 |
 |
 |
 |
|
||||
 |
|
||||
|
|
Workshop 3 Abstracts and Lecture Materials:
Presentation Materials: PPT Streaming Video: Real Media Synaptic plasticity is triggered by transient changes in Ca2+ concentration ([Ca2+]) within dendritic spines. This in turn induces molecular alterations including changes in the number and opening probability of various ion channels in the spine membrane that result in potentiation or depression of synaptic transmission. While the mechanisms induced by [Ca2+] variation are being elucidated, how the Ca2+ dynamics is governed remains mysterious. Ca2+ dynamic is influenced by sources and sinks in the spines, such as Ca2+ channels and pumps, but also by Ca2+ buffers and the spatiotemporal effects of diffusion. Here we investigate Ca2+ dynamics in dendritic spines duringback-propagating action potentials (BAPs). During depolarisation, voltage sensitive Ca2+ channels (VSCCs) are activated leading to a [Ca2+] increase. Using confocal laser scanning microscopy, we image BAP-induced Ca2+ transients in dendritic spines of CA1 hippocampal neurons in rat organotypic slices. Analysis of these transients under different conditions-including dye saturation, optical fluctuation analysis, and altered dye concentrations-enable us to estimate the increase and decay in [Ca2+], the number and open probability of VSCCs, and the endogenous Ca2+ buffering capacity. Our model predicts that, according both to our data and to previously published data, much bigger [Ca2+] elevations should be observed, from which we infer the operation of unknown Ca2+ sink mechanisms. We show that additional Ca2+ buffering could account for smaller [Ca2+] transients. However, we also show that, if spatiotemporal diffusion of Ca2+ is taken into account, very large [Ca2+] elevations can occur in microdomains although average transients in the spine are small. By combining experiments with physiology-based models, we can investigate the minimum elements required to explain published experimental observations, thereby clarifying the contribution of various molecular mechanisms to synaptic function and plasticity.
1 Mathematical Biosciences Institute,
The Ohio State University We construct simple mathematical models for the G-protein coupled
transduction machinery of olfactory receptor neurons. The models
all include descriptions of ligand-receptor interaction, intracellular
signaling via the second messenger cAMP, and effector ion channel
activity, although they differ in how calcium-dependent feedback
inhibition is incorporated. These models were parameterized with
respect to voltage-clamped current recordings from single cells
exposed to three different odorants over a family of concentrations
(Firestein, Picco, and Menini, 1993). We investigate how differences
in odorant identity are captured in the model parameterizations,
and test mechanisms to account for adaptation under repetitive or
prolonged stimulation.
Streaming Video: Real Media Inositol 1,4,5-trisphosphate (IP3) links activation of receptors at the plasma membane by extracellular agonists to the release of Ca2+ from the internal stores. IP3 binds to its receptor (IP3R) which is a Ca2+ channel gated by IP3 and cytoplasmic Ca2+ (Dufour JBC;1997). Local changes in the free cytoplasmic Ca2+ concentration regulate many enzymes and many aspects of the cellular economy, from metabolism and secretion to gene expression and cell proliferation. The IP3R exists in three isoforms with specific properties. We studied the expression of these isoforms during liver cirrhosis and regeneration. Methods. Cirrhosis was induced in rats by chronic exposure to CCl4/Phenobarbital (C/P) or by bile duct ligation (BDL), hepatic regeneration by 2/3 hepatectomy. Real time quantitative PCR (TaqMana) and Western blots with specific antibodies measured the expression at the mRNA and protein level. We used the 2-hybrid assay to search for interactions of the IP3R with itslef. Results. IP3R isoform 2 represented the predominant isoform (70%) in normal liver and was localized by immunohisto-chemistry near the bile canaliculus. The expression of the isoform 3, which was very low in normal liver, increased many times after induction of liver cirrhosis by C/P and BDL and appeared localized by immunohistochemistry in cholangiocytes. In the hours following 2/3 hepatectomy the isoform 1 increased and the isoform 2 decreased leading to a transient inversion in the predominant isoform in the hours preceding the first round of cellular division. We determined that the IP3R can interact with itself at the level of its C-terminal part and that this interaction is isoform-independent. The same C-terminal domain interacted also in the context of this assay with the C-terminal part of the Ca2+ entry channel Trp-4. Conclusion. The C-terminal part of the IP3R plays probably an important role in the formation and regulation of the tetrameric Ca2+ channel. It may also be the molecular interface between Ca2+ release from the stores and Ca2+ entry through the plasma membrane. The expression of the IP3R is tightly regulated at the isoform level during liver cirrhosis and regeneration.
Streaming Video: Real Media The fundamental role of fluctuations arising from the control of the IP3 receptor channel by calcium and IP3 became obvious in recent years. Fluctuations induce spatial and temporal structures in systems with deterministic dynamic regimes not able to produce these structures without fluctuations. I will present essential results from stochastic modelling. These results ensue questions concerning the dynamic regime of determinsitic models. Simulations of release through single clusters demonstrates the concentration gradients of four orders of magnitude around releasing channel clusters. These gradients are essential in understanding stability properties of determinsitic models of calcium dynamics. A determinsitic, spatially discrete model demonstrates the essential role of diffusion in local stability. I will discuss consequences of these basic results for future modelling and the interpretation of experimental findings.
The mammalian olfactory organ is arguably the best chemical detector on the planet. The first step in olfactory detection and discrimination is the interaction between an odor compound and a protein receptor on the membrane of specialized olfactory neurons in the nose. These receptors, each one encoded by a gene, are members of a superfamily of similar receptors that also play important roles in the brain and in other bodily functions. As a group they are known as G-Protein Coupled Receptors (GPCRs) and have many similar structural, genetic and functional features. The odor receptors are the most remarkable sub-group of this family in that they are encoded by more than 1000 genes in rodents, and as many as 350 in humans. This is an order of magnitude greater than the total of all other GPCRs. Two approaches to understanding how this large family of receptors act in concert to produce neural signals leading to complex perceptions have been pursued. Pharmacological investigation of the binding properties of ORs has allowed us to define molecular receptive fields and begin to appreciate the immense complexity of combinatorial encoding schemes. In parallel whole genome analysis has revealed an underlying organization of the enormous OR family of genes. Combining pharmacology and genomics provides insights into how the brain perceives a world of innumerable and complex chemical odors.
Ionized free Ca2+ is a ubiquitous signaling ion that serves as both an important charge carrier and a chemical intermediate that links a variety of physiological stimuli to their intracellular effectors. One of the most important steps in Ca2+ signaling is generation of the Ca2+ signal itself. This signal, which expresses the coordinated activity of multiple Ca2+ regulatory systems operating in different subcellular compartments, serves as the critical intermediate in the physiological control of all Ca2+-sensitive processes within cells. What defines this signal? How does it vary with stimulus strength? How is it influenced by the properties of individual Ca2+ channels, pumps, exchangers and binding proteins that may, during the life of a cell, undergo dramatic changes in expression level or modulatory state? What is the functional impact of mutations of the genes encoding proteins that participate in Ca2+ regulation? Answers to these fundamental questions have been elusive, largely because the system properties of cellular Ca2+ regulation are poorly understood. This is due, in large part, to a lack of quantitative functional descriptions of Ca2+ transporters that operate together in intact cells. I will describe an approach to obtaining this information that exploits basic mechanistic similarities between Ca2+ signaling and signaling through changes in membrane potential, and draws upon previous biophysical studies of membrane excitability. Along with a novel combination of experimental techniques, this approach has made it possible to obtain quantitative characterizations of intracellular Ca2+ transport and buffering systems in intact neurons. These characterizations, in turn, provide clear explanations of a number of puzzling properties of stimulus-evoked changes in neuronal Ca concentration, and reveal the existence of signaling regimes analogous to those found in excitable cells expressing multiple populations of voltage-gated ion channels.
The mechanism of receptor-induced activation of the ubiquitously expressed family of mammalian TRPC channels has been the focus of intense study. Primarily responding to phospholipase C (PLC)-coupled receptors, the channels are reported to receive modulatory input from diacylglycerol, endoplasmic reticulum (ER) inositol 1,4,5-trisphoshate receptors (InsP3R), and ER Ca2+ stores. Analysis of TRPC5 channels transfected within DT40 B cells and deletion mutants thereof, revealed efficient activation in response to PLC- or PLC-? activation which was independent of InsP3Rs or the content of stores. In both HEK293 cells and DT40 cells, TRPC5 and TRPC3 channel responses to PLC-activation were highly analogous, but only TRPC3 and not TRPC5 channels responded to addition of the permeant diacylglycerol (DAG) analogue, 1-oleoyl-2-acetyl-sn-glycerol (OAG). However, OAG application or elevated endogenous DAG resulting from either DAG-lipase or DAG-kinase inhibition, completely prevented TRPC5 or TRPC4 activation. This inhibitory action of DAG on TRPC5 and TRPC4 channels was clearly mediated by protein kinase C (PKC), in distinction to the stimulatory action of DAG on TRPC3 which is established to be PKC-independent. PKC activation totally blocked TRPC3 channel activation in response to OAG, and the activation was restored by PKC-blockade. PKC-inhibition resulted in decreased TRPC3 channel deactivation. Store-operated Ca2+ entry in response to PLC-coupled receptor activation was substantially reduced by OAG or DAG-lipase inhibition in a PKC-dependent manner. However, store-operated Ca2+ entry in response to the pump blocker, thapsigargin, was unaffected by PKC. The results reveal that each TRPC subtype is strongly inhibited by DAG-induced PKC activation reflecting a likely universal feed-back control on TRPCs, and that DAG-mediated PKC-independent activation of TRPC channels is highly subtype specific. The profound yet distinct control by PKC and DAG on the activation of TRPC channel subtypes is likely the basis of a spectrum of regulatory phenotypes of expressed TRPC channels.
Speaker: Li-Ping He The transcription factor NFAT controls expression of cytokine genes which are critical for the immune response. A series of 3,5-bistrifluoromethyl pyrazole (BTP) derivatives have been shown to be potent blockers of the nuclear import of NFAT and IL-2 production in T lymphocytes.. Reports have also indicated they can suppress Ca2+ responses in lymphocytes. We studied the effects of BTP-2 (N5-{4-[3,5-di(trifluoromethyl)-1H-pyrazol-1-yl]phenyl}-4-methyl-1,2,3-thiadi), one of the related pyrazole compounds, on the activation of store-operated Ca2+ entry channels (SOCs) and on the function of TRPC3 channels stably transfected in HEK 293 cells and transiently transfected in DT40 B lymphocytes. Whereas there was only a modest acute action of BTP-2 on SOC activation, preincubation of HEK 293 or DT40 cells with 10 \muM BTP-2 for 10 min caused complete inhibition of thapsigargin-induced Ca2+ entry through SOCs. With a lower concentration (1 \muM ), the inhibition was about 80% in both cell lines. However, with prolonged treatment (60 min), 1 \muM BTP-2 showed complete inhibition of SOC activation in both cell lines. A number of reports indicated that TRPC channels are store-operated and may be components of SOCs. We analyzed the actions of BTP-2 on TRPC3 channels stably expressed in HEK-293 cells (T3-65 clone) which, in distinction from the highly Ca2+-selective SOCs, allow Sr2+ entry in response to activation of the PLC-coupled muscarinic receptor by carbachol. Preincubation of the cells with 10 \muM BTP-2 for 10 min caused 90% blockade of carbachol-induced Sr2+ entry through TRPC3 channels. The action of OAG, a permeant diacylglycerol analogue (1-oleoyl-2-acetyl-sn-glycerol) and a highly effective "direct" activator of TRPC3 channels, was also blocked by 10 \muM BTP-2. However, without pre-incubation, the compound did not inhibit either thapsigargin-induced Ca2+ entry or carbachol-induced Sr2+entry. Theses results indicate that BTP-2 inhibits both thapsigargin-induced Ca2+ entry through SOCs and carbachol-induced Sr2+entry through TRPC3 channels. The level of inhibition depends on the concentration and the period of treatment, lower concentrations requiring longer time of treatment. It does not appear that BTP-2 is a direct "channel-blocker" since its action on SOCs or TRPC3 channels was not immediate. We conclude that BTP-2 is a modifier of capacitative Ca2+ through SOCs and TRP channels, and may be targeting the machinery coupling stores with activation of channels.
The regulation of the second messengers IP3 and calcium is closely
linked: IP3 activates calcium release from the endoplasmic reticulum,
while calcium ions can exert both positive and negative feedback
on IP3 metabolism by activating phospholipase C and IP3 3-kinase,
respectively. The generation of calcium oscillations and waves has
been much-studied theoretically for conditions of constant IP3 concentration.
We developed mathematical models of the IP3/calcium signal ing network
which extend the previous models by inclusion of IP3 dynamics. Carrying
out stability and bifurcation analyses alongside numerical simulations,
we found that calcium regulation of IP3 metabolism profoundly affects
both oscillations and wave propagation. Inclusion of IP3 3-kinase
is shown to result in a hitherto unidentified mechanism for the
generation of oscillations which gives rise to a wide frequency
range, as it is observed in a number of cell types. A similarly
extended frequency range for oscillations can also be supported
by positive feedback via calcium activation of PLC. Moreover, the
PLC-mediated feedback has implications for calcium waves, as it
provides an additional mechanism of partial or complete signal regeneration
in wave propagation. These findings raise the question whether it
is possible to identify if significant negative or positive feedback
of IP3 is present in a cell. We show that the response of calcium
oscillations to the ectopic expression of IP3 buffer, which has
recently become feasible experimentally, depends on the type of
feedback involved in the oscillator.
Presentation materials: PPT Streaming Video: Real Media Olfactory transduction comprises intracellular processes but also the extracellular 'perireceptor events'. The latter include the adsorptive uptake of odorant molecules (here the moth female pheromone) from the air space by the surface of the olfactory organ (here the male moths antenna), their diffusion towards the olfactory receptor cell, and a network of extracellular chemical reactions. The odorant reacts with odorant-binding proteins, receptor molecules of the receptor cell, and odorant-degrading enzymes. We discuss the structure of the pheromone-binding protein recently analysed by X-ray1 and NMR2, and its possible functions as carrier and scavenger of the pheromone. We developed a quantitative mathematical model (computerized by J. Thorson, Oxford) of the network reactions based on experimental data from several laboratories3. From these studies we conclude that the receptor potential, the first electrical response of the receptor cell, reflects the kinetics of the network rather than the one of intracellular transducer processes. The model allows for the moth Antheraea polyphemus to estimate the density of pheromone receptor molecules on the receptor cell membrane of 6,000/\mu m2 and a dissociation constant of the pheromone-receptor complex of 40 \muM. 1) Sandler, B.H., Nikonova, L., Leal, W.S., & Clardy, J. (2000).
Chemistry & Biology, 7, 143-151.
Speaker: Karl-Ernst Kaissling, PhD, apl. Prof.
Em. at the Ludwig-Maximilians-Universitaet Muenchen, Germany Max-Planck-Institut
fuer Verhaltensphysiologie Abstract for Poster Presentation: Olfactory receptor cells of silkmoths Bombyx mori respond to single pheromone molecules with "elementary" electrical events that appear as discrete "bumps" a few ms in duration, or bursts of bumps1. As revealed by simulation, one bump may result from a series of random openings of one or several ion channels, producing an average inward membrane current of 1.5 pA. For the simulation we developed an equivalent electrical circuit of the sensillum which took into account both the membrane resistances and capacitances of the sensory and auxiliary cells2,3. The distributions of durations of bumps and of gaps between bumps in a burst can be fitted by single exponentials with time constants of 10.2 ms and 40.5 ms, respectively. The distribution of burst durations is a sum of two exponentials; the number of bumps per burst obeyed a geometric distribution (mean 3.2 bumps per burst). Accordingly the elementary responses of the pheromone receptor cell could reflect transitions among three states of the pheromone receptor molecule: the vacant receptor (state 1), the pheromone-receptor complex (state 2), and the activated complex (state 3). The calculated rate constants of the transitions between states are for one cell k21 = 7.7/s, k23 = 16.8/s, and k32 = 98/s. The mean life time of the pheromone-receptor complex is Tc = 1/k21 + k23/(k21 x k32) = 152 ms.3 The rate constants were used for modeling perireceptor and receptor events in moth antennae4. 1)Kaissling, K.E. (1994). Int. Symp. Olfaction and Taste XI. In K. Kurihara, N. Suzuki, & H. Ogawa (Eds.), (pp. 812-815). Springer, Tokyo. 2) Kaissling, K.E. & Thorson, J. (1980). Receptors for Transmitters, Hormones and Pheromones in Insects. In D.B. Sattelle, L.M. Hall, & J.G. Hildebrand (Eds.), (pp. 261-282). Elsevier, Amsterdam. 3) Minor, A.V. & Kaissling, K.E. (2003). J. Comp. Physiol. A, 189, 221-230. 4) Kaissling, K.E. (2001). Chemical Senses, 26,125-150.
Streaming Video: Real Media Generally, two modes of fast communication between neurons are considered; Ca-dependent neurotransmitter release and electrical coupling via gap-junctions. There is, however, evidence for a third type of fast communication: ephaptic communication. Ephaptic comes from the Greek word Ephapse which means "to touch". In this type of communication, the extracellular potential instead of the intracellular potential is modulated. In the retina we have found evidence that horizontal cells project back to photoreceptors via an ephaptic mechanism (Kamermans et al., Science, 2001). In this talk Iwill present the evidence for ephaptic communication n the retina and, I will discuss the likelihood that this type of communication is a general phenomenon instead of being restricted to the retina.
Presentation Materials: PPT Streaming Video: Real Media When the olfactory system detects a very weak stimulus, it somehow discriminates it from false signals due to random fluctuations in the transduction cascade. At least two mechanisms have been found to facilitate this discrimination. First, the neuron maintains a small, optimal concentration of cAMP at rest. cAMP gates the primary transduction channels in the olfactory cilia. Because of non-linearities in the response to cAMP, a weak odorous stimulus can produce the greatest depolarization in a neuron that already contains 0.1-0.3 \muM cAMP. The second mechanism is a high-gain, low-noise amplification of the weak signal. A pair of receptor currents underlying vertebrate olfactory transduction constitutes such an amplifier. Calcium enters the cilium through the cAMP-gated channels. Calcium can in turn gate chloride channels, activating a secondary depolarizing receptor current. Because the chloride channels have a small unitary conductance, they amplify the primary current while introducing little additional noise.
Streaming Video: Real Media Abstract. The Virtual Cell computational framework provides a general approach to modeling complex biochemical pathways in the context of actual cellular geometry. Applications of the Virtual Cell to InsP3-mediated calcium signaling will be presented, highlighting how the interplay between theoretical modeling and experimental approaches provides a powerful tool in understanding complex biochemical processes. My colleagues and I combined a quantitative uncaging technique for generating controlled amounts of InsP3 within live cells, measurements of intracellular calcium dynamics, and modeling with the Virtual Cell software system, to determine the time-dependent concentrations of InsP3 in single cells. This technology was applied to the construction of a spatial model of bradykinin-induced calcium waves in differentiated N1E115 neuroblastoma. Using a comprehensive set of experimentally determined image and biochemical data as inputs to the model, we were able to generate simulations that reproduced the experimentally observed calcium waves in these cells. The model also enabled us to visualize the spatiotemporal behavior of InsP3 and gain an understanding of how the neuronal cell geometry controls the overall process. As additional examples of morphological control for very different spatial scales, I will describe the calcium signals following fertilization of frog eggs and how the unique morphology of dendritic spines can control InsP3 signals underlying long term depression.
Presentation Materials: PPT Streaming Video: Real Media Flexibility and robustness are important properties of biological systems. These properties are here investigated in a model for simple and complex intracellular calcium oscillations. Flexibility and robustness characterize responses of a system to various external perturbations, such as deterministic pulses or periodic signals, as well as noise. Here, the influence of external periodic forcing is studied. The main point of the study is to compare responses of the system in a chaotic regime with those obtained in a regular periodic regime. We show that the response to external signals in terms of the range of synchronization does not depend on the complexity of Ca2+ oscillations but on the local divergence. Parts of attractors with close to zero local divergence are highly susceptible to external perturbations, thus facilitating signal detection and transduction, whereas highly negative local divergence areas characterize extremely robust regions that are virtually impossible to modify even with strong external signals. The results of the study are applicable for studying cell coupling, robustness to white noise and give an explanation of the constructive and destructive role of noise in the sense of stochastic resonance.
Streaming Video: Real Media The Transient Receptor Potential (TRP) proteins constitute a large and diverse family of channel proteins, which is conserved through evolution. TRP channel proteins have critical functions in many tissues and cell types, but their gating mechanism is an enigma. I'll report on two important new properties of Drosophila TRP and TRP-like (TRPL) channels. We found that TRPL is translocated back and forth between the signaling membrane and an intracellular compartment by a light-regulated mechanism. A high level of rhabdomeral TRPL characteristic of dark-raised flies is functionally manifested in the properties of the light induced current and thus represents a novel mechanism to fine-tune visual responses. We also gained a new insight on the activation mechanism of the TRP and TRPL channels by which both Ca2+ and protein dephosphorylation are required for channel activation. ATP depletion or inhibition of protein kinase C activated the TRP channels, while photo-release of caged ATP or application of phorbol ester antagonized channels openings in the dark. Furthermore, Mg2+-dependent stable phosphorylation event or protein phosphatase inhibition abolished activation of the TRP and TRPL channels. While a high reduction of cellular Ca2+ abolished channel activation, subsequent application of Ca2+ combined with ATP depletion induced a robust dark current that was reminiscent of light responses. The results suggest that the combined action of Ca2+ and protein dephosphorylation activate the TRP and TRPL channels, while protein phosphorylation by PKC antagonized channels openings. The link of TRP gating to the metabolic state of cells makes cells expressing TRP extremely vulnerable to metabolic stress via a mechanism that may underlie retinal degeneration and neuronal cell death in mammals upon reduction in oxygen pressure in the tissue.
1Department of Neurobiology & Behavior,
University of California, Irvine Streaming Video: Real Media Cellular calcium buffers shape cytosolic calcium signals by regulating the availability and diffusional mobility of free calcium ions. Most experimental and theoretical studies of these effects have concentrated on signals arising from a fixed 'pulse' of calcium - for example, calcium entry through voltage-gated channels. A more complex case involves intracellular calcium liberation through inositol trisphosphate (IP3) receptors, which are themselves regulated by calcium. Thus, cytosolic buffers are expected to influence the successive cycles of diffusion and calcium-induced calcium release that act both between within clusters of IP3 receptors (to generate local 'puffs'), and between adjacent clusters (to generate global calcium waves). We studied such interactions in Xenopus oocytes, using confocal line-scan microscopy together with photo-release of IP3. Intracellular injections of buffers with 'slow' calcium-binding kinetics (EGTA and parvalbumin) speeded the decay of calcium signals (Fig. 1A) and 'balkanized' IP3-evoked calcium waves into discrete puffs. Contrastingly, equivalent concentrations 'fast' buffers (BAPTA and calretinin) slowed IP3-evoked calcium responses (Fig. 1B) and promoted 'globalization' of spatially uniform calcium signals. These observations likely reflect buffer actions on calcium feedback at IP3 receptors, since the effects of EGTA and BAPTA on calcium signals evoked by influx through expressed N-type voltage gated channels were markedly less pronounced. Moreover, they point to the importance of buffer kinetics in shaping IP3-evoked calcium signals; likely because fast buffers can influence calcium feedback between individual IP3R within a cluster, whereas slow buffers are able to modulate only cluster-cluster interactions. We propose that cell-specific expression of calcium binding proteins with distinct kinetics may shape the time course and spatial distribution of IP3-evoked calcium signals for specific physiological roles. Supported by NIH GM 48071 and GM 58329
In non-excitable cells agonist stimulation can induce, via production of inositol-1,4,5- trisphosphate (IP3), transient increases in cytoplasmic calcium so called calcium oscillations. Several mechanisms contribute to the generation and termination of these transients (e.g. calcium induced calcium release, calcium sequestration, inactivation of the IP3 receptors). Among these the calcium dependence of the IP3 metabolism is thought to be involved. Calcium affects the production, by activating phospholipase C (PLC), and the degradation, by activating IP3 3-kinase (IP3K), of IP3. Although there are indications that IP3 oscillates concomitantly to calcium, the role of these oscillations in shaping the calcium signal is still unclear. Starting from models that show calcium oscillations for constant IP3 levels (e.g. models including CICR and slow inactivation of the IP3R's by calcium), I included the calcium activation of PLC and IP3K (representing an indirect positive and negative feedback of calcium on itself, respectively). The presence of one of the feedbacks greatly increases the stimulatory range where one observes oscillations. Long period oscillations (> 5min) as well as short period oscillations (< 30 sec) are found. The analysis shows that the dynamics of IP3 can be very important in controlling the long period of the oscillations. To determine which of the feedbacks is the most predominant I simulated the expression of an IP3 buffer. In experiments the expression of the IP3 receptor ligand binding domain can strongly modify or stop the calcium oscillations (L.D. Gaspers, A.P. Thomas communication). I studied how IP3 buffer can abolish oscillations. Furthermore the effects of IP3 buffer on the characteristics of the calcium spikes, the period of the oscillations and the propagation of calcium waves have been investigated. Comparing the theoretical with the experimental results indicates that first at least one of the feedbacks is present in the studied cells, and second that the positive feedback of calcium on IP3 (via PLC) is the predominant one. This feedback turns out to be crucial for shaping the calcium signal. I thank A.P. Thomas and L.D. Gaspers from the New Jersey Medical School for the stimulating discussions.
1Institut für Biologische Informationsverarbeitung,
Forschungszentrum Jülich Presentation Materials: PPT Odorants activate sensory transduction in olfactory receptor neurons (ORNs) via a cAMP signalling cascade, which results in the opening of non-selective, cyclic nucleotide-gated (CNG) channels. The consequent Ca2+ influx through CNG channels activates Cl channels, which serve to amplify the transduction signal. We investigate here the functional interplay of the CNG channel with the Cl channel by using inside-out membrane patches excised from ORN dendritic knobs/cilia. The Cl channels on an excised patch could be activated by Ca2+ flux through the CNG channels which were opened by cytoplasmic application of cAMP. The magnitude of the Cl current depended on the strength of Ca-buffering in the bath solution, suggesting that the CNG and Cl channels were probably not organized as constituents of a local transducisome complex. Likewise, Cl channels and the Na/Ca exchanger, which extrudes Ca2+, appear to be spatially segregated. Based on the theory of buffered Ca2+ diffusion, we determined the Ca2+ diffusion coefficient and calculated that the CNG and Cl channel densities on the membrane were ca. 8 and 62 \mu m 2, respectively. These densities, together with the Ca2+ diffusion coefficient, demonstrate that a given Cl channel is activated by Ca2+ originating from multiple CNG channels, thus allowing low-noise amplification of the olfactory receptor current. Presentation Materials: PPT Streaming Video: Real Media Olfactory stimuli are complex chemical signals whose composition varies quantitatively and qualitatively in space and time. Olfactory receptor neurons (ORNs) can monitor these variations and convey raw data to the brain from which biologically meaningful information can be extracted. The primary purpose of this work is to investigate quantitatively how the intensitive properties of odors, measured by their odorant concentration in the air, are encoded in the spike trains delivered by ORNs. To this end, we studied ORN responses in the frog olfactory epithelium to four volatile compounds, anisole, camphor, isoamyl acetate and limonene, which are strongly and distinctively odorous for humans. All four odorants were applied as square pulses of 2-s duration at increasing concentrations. So, only the time variable was kept identical through all experiments. The neural activity was recorded one neuron at a time with extracellular electrodes from an intact epithelium in order to minimize the modifications due to experimental conditions [3, 4]. The spike train yielded by each stimulation was characterized by its latency, length (number of spikes), duration and median frequency. These variables were studied at the single neuron, neuron population and ciliary membrane levels. At the single neuron level, the dose-response plots for the four variables were fitted to specific functions (negative exponential, alpha and half-Hill functions). The location along the concentration axis (e.g. thresholds), width (dynamic ranges) and heights (maximum latency, duration or frequency) of the fitted curves were evaluated for each plot. At the neuron population level, the curves are extremely diverse. Both their dose characteristics (e.g. at threshold, at maximum response) and response characteristics (e.g. minimum latency, maximum frequency) were found to follow lognormal statistical distributions. Lognormal histograms are asymmetric with peaks close to the origin and long tails. The histogram of dynamic ranges in spike frequency is the most asymmetric, so that a significant fraction of neurons presents a much wider range than their one-decade peak, confirming the short dynamic range of the typical ORN but revealing the presence of a significant proportion of ORNs with a behavior very different from the average. From these histograms, response properties of the whole neuron population can be inferred. In general, thresholds, dynamic ranges and maximum responses are independent, which prevcnts their global categorization and can serve as a support for the qualitative coding of odorants. Some of the response characteristics can be interpreted in terms of molecular events (deactivation of odorant molecules, odorant-receptor interaction, transduction cascade) using a chemo-electrical model of the ORN [1] and its environment [2] based on known mucus biochemical and neuron morpho-electrical data. From this model both dose-conductance curves and the apparent dissociation equilibrium constants of odorant-receptor interaction were determined, as well as their statistical distributions in the neuron population. The values found are in broad agreement with independent determinations. References [1] Rospars, J.P., Lánský, P., Tuckwell, H.C., & Vermeulen, A. (1996). Coding of odor intensity in a steady-state deterministic model of an olfactory receptor cell neuron. Journal of Computational Neuroscience, 3, 51-72. [2] Rospars, J.P., Krivan, V., & Lansky, P. (2000a). Perireceptor and receptor events in olfaction. Comparison of concentration and flux detectors: a modeling study. Chemical Senses, 25, 293-311. [3] Rospars, J.P., Lánský, P., Duchamp-Viret, P., & Duchamp, A. (2000b). Spiking frequency vs. odorant concentration in olfactory receptor neurons. BioSystems, 58, 133-141. [4] Rospars, J.P., Lánský, P., Duchamp-Viret, P., & Duchamp, A. (2003). Relation between stimulus and response in frog olfactory receptor neurons in vivo. European Journal of Neuroscience, 18, 1135-1154.
Presentation Materials: PPT Streaming Video: Real Media The transition from a stationary regime to oscillations in the intracellular calcium concentration can occur in different ways. The mathematical tool to analyse this is bifurcation analysis. Calcium oscillations can emerge both by local bifurcations (e.g. supercritical and subcritical Hopf bifurcations) and global bifurcations (e.g. homoclinic and infinite-period bifurcations). The meaning of these terms is explained. Since calcium oscillations are relaxation oscillations (due to the presence of slow and fast processes), so-called canard orbits may arise near local bifurcation points. This means that quasi-harmonic oscillations with a very small amplitude grow very fast to become pulsed oscillations. The jump-like growth in amplitude, which also occurs in global bifurcations, may be of physiological advantage because "misinterpretation" of the signal is avoided. Near homoclinic and infinite-period bifurcations, the oscillations period can reach arbitrarily high values. This is interesting in the light of frequency encoding. These phenomona are illustrated by selected models of calcium oscillations.
Presentation Materials: PPT Streaming Video: Real Media An enhanced entry of extracellular Ca2+ is essentially a universal component of receptor-activated Ca2+ signals in cells. Until recently, it was generally assumed that this always involved the well-known, and apparently ubiquitous, capacitative mechanism of entry. In this, the activation of Ca2+ entry results solely from the depletion of intracellular Ca2+ stores. The channels responsible are generically described as store-operated Ca2+ channels (SOC channels), and the most thoroughly studied of these is the Ca2+-release activated Ca2+ channel (CRAC channel). However, in recent years, evidence has accumulated demonstrating an additional noncapacitative mode of receptor-activated Ca2+ entry involving a distinct arachidonic acid-regulated Ca2+ channel (ARC channel). The ARC channels and the SOC/CRAC channels co-exist in cells, but they are not simply alternative, mutually redundant, routes of entry - they are differentially activated at different agonist concentrations in a unique and coordinated fashion, and have distinct roles in the control of the resulting Ca2+ signals. In particular, it is the ARC channels that provide the predominant mode of Ca2+ entry at the low agonist concentrations that give rise to oscillatory Ca2+ signals, and its effect is to modulate oscillation frequency. In addition, the fact that the SOC channels and ARC channels represent spatially distinct entities activated at different agonist concentrations has potential implications for the specific activation of distinct targets within the cell. (Supported by NIH grant GM 40457).
Presentation Materials: PDF Streaming Video: Real Media Although there is consensus that Ca2+ puffs arise from the cooperative action of multiple IP3 receptors (IP3Rs), the precise relationship between single-channel kinetics and the collective phenomena of stochastic Ca2+ excitability is not well understood. Here we present a memory-efficient method by which mathematical models for IP3-sensitive Ca2+ release sites can be derived from Markov models of IP3R single-channel gating that include Ca2+ activation (IP3R-2 like), Ca2+ inactivation, or both (IP3R-1 like). Such models are essentially stochastic automata networks (SANs) that involve a large number of so-called `functional transitions,' that is, the transition probabilities of the infinitesimal generator matrix (or Q-matrix) of one automata (i.e, an individual channel) may depend on the local [Ca2+] and thus the state of the other channels. We present an iterative method for calculating hitting times that leads to a directly estimate of various puff statistics (e.g., puff duration and inter-puff-interval) from a SAN descriptor.
Presentation Materials: PDF
Streaming Video: Real Media Since the first observations showing that hormones coupled to the formation of inositol 1,4,5-trisphospate (IP3) elicit oscillations of cytosolic calcium, there has been much speculation on the mechanism underlying these calcium dynamics. As the properties of the IP3-receptor (IP3R) were elucidated, it became clear that positive and negative feedback effects of Ca2+ directly on the IP3R could yield oscillatory calcium signaling. However, there are a number of other potential sites of calcium feedback in the IP3-dependent calcium-signaling pathway, particularly through alterations in the generation and elimination of IP3. We have utilized GFP-pleckstrin homology domain fusion proteins to monitor intracellular changes in IP3, and a molecular buffer of IP3 based on the ligand binding domain of the IP3R fused to GFP, which serves as an IP3 buffer in the physiologically relevant range. These tools have allowed us to investigate how IP3 dynamics might participate in calcium oscillations. In particular, we suggest that IP3 oscillations may interact with the intrinsic calcium regulation of the IP3R to yield coupled oscillators. This type of system has the potential to enrich the properties of calcium oscillations, and extend the dynamic range of frequency-modulation in Ca2+ signaling. A third level of feedback control of calcium oscillations occurs through the calcium uptake and release properties of mitochondria, which can modify calcium-feedback regulation of the IP3R by virtue of their close proximity to ER calcium release sites. The complex interplay between different feedback loops in the generation of IP3-dependent calcium oscillations, together with the spatial organization of the underlying components, gives rise to diverse patterns of calcium signaling that are tailored to regulate a wide variety of different processes.
There have been numerous twists and turns in the road to understanding vertebrate phototransduction over the years. The phototransduction field has been characterized by great controversy surrounding numerous confusing and seemingly incompatible experimental results. Calcium has been at the heart of controversy on multiple occasions, including a recent one. In the dark, there is a circulating current across the rod membrane, which flows inward through channels in the outer segment and flows outward across the inner segment membrane. The modulation of the voltage across the rod membrane in response to light is a consequence of the reduction of this dark (light-sensitive) current. When rhodopsin absorbs a photon it is converted into an activated enzyme (R*) that initiates a cascade of biochemical reactions. These reactions lead to change in the concentration of an internal transmitter that gates the light-sensitive current. For many years the phototransduction community was divided between those who thought that this transmitter was calcium ions and those who thought it was cyclic-GMP (cGMP), until twenty years ago when definitive experimental evidence showed that the transmitter is cGMP. Other roles were found for calcium. Two salient features of rods are their ability to adapt to changes in ambient light level by adjusting the sensitivity and kinetics of their responses; and their remarkably reproducible responses to single photons. Calcium ions have been implicated in both these phenomena. A major outstanding problem in sensory physiology has been to understand how the rod's ingle-photon response (SPR) manages to have such little variation in amplitude and kinetics, despite the fact that it is mediated by the activity of a single molecule of activated rhodopsin (R*). Once R* is produced, it activates G-protein; G-protein activates phosphodiesterase; phosphodiesterase hydrolyzes cGMP; the cGMP concentration drops, resulting in closure of some of the light-sensitive channels; the reduction of inward current causes the rod membrane voltage to become hyperpolarized. The sequence of events following the activation of rhodopsin continues until R* is inactivated. The regularity of the single-photon response implies that the lifetime of R* is controlled with high precision-or does it? Closure of the light-sensitive channels blocks the entry of calcium, but it is still pumped out of the rod by a calcium exchanger. Therefore, the concentration of calcium is controlled by light, and it can serve as a feedback messenger to control sensitivity and the lifetime of R*. I will discuss the experimental evidence and mathematical theory for the role calcium in SPR reproducibility. Although evidence to support the calcium hypothesis seemed compelling, other experimental results and mathematical theory cast grave doubt on this hypothesis. Evidence on both sides can now be understood with the aid of a detailed stochastic biochemical kinetic model for rod phototransduction.
Streaming Video: Real Media Inositol 1,4,5-trisphosphate receptors (InsP3R) are the major route of intracellular calcium release in eukaryotic cells and as such are pivotal for stimulation of Ca2+ dependent effectors. Modulation of this release has important consequences for defining the particular spatio-temporal characteristics of Ca2+ signals. In this study, regulation of Ca2+ release by phosphorylation of type-1 InsP3R (InsP3R-1) by cAMP (PKA) and cGMP (PKG) dependent protein kinases was investigated in the two major splice variants of InsP3R-1. InsP3R-1 were expressed in DT-40 cells devoid of endogenous InsP3R. In cells expressing the neuronal, S2+ splice variant of the InsP3R-1, Ca2+ release was markedly enhanced when either PKA or PKG was activated. The sites of phosphorylation were investigated by mutation of serine residues present in two canonical phosphorylation sites present in the protein. Potentiated Ca2+ release was abolished when serine 1755 was mutated to alanine (S1755A) but was unaffected by a similar mutation of serine 1589 (S1589A). These data demonstrate that S1755 is the functionally important residue for phosphoregulation by PKA and PKG in the neuronal variant of the InsP3R-1. Activation of PKA also resulted in potentiated Ca2+ release in cells expressing the non-neuronal, S2- splice variant of the InsP3R-1. However, the PKA-induced potentiation was still evident in S1589A or S1755A InsP3R-1 mutants. The effect was abolished in the double (S1589A/S1755A) mutant, indicating both sites are phosphorylated and contribute to the functional effect. Indeed, mimicking phosphorylation by changing either S1589 or S1755 to a positively charged glutamate residue (S1589E or S1755E) resulted in InsP3R with apparent enhanced sensitivity to InsP3. Activation of PKG had no effect on Ca2+ release in cells expressing the S2- variant of InsP3R-1 Collectively these data indicate that phosphoregulation of InsP3R-1 has dramatic effects on Ca2+ release and defines the molecular sites phosphorylated in the major variants expressed in neuronal and peripheral tissues.
|
|
|||
