatic dynamic clamp resulted in faster repolarization and increased AP related calcium signals relative to the control (i. e. in the absence of the extra conductance) at the same membrane potential. In conclusion, our results revealed that activation of potassium currents can profoundly enhance dendritic bAP-evoked calcium signals in GC dendrites, thus providing a previously unknown state-dependent modulatory mechanism in dendritic signalization. Keywords: Hippocampus, Dentate Gyrus, granule cell, backpropagating action potential, dendritic calcium, calcium imaging Conference: 4th NAMASEN Training Workshop - Dendrites 2014, Heraklion, Greece, 1 Jul - 4 Jul, 2014. Presentation Type: Poster presentation Topic: dendritic integration and compartmentalization Citation: Brunner J and Szabadics J (2014). Potassium conductances mediate bidirectional state-dependent modulation of action potential evoked dendritic calcium signals in dentate gyrus granule cells. Front. Syst. Neurosci. Conference Abstract: 4th NAMASEN Training Workshop - Dendrites 2014. doi: 10.3389/conf.fnsys.2014.05.00019 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 11 Apr 2014; Published Online: 12 Jun 2014. * Correspondence: Mr. János Brunner, Institute of Experimental Medicine Hungarian Academy of Sciences, Department of Pharmacology, Budapest, 1083, Hungary, brunnerj@koki.hu Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers János Brunner János Szabadics Google János Brunner János Szabadics Google Scholar János Brunner János Szabadics PubMed János Brunner János Szabadics Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

A Population Density and Moment-Based Approach to Modeling Domain Calcium-Mediated Inactivation of L-Type Calcium Channels

We present a population density and moment-based description of stochastic domain calcium-mediated inactivation of L-type calcium channels. Our approach accounts for the effect of heterogeneity of local calcium signals on whole cell calcium currents; however, in contrast with prior work by Sherman et al. [Biophys J. 58(4):985, 1990], we do not assume that calcium domain formation and collapse are fast compared to channel gating. We demonstrate the population density and moment-based modeling approach using a12-state Markov chain model of an L-type calcium channel [Greenstein and Winslow, Biophys J. 83(6):2918, 2002]. Simulated whole cell voltage clamp responses yield an inactivation function for the whole cell calcium current that agrees or disagrees with the classic result of Sherman et al. when domains dynamics are fast or slow, respectively. We analyze the voltage-dependence of calcium inactivation that occurs via slow heterogeneous domains and find that when channel permeability is held constant, calcium inactivation increases as the domain time constant increases. However, when this parameter study is repeated for fixed maximum domain calcium concentration, inactivation decreases as the domain time constant increases. Comparison of simulation results using population densities and moment equations confirms the computational efficiency of the moment-based approach, and enables the validation of several distinct methods of truncating and closing the open system of moment equations. In general, a slow domain time constant requires higher order moment truncation for agreement between moment-based and population density simulations.

TRPC3 Modulates Association of Orai1 with Immunophilin FKBP12 and Orai-Mediated Ca2+-Transcription Coupling in Mast Cells

Various Ca2+-permeable channels were demonstrated to associate with immunophilins (FKBP12) as well as calcineurin in a wide range of cellular systems. A possible task of such signalosomes may be efficient translation of local calcium signals into control of gene expression. In mast cells, transcriptionally relevant Ca2+-entry is mainly mediated by store-operated Orai channels or by the canonical transient receptor potential channel (TRPC) family. However, as yet little is known as to how these Ca2+-permeable channels interact with FKBP12 and calcineurin and how they contribute to NFAT signaling in mast cells.

Activation of the Cl- channel ANO1 by localized calcium signals in nociceptive sensory neurons requires coupling with the IP3 receptor.

We report that anoctamin 1 (ANO1; also known as TMEM16A) Ca(2+)-activated Cl(-) channels in small neurons from dorsal root ganglia are preferentially activated by particular pools of intracellular Ca(2+). These ANO1 channels can be selectively activated by the G protein-coupled receptor (GPCR)-induced release of Ca(2+) from intracellular stores but not by Ca(2+) influx through voltage-gated Ca(2+) channels. This ability to discriminate between Ca(2+) pools was achieved by the tethering of ANO1-containing plasma membrane domains, which also contained GPCRs such as bradykinin receptor 2 and protease-activated receptor 2, to juxtamembrane regions of the endoplasmic reticulum. Interaction of the carboxyl terminus and the first intracellular loop of ANO1 with IP3R1 (inositol 1,4,5-trisphosphate receptor 1) contributed to the tethering. Disruption of membrane microdomains blocked the ANO1 and IP3R1 interaction and resulted in the loss of coupling between GPCR signaling and ANO1. The junctional signaling complex enabled ANO1-mediated excitation in response to specific Ca(2+)signals rather than to global changes in intracellular Ca(2+).

Diffusive spatio-temporal noise in a first-passage time model for intracellular calcium release

The intracellular release of calcium from the endoplasmic reticulum is controlled by ion channels. The resulting calcium signals exhibit a rich spatio-temporal signature, which originates at least partly from microscopic fluctuations. While stochasticity in the gating transition of ion channels has been incorporated into many models, the distribution of calcium is usually described by deterministic reaction-diffusion equations. Here we test the validity of the latter modeling approach by using two different models to calculate the frequency of localized calcium signals (calcium puffs) from clustered IP3 receptor channels. The complexity of the full calcium system is here limited to the basic opening mechanism of the ion channels and, in the mathematical reduction simplifies to the calculation of a first passage time. Two models are then studied: (i) a hybrid model, where channel gating is treated stochastically, while calcium concentration is deterministic and (ii) a fully stochastic model with noisy channel gating and Brownian calcium ion motion. The second model utilises the recently developed two-regime method [M. B. Flegg, S. J. Chapman, and R. Erban, "The two-regime method for optimizing stochastic reaction-diffusion simulations," J. R. Soc., Interface 9, 859-868 (2012)] in order to simulate a large domain with precision required only near the Ca(2+) absorbing channels. The expected time for a first channel opening that results in a calcium puff event is calculated. It is found that for a large diffusion constant, predictions of the interpuff time are significantly overestimated using the model (i) with a deterministic non-spatial calcium variable. It is thus demonstrated that the presence of diffusive noise in local concentrations of intracellular Ca(2+) ions can substantially influence the occurrence of calcium signals. The presented approach and results may also be relevant for other cell-physiological first-passage time problems with small ligand concentration and high cooperativity.

Ecto-Nucleoside Triphosphate Diphosphohydrolase 2 Modulates Local ATP-Induced Calcium Signaling in Human HaCaT Keratinocytes

Keratinocytes are the major building blocks of the human epidermis. In many physiological and pathophysiological conditions, keratinocytes release adenosine triphosphate (ATP) as an autocrine/paracrine mediator that regulates cell proliferation, differentiation, and migration. ATP receptors have been identified in various epidermal cell types; therefore, extracellular ATP homeostasis likely determines its long-term, trophic effects on skin health. We investigated the possibility that human keratinocytes express surface-located enzymes that modulate ATP concentration, as well as the corresponding receptor activation, in the pericellular microenvironment. We observed that the human keratinocyte cell line HaCaT released ATP and hydrolyzed extracellular ATP. Interestingly, ATP hydrolysis resulted in adenosine diphosphate (ADP) accumulation in the extracellular space. Pharmacological inhibition by ARL 67156 or gene silencing of the endogenous ecto-nucleoside triphosphate diphosphohydrolase (NTPDase) isoform 2 resulted in a 25% reduction in both ATP hydrolysis and ADP formation. Using intracellular calcium as a reporter, we found that although NTPDase2 hydrolyzed ATP and generated sustainable ADP levels, only ATP contributed to increased intracellular calcium via P2Y2 receptor activation. Furthermore, knocking down NTPDase2 potentiated the nanomolar ATP-induced intracellular calcium increase, suggesting that NTPDase2 globally attenuates nucleotide concentration in the pericellular microenvironment as well as locally shields receptors in the vicinity from being activated by extracellular ATP. Our findings reveal an important role of human keratinocyte NTPDase2 in modulating nucleotide signaling in the extracellular milieu of human epidermis.

Metabotropic Glutamate Receptors Drive Global Persistent Inhibition in the Visual Thalamus

Within the dorsal lateral geniculate nucleus (dLGN) of the thalamus, retinal ganglion cell (RGC) projections excite thalamocortical (TC) cells that in turn relay visual information to the cortex. Local interneurons in the dLGN regulate the output of TC cells by releasing GABA from their axonal boutons and specialized dendritic spines. Here we examine the functional role of these highly specialized interneurons and how they inhibit TC cells in mouse brain slices. It was widely thought that activation of metabotropic glutamate receptor type 5 (mGluR5) on interneuron spines leads to local GABA release restricted to sites receiving active RGC inputs. We reexamined experiments that supported this view, and found that in the presence of TTX, mGluR5 agonists evoked GABA release that could instead be explained by interneuron depolarization and widespread intracellular calcium increases. We also examined GABA release evoked by RGC activation and found that high-frequency stimulation induces a long-lasting subthreshold afterdepolarization, persistent firing, or prolonged plateau potentials in interneurons and evokes sustained GABA release. mGluR5 antagonists virtually eliminated sustained spiking and the resulting widespread calcium-signals, and reduced inhibition by >50%. The remaining inhibition appeared to be mediated by a fraction of interneurons in which plateau potentials produced large and widespread calcium increases. Local calcium signals required for local GABA release were not observed. These findings indicate that, contrary to the previous view, RGC activation does not simply evoke localized GABA release by activating mGluR5, rather, synaptic activation of mGluR5 acts primarily by depolarizing interneurons and evoking widespread dendritic GABA release.

Calcium Concentration Fluctuations and Subspace Volume Influence Calcium-Regulated Calcium Channel Gating and Subspace Dynamics

Cardiac myocyte calcium signaling is often modeled using deterministic ordinary differential equations (ODEs) and mass-action kinetics. However, spatially restricted “domains” associated with calcium influx are small enough that local signaling may involve 1-100 calcium ions. Therefore, the question arises: is it appropriate to model the dynamics of subspace calcium using deterministic ODEs or, alternatively, do we require stochastic descriptions that account for the fundamentally discrete nature of these local calcium signals? To address this question, we constructed a minimal Markov model of a calcium-regulated calcium channel and associated subspace. We compared the expected value of subspace calcium concentration and channel open probability (a result that accounts for the small subspace volume and concentration fluctuations) with the corresponding deterministic model (an approximation that assumes large system size and ignores concentration fluctuations). When subspace calcium did not regulate calcium influx, the deterministic and stochastic descriptions agreed. However, when calcium-binding altered channel activity in the model, the continuous deterministic description often deviated significantly from the discrete stochastic model, unless the subspace volume is unrealistically large and/or the kinetics of the calcium binding are sufficiently fast, demonstrating that the calcium concentration fluctuations and subspace volume influence channel gating and subspace dynamics. This principle was also demonstrated using a physiologically realistic model of calmodulin regulation of L-type calcium channels introduced by Yue and coworkers [Tadross, Dick, Yue. Cell 133: 1228-40, 2008]. Additional work will consider the influence of slow and rapid buffers present in the subspace and whether and under what conditions these buffers mitigate the effects of concentration fluctuations on channel gating and subspace dynamics.

Imaging the Motility of Inositol Trisphosphate Receptors in Intact Mammalian Cells using Single Particle Tracking Photoactivated Localization Microscopy (Sptpalm)

Inositol trisphosphate receptors (IP3Rs) are calcium-permeable channels in the membrane of the endoplasmic reticulum (ER) that liberate calcium to generate cytosolic calcium signals that control diverse cellular functions including gene expression, secretion and synaptic plasticity. The spatial distribution of these channels is crucial in determining the patterning of intracellular calcium signals. The mechanisms underlying the aggregation and maintenance of IP3Rs into clusters are controversial. Local calcium puffs reflecting concerted openings of clustered IP3Rs arise at just a few, fixed locations within a cell, suggesting clusters are stable entities; and calcium blips generated by ‘lone’ IP3Rs are also immotile. In contrast, GFP-tagged or immunostained IP3Rs show a dense distribution throughout a cell. Moreover, the majority IP3Rs can diffuse freely within the ER membrane, and aggregate into clusters following activation of IP3 signaling and/or cytosolic calcium elevation. These apparently different behaviors may be explained because calcium imaging detects only functional IP3Rs, whereas immunostained or GFP-tagged IP3Rs report the entire population of IP3R proteins. We therefore hypothesize that most IP3Rs are motile, but functionally unresponsive. Local calcium signals arise, instead, from a small subset of IP3Rs that are anchored, individually or in clusters, by association with static cytoskeletal structures and possibly as a consequence of this anchoring, display high sensitivity to IP3 to produce calcium signals. To test this hypothesis we expressed type 1 IP3R tagged with a photoactivatable genetically encoded protein to track the motility of thousands of individual IP3R molecules with nanoscale spatial and millisecond temporal resolution. We find that IP3Rs can be distinguished into two groups with relatively high or low motility, and that the apparently immotile IP3Rs are preferentially grouped within tight clusters. Support: NIH GM40871 (IP) and GM100201 (IFS).

Semaphorin3A facilitates axonal transport through a local calcium signaling and tetrodotoxin-sensitive voltage-gated sodium channels

Semaphorin3A (Sema3A), a secreted factor that navigates axons and dendrites of developing neurons, facilitates axonal transport. However, little is known about the mechanism underlying Sema3A-induced facilitation and its functional implications. Here we show that Sema3A induces facilitation of axonal transport via local calcium signaling in growth cone. The facilitation of axonal transport was blocked by inhibitors of voltage-gated sodium channels (tetrodotoxin, TTX), L-type voltage-gated calcium channel, and ryanodine receptor (RyR). Sema3A evoked intracellular Ca2+ elevation in growth cone by local application of Sema3A to growth cone. Sema3A also activated RyR in growth cone as well as cell body. Notably, TTX suppressed Sema3A-induced RyR activation in cell body but not in growth cone. Our results identify a novel mechanism of Sema3A-induced axonal transport, and further suggest that Sema3A-induced local calcium signaling in growth cone is propagated to cell body in a TTX-sensitive manner.

Inositol 1,4,5-Trisphosphate 3-Kinase A Is a Novel Microtubule-associated Protein

Inositol 1,4,5-trisphosphate 3-kinase A (IP(3)K-A) is a brain specific and F-actin-binding protein. We recently demonstrated that IP(3)K-A modulates a structural reorganization of dendritic spines through F-actin remodeling, which is required for synaptic plasticity and memory formation in brain. However, detailed functions of IP(3)K-A and its regulatory mechanisms involved in the neuronal cytoskeletal dynamics still remain unknown. In the present study, we identified tubulin as a candidate of IP(3)K-A-binding protein through proteomic screening. By various in vitro and in vivo approaches, we demonstrated that IP(3)K-A was a novel microtubule-associated protein (MAP), and the N terminus of IP(3)K-A was a critical region for direct binding to tubulin in dendritic shaft of hippocampal neurons. Moreover, PKA phosphorylated Ser-119 within IP(3)K-A, leading to a significant reduction of microtubule binding affinity. These results suggest that PKA-dependent phosphorylation and microtubule binding of IP(3)K-A are involved in its regulatory mechanism for activity-dependent neuronal events such as local calcium signaling and its synaptic targeting.

Characterization of NAADP‐Induced Global and Local Calcium Signals in Rat Pulmonary Arterial Smooth Muscle Cells

Ca2+ signaling in vascular smooth muscle cells involves global and local interactions between multiple Ca2+ influx and release pathways. Ca2+ release pathways include IP3 receptor‐, ryanodine receptor‐, and nicotinic acid adenine dinucleotide phosphate (NAADP) receptor‐mediated mechanisms. Recent studies demonstrated that the two‐pore channels (TPC1 and TPC2) are NAADP receptors located in the endo‐/lysosomes. Here we characterized the NAADP‐mediated Ca2+ signals in rat pulmonary arterial smooth muscle cells (PASMCs). Application of the membrane permeable NAADP‐AM (0.25–1 μM) to PASMCs elicited concentration‐dependent increase in global [Ca2+]i. It was blocked by the NAADP antagonist NED‐19 or the acidic vacuolar H+‐ATPase bafilomycin A, suggesting Ca2+ release from the lysosomal Ca2+ stores. The Ca2+ response was independent of extracellular Ca2+ influx, and was unaffected by the IP3 receptor blocker xestospongin C; but was partially inhibited by ryanodine or thapsigargin. Moreover, NAADP caused dramatic increase in local Ca2+ release events, which was inhibited by ryanodine or NED‐19. The temporal and spatial properties of the NAADP‐induced local release events were comparable to the spontaneous Ca2+ sparks, the elementary Ca2+ events mediated via the ryanodine receptors. Our results, hence, show that the NAADP‐sensitive release channels are functionally expressed and Ca2+ signals from the NAADP channel‐gated lysosomal stores cross‐activate ryanodine receptors to amplify Ca2+ release in PASMCs.

Cyclic Nucleotides Cause Divergent Ryanodine Receptor Modulation in Pulmonary Arterial Myocytes from Immature Chronic Hypoxic Sheep

Calcium sparks are due to ryanodine receptor (RyR) activation and are intimate to local and global calcium signals in pulmonary arterial (PA) myocytes. RyRs are heavily regulated and cyclic nucleotides, including cAMP and cGMP, can increase RyR activity and thereby augment Ca2+ sparks and global Ca2+ waves. One possibility is that enhanced RyR activity contributes to altered pulmonary arterial reactivity responses found in sheep born at high altitude. Yet, the influence of chronic hypoxia (CH) on cyclic nucleotide regulation of RyRs is unknown. In these studies, we tested the hypothesis that cAMP and cGMP increase Ca2+ sparks and waves in PA myocytes from hypoxic sheep. Spark and wave activity was determined by visual analysis of line‐scan confocal recordings of Fluo‐4 or Fluo‐8 loaded PA from term‐fetal, ~ 10 day old, or adult sheep that lived at 3,200 meters for <100 days. Similar percentages of fetal, newborn, and adult myocytes had sparks and waves before cyclic nucleotide treatment, although newborns had more sparks per recording. cAMP and cGMP increased the number of adult cells with sparks and waves, yet, they respectively decreased the number of newborn and fetal myocytes with sparks. Spark activity was increased in adult myocytes by cGMP. The data suggest that cyclic nucleotides cause divergent regulation of RyRs from PA myocytes of immature CH sheep. NSF MRI 092355 (SMW), NIH P01HD031226, R01HD003807 (LDL)

Lobe-Specific Functions of Calcium.Calmodulin in Alpha-Calcium. Calmodulin-Dependent Protein Kinase II Activation

N-methyl-D-aspartic acid receptor-dependent long-term potentiation (LTP), a model of memory formation requires calcium.calmodulin-dependent protein kinase II (αCaMKII) activity and Thr286-autophosphorylation via both global and local calcium signalling but the mechanisms of signal transduction are not understood. We tested the hypothesis that the calcium-binding activator-protein calmodulin (CaM) is the primary decoder of calmodulin signals thereby determining the output, e.g. LTP. Thus we investigated the function of CaM mutants, deficient in calcium binding at sites 1, 2 of the N terminal lobe or sites 3, 4 of the C terminal CaM lobe, in the activation of αCaMKII. Occupancy of CaM calcium binding sites 1, 3 and 4 is necessary and sufficient for full activation. Moreover, the N and C terminal CaM lobes have distinct functions: calcium binding to N lobe calcium binding site 1 increases the turnover rate of the enzyme 5-fold, while the C lobe plays a dual role: it is required for full activity, but in addition, via calcium binding site 3, it stabilizes ATP binding to αCaMKII 4-fold. Thr286-autophosphorylation too is dependent on calcium binding sites on both the N and C lobes of CaM. As the CaM C lobe sites are populated by low amplitude/low frequency (global) calcium signals, but occupancy of N lobe site 1 and thus activation of αCaMKII requires high amplitude/high frequency (local) calcium signals, lobe-specific sensing of calcium signalling patterns by CaM is proposed to explain the requirement for both global and local calcium signalling in the induction of LTP via αCaMKII.

Discrete-State Stochastic Models of Calcium-Regulated Calcium Influx and Subspace Dynamics Are Not Well-Approximated by ODEs That Neglect Concentration Fluctuations

Cardiac myocyte calcium signaling is often modeled using deterministic ordinary differential equations (ODEs) and mass-action kinetics. However, spatially restricted “domains” associated with calcium influx are small enough (e.g., 10−17 liters) that local signaling may involve 1–100 calcium ions. Is it appropriate to model the dynamics of subspace calcium using deterministic ODEs or, alternatively, do we require stochastic descriptions that account for the fundamentally discrete nature of these local calcium signals? To address this question, we constructed a minimal Markov model of a calcium-regulated calcium channel and associated subspace. We compared the expected value of fluctuating subspace calcium concentration (a result that accounts for the small subspace volume) with the corresponding deterministic model (an approximation that assumes large system size). When subspace calcium did not regulate calcium influx, the deterministic and stochastic descriptions agreed. However, when calcium binding altered channel activity in the model, the continuous deterministic description often deviated significantly from the discrete stochastic model, unless the subspace volume is unrealistically large and/or the kinetics of the calcium binding are sufficiently fast. This principle was also demonstrated using a physiologically realistic model of calmodulin regulation of L-type calcium channels introduced by Yue and coworkers.

Synaptically evoked Ca2+release from intracellular stores is not influenced by vesicular zinc in CA3 hippocampal pyramidal neurones

The co-release of neuromodulatory substances in combination with classic neurotransmitters such as glutamate and GABA from individual presynaptic nerve terminals has the capacity to dramatically influence synaptic efficacy and plasticity. At hippocampal mossy fibre synapses vesicular zinc is suggested to serve as a cotransmitter capable of regulating calcium release from internal stores in postsynaptic CA3 pyramidal cells. Here we investigated this possibility using combined intracellular ratiometric calcium imaging and patch-clamp recording techniques. In acute hippocampal slices a brief train of mossy fibre stimulation produced a large, delayed postsynaptic Ca(2+) wave that was spatially restricted to the proximal apical dendrites of CA3 pyramidal cells within stratum lucidum. This calcium increase was sensitive to intracellularly applied heparin indicating reliance upon release from internal stores and was triggered by activation of both group I metabotropic glutamate and NMDA receptors. Importantly, treatment of slices with the membrane-impermeant zinc chelator CaEDTA did not influence the synaptically evoked postsynaptic Ca(2+) waves. Moreover, mossy fibre stimulus evoked postsynaptic Ca(2+) signals were not significantly different between wild-type and zinc transporter 3 (ZnT3) knock-out animals. Considered together our data do not support a role for vesicular zinc in regulating mossy fibre evoked Ca(2+) release from CA3 pyramidal cell internal stores.

Abstract A30: Inhibition of PMCA4 as a strategy to increase the effectiveness of Bcl-2 inhibitor-mediated cell death in breast cancer cells.

Abstract Intracellular calcium is a vital cell signaling molecule that can influence a myriad of important cellular functions, including those deregulated in the cancer cell. Consequently Ca2+ levels within the cell must be precisely controlled. Amongst the suite of proteins controlling calcium homeostasis are the plasma membrane calcium ATPases (PMCAs), which belong to a family of druggable proteins. PMCAs are pumps that reside on the plasma membrane and are responsible for the efflux of intracellular Ca2+ and thus are important in maintaining basal cellular calcium levels. Sustained increases in intracellular Ca2+ can be a trigger for cell death and agents that induce alterations in calcium signaling could be a strategy to restore the sensitivity of a cancer cell to death stimuli. The aim of this study was to explore the effectiveness of inhibiting two PMCA isoforms, PMCA1 and PMCA4, on the augmentation of cell death initiated by a Ca2+ionophore (ionomycin) and a Bcl-2 inhibitor (ABT-263) in MDA-MB-231 breast cancer cells. Ionomycin represents a caspase-independent mechanism of cell death and ABT-263 enhances cell death via a caspase-dependent mechanism. PMCA1 and PMCA4 were inhibited using siRNA and the effect on cell death was measured by staining with Hoechst 33342 and propidium iodide using high content imaging (ImageXpress). Intracellular Ca2+ was determined using a FLIPRTETRA with cells loaded with the calcium sensor Fluo4-AM. Inhibition of PMCA1 was associated with significant changes in global cytosolic free Ca2+ induced by purinergic receptor activation, whereas the same effect was not seen with PMCA4 inhibition. Consistent with effects on global calcium levels, ionomycin (3 μM)-induced cell death was significantly augmented by PMCA1 but not PMCA4 siRNA. However, ABT-263 (3 μM)-induced cell death was augmented only by inhibition of PMCA4, suggesting that PMCA4 siRNA-mediated inhibition of ABT-263 apoptosis is mediated through effects on local and/or specific calcium signaling processes. PMCA4 can alter the activity of calcium-dependent transcription factors in cardiac myocytes independent of global cytosolic calcium. In MDA-MB-231 cells, the effects of PMCA4 inhibition appear to be associated with alterations in the activity of NFkappaB. These results highlight the specificity of different isoforms of PMCA in the modulation of cell death pathways in MDA-MB-231 breast cancer cells. Inhibition of PMCA4 may be a viable adjuvant therapy when combined with Bcl-2 family inhibitors for the treatment of breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A30.

Associate editor: Sharona Gordon

The JGP is pleased to announce that Sharona Gordon, associate professor of physiology and biophysics at the University of Washington, has become an associate editor. Sharona has held a Jules and Doris Stein Research to Prevent Blindness Professorship and served with distinction on many national and institutional committees, including the National Institutes of Health/CSR Neurotransmitter Transporter, Receptors and Channels study section, the Biophysical Society Council, and the Biophysical Society Program Committee, which she has chaired for the 2012 meeting. She has been a member of the JGP Editorial Board since 2000, and in 2010 was the organizer and editor of the excellent Perspectives on Local Calcium Signaling (Gordon, 2010; JGP 136:117). Sharona brings to the editorial position a depth of research experience with cyclic nucleotide–activated and TRP channels, and a broad knowledge of neuronal signaling. She also brings to the job a special passion for the recruiting, training, and education of young scientists. Experiment in publishing: Video summaries of articles The editors want to notify the Journal’s authors of an experiment that we will undertake in the upcoming months: the publication of video summaries of JGP articles. The Journal’s goal for this voluntary effort is to enhance access to the scientific content of articles, as though the author were presenting a poster on the work to non-specialists at a general scientific meeting. As a rough guide, the Video Summary will encompass five to seven slides, including an introductory slide and one summary (or future directions) slide and will last 5 minutes. The spoken portion of the presentation should be 650–700 words, presented at a pace making it accessible to nonnative English speakers. The authors’ objective in the Video Summary should be to present the question posed in the work and the main results, for example, as described in the article’s abstract. Authors will be able to submit a Video Summary up to two months after an article’s publication. Video Summaries will remain indefinitely accessible on the JGP website. A Video Summary submission will comprise a written script and PowerPoint presentation that will be submitted by the authors and vetted by the editors around the time of acceptance of a paper. The authors of all accepted articles will be invited to submit a Video Summary, but the decision to do so will lie completely with the article’s authors. The presenter should introduce him/herself along with the title of the paper (if desired, by talking directly to the camera), and then proceed to the introductory slide(s), which should ensure that the non-specialist can understand the point of the research and the specific question(s) it addresses. The presenter will then proceed to the key results, making sure that each item presented is clearly and concisely explained, and keeping in mind that the interested reader can always turn to the published paper to examine control experiments, statistics, etc., to assess the validity of the conclusions. The final slide of the video summary should provide bullet points and or a diagram explaining the main conclusions, and if possible some questions for future research that may have arisen from the study. The JGP will publish a complete description of the Video Summary in the Instructions for Authors. We hope that authors will take advantage of this new option and provide the Journal’s “viewership” with many stimulating and informative presentations.

Alison Munro Elliman

Within the dorsal lateral geniculate nucleus (dLGN) of the thalamus, retinal ganglion cell (RGC) projections excite thalamocortical (TC) cells that in turn relay visual information to the cortex. Local interneurons...