Impact of Geometrical Characteristics of the Organellar Store and Organelle-Free Cytosol on Intracellular Calcium Dynamics in the Dendrite: a Simulation Study

The dependence of intracellular calcium dynamics on geometrical size relations between calcium-exchanging parts of the intracellular space was studied in mathematical models corresponding to a thin fragment of the Purkinje neuron spiny dendrite. The plasma membrane contained ion channels typical of this cell type, including channels that conduct an excitatory synaptic current, and ion pumps. The model equations took into account calcium exchange between the cytosol, extracellular medium, intracellular store (a cistern of the endoplasmic reticulum, ER), endogenous calcium buffers, and an exogenous buffer (fluorescent dye used in the experiments). The ER membrane contained the calcium pump and channels of calcium-dependent and inositol-3-phosphate-dependent calcium release, as well as leakage channels. With the compartment size fixed, the ER cistern diameter was varied so that the proportion of the organelle in the total volume changed from 1 to 36%. Under these conditions, identical synaptic excitation caused similar electrical reactions (calcium spikes) but different concentration responses. Equal increments in the ER diameter led to unequal, more pronounced at thicker diameters, increments of the peak cytosolic concentrations of Са²⁺ ([Ca²⁺] _i ) and of a Са²⁺-fluorescent dye complex [CaD], as well as those of the Са²⁺ concentration in the dendrite ER (characterized by a shift from the basal level, Δ[Ca²⁺]_ER). The changes in [Ca²⁺] _i and [CaD] followed more adequately those in the volume of the organelle-free cytosol, while Δ[Ca²⁺]_ER changes were more similar to those in the ER membrane area. Therefore, the relative occupancy of the intracellular volume by organellar calcium stores and their sizes in a dendritic compartment are important structural factors that essentially modulate the calcium dynamics, and this structural dependence can be adequately reflected in the experiments using fluorophores. Neirofiziologiya/Neurophysiology, Vol. 41, No. 1, pp. 19–31, January–February, 2009.     

Structure Dependence of the Calcium Dynamics in Purkinje Neuron Dendrites during Generation of Bursting Discharges: a Simulation Study

In the model of a cerebellar Purkinje neuron with reconstructed active dendrites, we investigated the impact of the ratio between volumes of the endoplasmic reticulum (organellar calcium store) and cytosol on the Ca²⁺ dynamics in asymmetrical parts of the dendritic arborization during generation of different structure-dependent patterns of bursting activity. Tonic synaptic excitation homogeneously distributed over the dendrites (a spatially homogeneous stationary input signal) caused spatially heterogeneous variations of the dendritic membrane potential (MP) accompanied by periodical or nonperiodical bursts of action potentials at the cell output. The MP waveforms recorded from the segments of asymmetrical dendrites were then applied to the membrane of selected dendrite segments as command voltages in a dynamic clamp mode. In these segments, the relative size of the stores was varied. This provided equal to each other local calcium currents and influxes into the cytosol of the segment differently filled with the organellar store. Regardless of the impulse pattern, microgeometry of the segment and the store modulated calcium transients exactly in the same way as in previous studies of electrical and concentration responses to local phasic synaptic excitation of the modeled neuron. Peak values of depolarization-induced elevations of the cytosolic Ca²⁺ concentration increased with the portion of the intracellular volume occupied by the store. The most important factor defining this dependence was the ratio of the membrane area vs the organelle-free cytosol volume of the dendritic segment. Concentrations of Са²⁺ deposited in equal-sized segments of asymmetrical parts of the dendritic arborization where asynchronous unequal variations of the MP were observed during generation of nonperiodical bursting at the output demonstrated considerable specificity. A greater amount of calcium was deposited in the segments staying, on average, in a high-depolarization state for a longer time (this intensified activation of calcium channels and amplified the corresponding Ca²⁺ influx into the cytosol). Hence, local dynamics of the Ca²⁺ concentration depend directly on local microgeometry and indirectly on global macrogeometry of the dendrite arborization, as the latter determines spatial asymmetry-related unequal transients in different parts of the dendritic arborization having active membrane properties.     

Comparative Model Analysis of Calcium Exchange between the Cytosol and Stores of Mitochondria or Endoplasmic Reticulum

The objects of the study were single-compartment mathematical models corresponding to a fragment of the dendrite of a cerebellar Purkinje neuron containing the mitochondria (model 1) or a cistern of the endoplasmic reticulum, ER, (model 2) as the calcium stores. We investigated the dependence of the intracellular Ca²⁺ dynamics on geometrical sizes of calcium exchanging parts of the intracellular space and the difference between the kinetic characteristics of storing in two types of stores occupying different portions of the compartment volume. The plasma membrane of the compartment bore the ion channels, particularly those conducting excitatory synaptic current, and the calcium pump typical of this neuron type. The model equations took into account Ca²⁺ exchange between the cytosol, extracellular medium, organelle stores, non-organelle endogenous buffers, and an exogenous buffer (fluorescent dye), and also the diffusion of Са²⁺ into adjacent regions of the dendrite. In model 1, the mitochondria exchanged Са²⁺ with the cytosol via the uniporter and sodium/calcium exchanger; mitochondrial processes, such as the tricarboxylic acid cycle and aerobic cellular respiration, were also taken into account. In model 2, the ER membrane contained the calcium pump, channels of passive leak, and channels of calcium-induced and inositol-3-phosphate-dependent release of Са²⁺. Increases in the portion of the stores in the total volume of the compartment from 1 to 36% led to a proportional increase in the peak values of the cytosolic calcium concentration ([Ca²⁺] _i ); the concentration of Са²⁺ in the mitochondria ([Ca²⁺]_mit) or ER ([Ca²⁺]_ER) increased correspondingly. During generation of bell-shaped cytosolic calcium signals of equal intensity and duration, the ER (due to a greater rate of storing, as compared with that in the mitochondria) was able to uptake several times more Са²⁺ (four times at 36% filling of the volume by the organelles). It is suggested that the revealed different kinetic characteristics of Са²⁺ storing by different organelles are determined by the rates of binding to transport molecules present in the store membrane and, therefore, are defined by concentrations (surface densities) of these molecules and their saturation at certain levels of [Ca²⁺]i. It has been shown that the occupancy of the intracellular volume by organelle stores of any type is a structural factor, which is able to essentially modulate the values of Ca²⁺ concentration.     

A Simulation Study of Calcium Dynamics Features Caused by Exchange between the Cytosol and Organellar Stores of Neurons

The objects of the study were single-compartment mathematical models corresponding to a fragment of the dendrite of a cerebellar Purkinje neuron. The fragments contained the mitochondria (model 1) or a cistern of the endoplasmic reticulum, ER (model 2), functioning as calcium stores. With simulating single excitatory synaptic actions, we examined the dependence of the dynamics of intracellular Ca²⁺ levels on the maximum rate of Ca²⁺ exchange between the cytosol and these stores, as well as on the intensity of the diffusion flow into adjacent organelle-free regions. The plasma membrane of the compartment had ion channels (including those of the synaptic current) and the calcium pump characteristic of the mentioned neurons. The model equations took into account Ca²⁺ exchange between the cytosol, extracellular environment, and organellar stores, as well as the diffusion process. In model 1, the mitochondria exchanged Ca²⁺ with the cytosol through the uniporter and sodium-calcium exchanger; mitochondrial processes, such as the tricarboxylic acid cycle and aerobic cellular respiration, were also included. In model 2, the ER membrane had the calcium pump, leak channels, and channels of calcium-induced and inositol-3-phosphate-dependent Ca²⁺ release. The stores (mitochondria or ER) occupied 36% of the total volume of the compartment. An increase in the maximum rate of calcium exchange with the stores led to a proportional decrease in the peak Ca²⁺ concentrations in the cytosol ([Ca²⁺]_i), more pronounced in the case of the ER; the Ca²⁺ concentration in both types of stores increased significantly. Due to the higher storage rate, the ER was able to absorb several times greater amounts of Ca²⁺ than the mitochondria did. With smaller diffusion flux (e.g., similarly to the case of diffusion from a larger-sized head into the neck of the dendritic spine), the intensity of cytosolic transients increased at fixed kinetics of flux exchange with the stores. Therefore, the organellar stores can significantly modulate not only the intensity but also the time course of changes in the intracellular Ca²⁺ levels.     

Calcium Domains around Single and Clustered IP3 Receptors and Their Modulation by Buffers

We study Ca²⁺ release through single and clustered IP₃ receptor channels on the ER membrane under presence of buffer proteins. Our computational scheme couples reaction-diffusion equations and a Markovian channel model and allows our investigating the effects of buffer proteins on local calcium concentrations and channel gating. We find transient and stationary elevations of calcium concentrations around active channels and show how they determine release amplitude. Transient calcium domains occur after closing of isolated channels and constitute an important part of the channel's feedback. They cause repeated openings (bursts) and mediate increased release due to Ca²⁺ buffering by immobile proteins. Stationary domains occur during prolonged activity of clustered channels, where the spatial proximity of IP₃Rs produces a distinct [Ca²⁺] scale (0.5-10 μM), which is smaller than channel pore concentrations (>100 μM) but larger than transient levels. While immobile buffer affects transient levels only, mobile buffers in general reduce both transient and stationary domains, giving rise to Ca²⁺ evacuation and biphasic modulation of release amplitude. Our findings explain recent experiments in oocytes and provide a general framework for the understanding of calcium signals.     

Competitive Calcium Binding: Implications for Dendritic Calcium Signaling

Action potentials evoke calcium transients in dendrites of neocortical pyramidal neurons with time constants of <100 ms at physiological temperature. This time period may not be sufficient for inflowing calcium ions to equilibrate with all present Ca²⁺-binding molecules. We therefore explored nonequilibrium dynamics of Ca²⁺ binding to numerous Ca²⁺ reaction partners within a dendritelike compartment using numerical simulations. After a brief Ca²⁺ influx, the reaction partner with the fastest Ca²⁺ binding kinetics initially binds more Ca²⁺ than predicted from chemical equilibrium, while companion reaction partners bind less. This difference is consolidated and may result in bypassing of slow reaction partners if a Ca²⁺ clearance mechanism is active. On the other hand, slower reaction partners effectively bind Ca²⁺ during repetitive calcium current pulses or during slower Ca²⁺ influx. Nonequilibrium Ca²⁺ distribution can further be enhanced through strategic placement of the reaction partners within the compartment. Using the Ca²⁺ buffer EGTA as a competitor of fluo-3, we demonstrate competitive Ca²⁺ binding within dendrites experimentally. Nonequilibrium calcium dynamics is proposed as a potential mechanism for differential and conditional activation of intradendritic targets.     

Modeling of the Modulation by Buffers of Ca Release through Clusters of IP3 Receptors

Intracellular Ca²⁺ release is a versatile second messenger system. It is modeled here by reaction-diffusion equations for the free Ca²⁺ and Ca²⁺ buffers, with spatially discrete clusters of stochastic IP₃ receptor channels (IP₃Rs) controlling the release of Ca²⁺ from the endoplasmic reticulum. IP₃Rs are activated by a small rise of the cytosolic Ca²⁺ concentration and inhibited by large concentrations. Buffering of cytosolic Ca²⁺ shapes global Ca²⁺ transients. Here we use a model to investigate the effect of buffers with slow and fast reaction rates on single release spikes. We find that, depending on their diffusion coefficient, fast buffers can either decouple clusters or delay inhibition. Slow buffers have little effect on Ca²⁺ release, but affect the time course of the signals from the fluorescent Ca²⁺ indicator mainly by competing for Ca²⁺. At low [IP₃], fast buffers suppress fluorescence signals, slow buffers increase the contrast between bulk signals and signals at open clusters, and large concentrations of buffers, either fast or slow, decouple clusters.     

Extension of Rapid Buffering Approximation to Ca2+ Buffers with Two Binding Sites

Fundamental cell processes such as synaptic neurotransmitter release, endocrine hormone secretion, and myocyte contraction are controlled by highly localized calcium (Ca²⁺) signals resulting from brief openings of trans-membrane Ca²⁺ channels. On short temporal and spatial scales, the corresponding local Ca²⁺ nanodomains formed in the vicinity of a single or several open Ca²⁺ channels can be effectively approximated by quasi-stationary solutions. The rapid buffering approximation (RBA) is one of the most powerful of such approximations, and is based on the assumption of instantaneous equilibration of the bimolecular Ca²⁺ buffering reaction, combined with the conservation condition for the total Ca²⁺ and buffer molecule numbers. Previously, RBA has been generalized to an arbitrary arrangement of Ca²⁺ channels on a flat membrane, in the presence of any number of simple Ca²⁺ buffers with one-to-one Ca²⁺ binding stoichiometry. However, many biological buffers have multiple binding sites. For example, buffers and sensors phylogenetically related to calmodulin consist of two Ca²⁺-binding domains (lobes), with each domain binding two Ca²⁺ ions in a cooperative manner. Here we consider an extension of RBA to such buffers with two interdependent Ca²⁺ binding sites. We show that in the presence of such buffers, RBA solution is given by the solution to a cubic equation, analogous to the quadratic equation describing RBA in the case of a simple, one-to-one Ca²⁺ buffer. We examine in detail the dependence of RBA accuracy on buffering parameters, to reveal conditions under which RBA provides sufficient precision.     

Cell proliferation, calcium influx and calcium channels

Both increases in the basal cytosolic calcium concentration ([Ca²⁺]_cyt) and [Ca²⁺]_cyt transients play major roles in cell cycle progression, cell proliferation and division. Calcium transients are observed at various stages of cell cycle and more specifically during late G₁ phase, before and during mitosis. These calcium transients are mainly due to calcium release and reuptake by the endoplasmic reticulum (ER) and are observed over periods of hours in oocytes and mammalian cells. Calcium entry sustains the ER Ca²⁺ load and thereby helps to maintain these calcium transients for such a long period. Calcium influx also controls cell growth and proliferation in several cell types. Various calcium channels are involved in this process and the tight relation between the expression and activity of cyclins and calcium channels also suggests that calcium entry may be needed only at particular stages of the cell cycle. Consistent with this idea, the expression of l-type and T-type calcium channels and SOCE amplitude fluctuate along the cell cycle. But, as calcium influx regulates several other transduction pathways, the presence of a specific connection to trigger activation of proliferation and cell division in mammalian cells will be discussed in this review.     

Estimation of the Capacity of Heterogeneously Distributed Endogenous Calcium Buffers in a Neuron

The value of the ratio of half-decay times of calcium transients recorded using calcium sensitive fluorescent dyes with low and high ffinities can be used for estimating the cytosolic endogenous Ca²⁺ buffer capacity (k _buf); for this purpose, the parameters obtained in the model and in real experiments are compared. However, if the distribution of endogenous buffers is characterized by a high heterogeneity, the ratio of half-decay times of fluorescence transients depends not only on the k _buf but also on the pattern of distribution of Ca²⁺ channels. Our simulations showed that in spite of a considerable slowing down of fluorescence responses, when Oregon Green BAPTA-1 Ca²⁺ indicator is used, the k _buf value in the dendritic endings of cerebellar granule cells (GrCs) can reach 300 to 500. This can happen in the cases where, according to our suggestion [1], a high-capacity calcium buffer is localized in the apical parts of dendritic endings of the above cells.     

Mutual influence of plasma membrane structures and intracellular calcium stores on the formation of calcium signals in primary nociceptive neurons

Participation of different calcium-regulating mechanisms in the formation of intracellular calcium signals in rat primary sensory neurons was studied using two-wavelength fluorescent microscopy. Mitochondria were shown to be the most powerful intracellular calcium-regulating structures in the investigated neurons. These organelles were involved in the modulation of calcium signals induced either by Ca²⁺ entry from the extracellular medium or by Ca²⁺ release from endoplasmic reticulum (ER). Analysis of the mitochondrial calcium exchange showed that the efficiency of mitochondria depended on whether calcium entered the cytosol from ER or from the extracellular solution. Depletion of ER by activation of ryanodine-sensitive, inositol-3-phosphate-sensitive receptors of ER or by activation of the leak channels via the block of ATPases in ER activated the store-operated calcium entry from the extracellular medium to cytosol. The kinetics of the rising phase of these Ca²⁺ transients depended on the way of ER depletion. This allows suggesting the existence of different activation mechanisms for the studied signals. The block of the mitochondrial calcium uniporter resulted in a rapid recovery of the intracellular calcium concentration after the Ca²⁺ transient induced by store-operated calcium influx. We conclude that mitochondrial calcium uptake can prevent calcium-dependent inactivation of store-operated calcium channels.     

Computational study of non-homogeneous distribution of Ca handling systems in cerebellar granule cells

The spatiotemporal distribution of cytosolic free calcium concentration ([Ca²⁺]_i) in cerebellar granule cells (GrCs) is thought to be critical in defining the occurrence and direction of long-term changes in synaptic strength at cerebellar mossy fiber-GrC synapses. Despite this, the mechanisms responsible for shaping Ca²⁺ transients in GrCs are not well understood. To investigate the interplay between Ca²⁺ entry, extrusion, buffering and dendritic morphology in shaping Ca²⁺ elevations in GrCs, we developed a model of Ca²⁺ regulation in these cells and examined the requirements for reproducing fluorescence responses to depolarization and synaptic stimulation previously described in the literature. Two conclusions can be drawn from our simulation results. First, a significant progressive decrease in the amplitudes of depolarization-evoked fluorescence transients from the dendritic endings (digits) toward the soma of GrCs, can be reproduced in the model only if the density of Ca²⁺ channels is considerably higher or the concentration of endogenous buffers is much lower in the digits than in the parent dendrites. In contrast, heterogeneities in the distribution of Ca²⁺ pumps or in cytosolic fractional volume cannot account for the formation of [Ca²⁺]_i gradients in GrCs. Second, much lower amplitudes of fluorescence transients induced by depolarization and synaptic stimulation than expected from typical measurements of Ca²⁺ and NMDA receptor-mediated currents can be reconciled with a pronounced slowing of the decay of fluorescence responses in the digits of GrCs after introducing a high-affinity Ca²⁺ indicator if a high-capacity immobile Ca²⁺ buffer (presumably plasma membrane-associated) is suggested to be present in the soma and apical part of digits. Mitochondria also are likely to modulate synaptically evoked Ca²⁺ responses in GrCs. The alternative hypotheses are thoroughly discussed and research avenues for their testing in future experiments are proposed.     

Involvement of the Endoplasmic Reticulum of Chromaffin Cells of the Rat Adrenal Gland in Calcium Signaling

We studied the involvement of the endoplasmic reticulum (ER) in calcium signaling in rat chromaffin cells. For this purpose, the following agents influencing the activity of the ER were used: (i) Caffeine that activates the release of Ca²⁺ from the endoplasmic store and (ii) thapsigargin that suppresses accumulation of calcium in the ER. The intracellular Ca²⁺ concentration was measured with the help of a calcium-sensitive dye, Fura-2AM, using the microfluorescent technique. Applications of caffeine led to a rise in the level of free Ca²⁺ in the cell cytosol and also to a decrease in the amplitude of calcium transients induced by depolarization of the plasma membrane under the action of a hyperpotassium solution. Under conditions of repeated caffeine applications, the amplitude of transients decreased to 9% of its initial value, which is explained by exhaustion of the calcium stores. The action of caffeine was restored when the calcium stores were re-filled under the action of depolarization of the plasma membrane. Thapsigargin completely removed the effect of caffeine and did not influence KCl-induced transients. Therefore, our experiments are indicative of a significant importance of the ER calcium stores for calcium signaling in chromaffin cells, which allows us to hypothesize that these stores play an important role in the control of secretion of catecholamines.     

Activation of galanin receptor 2 stimulates large conductance Ca-dependent K (BK) channels through the IP3 pathway in human embryonic kidney (HEK293) cells

The large conductance Ca²⁺-activated K⁺ (BK) channels are widely distributed in the brain, and act as intracellular calcium sensors in neurons. They play an important feedback role in controlling Ca²⁺ flux and Ca²⁺-dependent processes, including neurotransmitter release and cellular excitability. In this study, the effects of the neuropeptide galanin on BK channels were examined by determining the whole-cell currents and single-channel activities in human embryonic kidney (HEK293) cells co-expressing GalR2 and the BK alpha subunit. Galanin enhanced the currents of BK channels, in a concentration-dependent and PTX-independent manner, with an ED50 value of 71.8 ± 16.9 nM. This activation was mediated by GalR2, since its agonist AR-M1896 mimicked the effect of galanin, and since galanin did not facilitate BK currents in cells co-expressing cDNAs of BK and GalR1 or GalR3. The galanin-induced BK current persisted after replacement with Ca²⁺-free solution, suggesting that extracellular Ca²⁺ is not essential. Chelating intracellular Ca²⁺ by either the slow Ca²⁺ buffer EGTA or the fast Ca²⁺ buffer BAPTA abolished galanin-mediated activation of BK channels, indicating the important role of intracellular Ca²⁺. The role of Ca²⁺ efflux from the sarcoplasmic reticulum/endoplasmic reticulum (SR/ER) was confirmed by application of thapsigargin, an irreversible inhibitor that depletes Ca²⁺ from SR/ER. Moreover, the inositol-1,4,5-triphosphate receptor (IP3R) was identified as the mediator responsible for increased intracellular Ca²⁺ activating BK channels. Taken together, activation of GalR2 leads to elevation of intracellular Ca²⁺ is due to Ca²⁺ efflux from ER through IP3R sequentially opening BK channels.     

Calcium signalling in cortical and thalamic neurons of rats with streptozotocin-induced diabetes

Intracellular Ca²⁺ transients were measured with the use of a Ca²⁺-sensitive fluorescent indicator, fura-2, in neocortical and thalamic neurons in brain slices from control rats and rats with uncompensated streptozotocin-induced diabetes. The transients were evoked by high-potassium (50 mM)-induced membrane depolarization. The amplitude of depolarization-induced Ca²⁺ transients demonstrated a tendency to increase under diabetic conditions, beeing more expressed in cortical neurons compared with thalamic ones. The transients in cortical neurons from diabetic animals became also more susceptible to the blocking action of nifedipine (100μM) and less sensitive to Ni²⁺ (50μM), indicating that diabetic changes affect mostly Ca²⁺ transients triggered by high-voltage activated (L-type) calcium channels. The duration of a statistically significant increase was observed in the residual elevation of intracellular Ca²⁺ changes. However, a statistically significant increase was observed in the residual elevation of intracellular Ca²⁺ measured 60 sec after termination of membrane depolarization in both cortical and thalamic neurons, indicating alterations in the mechanisms that restore the resting level of Ca²⁺ in the cytosol. It is concluded that uncomensated insulin-dependent diabetes, which according to earlier data substantially alters calcium signalling in primary sensory neurons, also affects such signalling in the neurons of higher brain structures including the thalamus and cortex.     

Pacemaking activity is regulated by membrane stretch via the CICR pathway in cultured interstitial cells of Cajal from murine intestine

Membrane stretch is an important stimulus in gastrointestinal (GI) motility regulation, but the relationship between membrane stretch and the pacemaking activity of GI smooth muscle is poorly understood. We examined the effect of intestinal distension on slow waves and the effect of membrane stretch on pacemaker currents in cultured intestinal interstitial cells of Cajal (ICCs) from murine small intestine. At organ level, intestinal distension significantly increased amplitude of slow and fast waves, and enhanced frequencies of fast but not slow waves. At the cellular level, membrane stretch-induced by hyposmotic cell swelling (MSHC) depolarized membrane potential and activated large inward holding current, but suppressed amplitude of pacemaker potential or pacemaking current. External Ca²⁺-free solution abolished pacemaker current and blocked MSHC-induced inward holding current. However, a sustained inward holding current was activated and the amplitude of pacemaker current was increased by high ethylene glycol tetraacetic acid (EGTA) in pipette. Then MSHC also potentiated the inward holding current. MSHC significantly increased amplitude of rhythmic Ca²⁺ transients and basal intracellular Ca²⁺ concentration ([Ca²⁺]_i). 2-APB blocked both pacemaker current and Ca²⁺ transients but did not alter the effect of MSHC on pacemaker current and Ca²⁺ transients. In contrast, ryanodine inhibited Ca²⁺ transients but not pacemaker current, and completely blocked MSHC-induced inward holding current and MSHC-induced increase of basal [Ca²⁺]_i. These results suggest that intestinal distension potentiates intestinal motility by increasing the amplitude of slow waves. Membrane stretch potentiates pacemaking activity via releasing Ca²⁺ from calcium-induced calcium release (CICR) in cultured intestinal ICCs.     

Calcium Movement in Cardiac Mitochondria

Existing theory suggests that mitochondria act as significant, dynamic buffers of cytosolic calcium ([Ca²⁺]_i) in heart. These buffers can remove up to one-third of the Ca²⁺ that enters the cytosol during the [Ca²⁺]_i transients that underlie contractions. However, few quantitative experiments have been presented to test this hypothesis. Here, we investigate the influence of Ca²⁺ movement across the inner mitochondrial membrane during both subcellular and global cellular cytosolic Ca²⁺ signals (i.e., Ca²⁺ sparks and [Ca²⁺]_i transients, respectively) in isolated rat cardiomyocytes. By rapidly turning off the mitochondria using depolarization of the inner mitochondrial membrane potential (ΔΨ_m), the role of the mitochondria in buffering cytosolic Ca²⁺ signals was investigated. We show here that rapid loss of ΔΨ_m leads to no significant changes in cytosolic Ca²⁺ signals. Second, we make direct measurements of mitochondrial [Ca²⁺] ([Ca²⁺]_m) using a mitochondrially targeted Ca²⁺ probe (MityCam) and these data suggest that [Ca²⁺]_m is near the [Ca²⁺]_i level (∼100 nM) under quiescent conditions. These two findings indicate that although the mitochondrial matrix is fully buffer-capable under quiescent conditions, it does not function as a significant dynamic buffer during physiological Ca²⁺ signaling. Finally, quantitative analysis using a computational model of mitochondrial Ca²⁺ cycling suggests that mitochondrial Ca²⁺ uptake would need to be at least ∼100-fold greater than the current estimates of Ca²⁺ influx for mitochondria to influence measurably cytosolic [Ca²⁺] signals under physiological conditions. Combined, these experiments and computational investigations show that mitochondrial Ca²⁺ uptake does not significantly alter cytosolic Ca²⁺ signals under normal conditions and indicates that mitochondria do not act as important dynamic buffers of [Ca²⁺]_i under physiological conditions in heart.     

Ca signaling and STIM1

An increase in the intracellular calcium ion concentration ([Ca²⁺]) impacts a diverse range of cell functions, including adhesion, motility, gene expression and proliferation. Elevation of intracellular calcium ion (Ca²⁺) regulates various cellular events after the stimulation of cells. Initial increase in Ca²⁺ comes from the endoplasmic reticulum (ER), intracellular storage space. However, the continuous influx of extracellular Ca²⁺ is required to maintain the increased level of Ca²⁺ inside cells. Store-operated Ca²⁺ entry (SOCE) manages this process, and STIM1, a newly discovered molecule, has a unique and essential role in SOCE. STIM1 can sense the exhaustion of Ca²⁺ in the ER, and activate the SOC channel in the plasma membrane, leading to the continuous influx of extracellular Ca²⁺. STIM1 senses the status of the intracellular Ca²⁺ stores via a luminal N-terminal Ca²⁺-binding EF-hand domain. Dissociation of Ca²⁺ from this domain induces the clustering of STIM1 to regions of the ER that lie close to the plasma membrane, where it regulates the activity of the store-operated Ca²⁺ channels/entry (calcium-release-activated calcium channels/entry). In this review, we summarize the mechanism by which STIM1 regulates SOCE, and also its role in the control of mast cell functions and allergic responses.     

Spike‐triggered dendritic calcium transients depend on synaptic activity in the cricket giant interneurons

The relationship between electrical activity and spike‐induced Ca²⁺ increases in dendrites was investigated in the identified wind‐sensitive giant interneurons in the cricket. We applied a high‐speed Ca²⁺ imaging technique to the giant interneurons, and succeeded in recording the transient Ca²⁺ increases (Ca²⁺ transients) induced by a single action potential, which was evoked by presynaptic stimulus to the sensory neurons. The dendritic Ca²⁺ transients evoked by a pair of action potentials accumulated when spike intervals were shorter than 100 ms. The amplitude of the Ca²⁺ transients induced by a train of spikes depended on the number of action potentials. When stimulation pulses evoking the same numbers of action potentials were separately applied to the ipsi‐ or contra‐lateral cercal sensory nerves, the dendritic Ca²⁺ transients induced by these presynaptic stimuli were different in their amplitude. Furthermore, the side of presynaptic stimulation that evoked larger Ca²⁺ transients depended on the location of the recorded dendritic regions. This result means that the spike‐triggered Ca²⁺ transients in dendrites depend on postsynaptic activity. It is proposed that Ca²⁺ entry through voltage‐dependent Ca²⁺ channels activated by the action potentials will be enhanced by excitatory synaptic inputs at the dendrites in the cricket giant interneurons. © 2002 Wiley Periodicals, Inc. J Neurobiol 50: 234–244, 2002; DOI 10.1002/neu.10032     

Functional organization of dendritic Ca2+ signals in midbrain dopamine neurons

Dendritic Ca²⁺ plays an important role not only in synaptic integration and synaptic plasticity, but also in dendritic excitability in midbrain dopamine neurons. However, the functional organization of dendritic Ca²⁺ signals in the dopamine neurons remains largely unknown. We therefore investigated dendritic Ca²⁺ signals by measuring glutamate-induced Ca²⁺ increases along the dendrites of acutely isolated midbrain dopamine neurons.Maximal doses of glutamate induced a [Ca²⁺]_c rise with similar amplitudes in proximal and distal dendritic regions of a dopamine neuron. Glutamate receptors contributed incrementally to the [Ca²⁺]_c rise according to their distance from the soma, with a reciprocal decrement in the contribution of voltage-operated Ca²⁺ channels (VOCCs). The contribution of AMPA and NMDA receptors increased with dendritic length, but that of metabotropic glutamate receptors decreased. At low doses of glutamate at which spontaneous firing was sustained, the [Ca²⁺]_c rise was higher in the distal than the proximal regions of a dendrite, possibly due to the increased spontaneous firing rate.These results indicate that functional organization of Ca²⁺ signals in the dendrites of dopamine neurons requires different combination of VOCCs and glutamate receptors according to dendritic length, and that regional Ca²⁺ rises in dendrites respond differently to applied glutamate concentration.