Ca Buffering Research Articles

Sirt3 attenuates hydrogen peroxide-induced oxidative stress through the preservation of mitochondrial function in HT22 cells.

Sirtuins (Sirt) are a family of phylogenetically conserved nicotinamide adenine nucleotide (NAD(+))-dependent protein deacetylases, among which Sirt3 resides primarily in the mitochondria and serves as a stress responsive deacetylase, playing a role in protecting cells from damage under stress conditions. The present study aimed to investigate the role of Sirt3 in hydrogen peroxide (H(2)O(2))-induced oxidative neuronal injury in HT22 mouse hippocampal cells. Treatment with H(2)O(2) increased the expression of Sirt3 in a dose- and time-dependent manner, and the knockdown of Sirt3 using specific small interfering RNA (siRNA) exacerbated the H(2)O(2)-induced neuronal injury. The overexpression of Sirt3 induced by lentiviral transfection significantly reduced the generation of reactive oxygen species (ROS) and lipid peroxidation following injury, whereas the activities of endogenous antioxidant enzymes were not affected. Further experiments revealed that the H(2)O(2)-induced inhibition of mitochondrial complex activity and adenosine triphosphate (ATP) synthesis, the decrease in mitochondrial Ca(2+) buffering capacity and mitochondrial swelling were all partly reversed by Sirt3. Furthermore, the overexpression of Sirt3 attenuated the release of cytochrome c, the increase in the Bax/Bcl-2 ratio, as well as caspase-9/caspase-3 activity induced by H(2)O(2), and eventually inhibited apoptotic neuronal cell death. These results suggest that Sirt3 acts as a prosurvival factor, playing an essential role in protecting HT22 cells under H(2)O(2)-induced oxidative stress, possibly by inhibiting ROS accumulation and the activation of the mitochondrial apoptotic pathway.

Model Driven Process Design and Development for a Continuous Process

This paper presents a case study where kinetic models were used to drive process development of an amination reaction in a continuous process. The kinetic model was first used to optimize the process by minimizing impurity formation. Then, the kinetic parameters were put into a two-in-series CSTR (continuous stirred tank reactor) model and a separate PFR (plug flow reactor) model to guide reactor selection and optimize the reactor design. Once the model predictions were validated with experimental data, the simulated impurity profile from each reactor type was compared. While the results showed that both reactors delivered similar impurity profiles at steady state, the model was used to conduct perturbation analysis on the two reactors to assess the impurity and operational control strategy. The simulations showed that the CSTR can tolerate a process upset time up to 10% of its residence time with materials meeting product specification as compared to the PFR. Since the CSTR offers additional buffering ca...

Calcium regulation of HCN supports persistent activity associated with working memory: a multiscale model of prefrontal cortex

The Hyperpolarization-activated cyclic-nucleotide gated channel (HCN; Ih current) has been hypothesized to facilitate short-term memory storage in the prefrontal cortex (PFC) via a cascade of signalling events involving second messengers including protein kinases and intracellular calcium (Ca) accumulation [1]. This memory mechanism is effected via alterations in the dynamics of the network, two scales above the underlying molecular level. To determine the factors that enable persistent activity to emerge, we developed a multiscale model (molecular to network level) of PFC that displays the persistent activity associated with working memory. The network contained 800 cells arranged in 6 neocortical layers. Cell classes included pyramidal cells, and fast-spiking and low-threshold-spiking interneurons. Each neuron contained Na, K, Ca, and HCN channels. Cells were interconnected probabilistically using detailed anatomical data from M1, with AMPA/NMDA, and GABAA synapses. Intracellular Ca was admitted via NMDA and L-type Ca channels. Intracellular signaling components which contributed to persistent activity included: diffusible Ca, diffusible Ca buffering proteins, intracellular endoplasmic reticulum (ER) compartments for sequestering Ca, diffusible inositol triphosphate (IP3), ER IP3 receptors (IP3R) which release Ca from ER upon IP3/Ca binding, sarco/endoplasmic reticulum Ca-ATP-ase pumps (SERCA) which pump cytosolic Ca into the ER, Ca/IP3 degradation reactions. Pyramidal cells had HCN channels regulated by protein kinases bound to intracellular Ca. The model was implemented in parallel NEURON with reaction-diffusion [2]. 18 seconds of simulation time ran over 24 Intel XEON CPUs in ~7 minutes. We assessed influences between intracellular signaling and network dynamics to isolate sources needed for emergence of short-term memory representations. We provided excitatory stimuli to a subset of pyramidal cells which represented the memory. Stimulus-induced depolarization led to Ca influx which increased HCN conductance and excitability of the stimulated population (firing increased from 0.5-2 Hz). Activation of the stimulated population lasted for 5-10 seconds and also increased beta/gamma (~15-35 Hz) oscillations. In contrast, non-stimulated populations were suppressed (firing rate reduction up to 50%) due to higher inhibition caused by the activated cells providing stronger drive to the interneurons. Knocking out specific pathways showed that persistent activity of activated cells was only viable in the presence of Ca influx, Ca regulation of HCN channels, and interactions between excitatory and inhibitory populations, which led to network-derived inhibition activating HCN. Lowering concentrations and binding rates of Ca buffering proteins reduced the network’s ability to represent specific inputs, due to the higher levels of intracellular Ca saturating HCN channels in all pyramidal neurons. This predicts that Ca buffering proteins contribute to regulation of Ca at time-scales relevant for maintaining distinct short-term memory representations. We also used our model to demonstrate nonsynaptic plasticity - initial stimuli cause Ca sequestration in the ER of activated neurons, allowing for later, selective modulation of their excitability via metabotropic glutamate receptor activation of IP3Rs, which releases the stored Ca. Our model demonstrates how electro-chemical interactions at the nanoscale may lead to network consequences that produce persistent activity associated with working memory.

Kinetic Organization of Ca2+ Signals That Regulate Synaptic Release Efficacy in Sympathetic Neurons

Calcium regulation of neurotransmitter release is essential for maintenance of synaptic transmission. However, the temporal and spatial organization of Ca(2+) dynamics that regulate synaptic vesicle (SV) release efficacy in sympathetic neurons is poorly understood. Here, we investigate the N-type Ca(2+) channel-mediated kinetic structure of Ca(2+) regulation of cholinergic transmission of sympathetic neurons. We measured the effect of Ca(2+) chelation with fast 1,2-bis(2-aminophenoxy) ethane-tetraacetic acid (BAPTA) and slow ethyleneglycol-tetraacetic acid (EGTA) buffers on exocytosis, synaptic depression, and recovery of the readily releasable vesicle pool (RRP), after both single action potential (AP) and repetitive APs. Surprisingly, postsynaptic potentials peaking at ~12 milliseconds after the AP was inhibited by both rapid and slow Ca(2+) buffers suggests that, in addition to the well known fast Ca(2+) signals at the active zone (AZ), slow Ca(2+) signals at the peak of Ca(2+) entry also contribute to paired-pulse or repetitive AP responses. Following a single AP, discrete Ca(2+) transient increase regulated synaptic depression in rapid (<30-millisecond) and slow (<120-millisecond) phases. In contrast, following prolonged AP trains, synaptic depression was reduced by a slow Ca(2+) signal regulation lasting >200 milliseconds. Finally, after an AP burst, recovery of the RRP was mediated by an AP-dependent rapid Ca(2+) signal, and the expansion of releasable SV number by an AP firing activity-dependent slow Ca(2+) signal. These data indicate that local Ca(2+) signals operating near Ca(2+) sources in the AZ are organized into discrete fast and slow temporal phases that remodel exocytosis and short-term plasticity to ensure long-term stability in acetylcholine release efficacy.

Computational analysis of Ca2+ dynamics in isolated cardiac mitochondria predicts two distinct modes of Ca2+ uptake

Cardiac mitochondria can act as a significant Ca(2+) sink and shape cytosolic Ca(2+) signals affecting various cellular processes, such as energy metabolism and excitation-contraction coupling. However, different mitochondrial Ca(2+) uptake mechanisms are still not well understood. In this study, we analysed recently published Ca(2+) uptake experiments performed on isolated guinea pig cardiac mitochondria using a computer model of mitochondrial bioenergetics and cation handling. The model analyses of the data suggest that the majority of mitochondrial Ca(2+) uptake, at physiological levels of cytosolic Ca(2+) and Mg(2+), occurs through a fast Ca(2+) uptake pathway, which is neither the Ca(2+) uniporter nor the rapid mode of Ca(2+) uptake. This fast Ca(2+) uptake component was explained by including a biophysical model of the ryanodine receptor (RyR) in the computer model. However, the Mg(2+)-dependent enhancement of the RyR adaptation was not evident in this RyR-type channel, in contrast to that of cardiac sarcoplasmic reticulum RyR. The extended computer model is corroborated by simulating an independent experimental dataset, featuring mitochondrial Ca(2+) uptake, egress and sequestration. The model analyses of the two datasets validate the existence of two classes of Ca(2+) buffers that comprise the mitochondrial Ca(2+) sequestration system. The modelling study further indicates that the Ca(2+) buffers respond differentially depending on the source of Ca(2+) uptake. In particular, it suggests that the Class 1 Ca(2+) buffering capacity is auto-regulated by the rate at which Ca(2+) is taken up by mitochondria.

Isoflurane induces substrate‐dependent early mitochondrial PTP opening (648.13)

The volatile anesthetic isoflurane (ISO) may increase permeability of mitochondria to protect against mitochondrial injury. Prolonged opening of the mitochondrial permeability transition pore (mPTP) inevitably causes mitochondrial demise, whereas transient mPTP opening may release accumulated Ca2+ from the matrix to avoid subsequent irreversible mPTP opening due to Ca2+ overload. To test if ISO alters mPTP opening, we isolated mitochondria from Wistar rat hearts to assess buffer Ca2+ uptake by Fura 4F. We found that: (a) ISO (1 mM) induced earlier mPTP opening by increasing Ca2+ uptake rate with succinate (SUC). (b) ISO did not alter mPTP opening but reduced Ca2+ uptake with pyruvate/malate. (c) 10 µM ryanodine or 10 nM imperatoxin A with SUC increased Ca2+ uptake and induced earlier mPTP opening; this suggests that ISO’s effects on mPTP may be due to increased conductance via mitochondrial ryanodine receptors (mRyR). We have patch‐clamped single mRyRs in mitoplasts. Our data infer indirectly that SUC substrate ‐dependent transient mPTP opening may be due to enhanced RyR Ca2+ uptake. Transient Ca2+ release could be a mechanism underlying ISO –induced cardiac protection in ischemia‐reperfusion injury.Grant Funding Source: P01‐GM066730, R01‐HL095122, R01‐HL089514

Disrupted WNT Signaling in Mouse Embryonic Stem Cells in the Absence of Calreticulin

The role of endoplasmic reticulum (ER) homeostasis and protein quality control in the regulation of WNT signaling is not understood. Here we provide evidence for a role of calreticulin in the regulation of WNT signaling. We show that a deficiency in calreticulin disrupted WNT signaling, and prevented cell cycle progression via the miR-302 microRNA family. These effects were dependent on the Ca(2+) buffering capacity of calreticulin, as the protein is important in regulating ER Ca(2+) release and activation of Ca(2+)-dependent kinase and phosphatase cascades (including c-Src, Akt, and PTP1B). We also show that calreticulin plays a role in the secretion and ER retention of WNT3a, thereby affecting downstream WNT signaling. In calreticulin-deficient ES cells, the WNT and miR-302 dependent maintenance of the naïve ES cell state and the transition to primed pluripotency transition were lost, preventing cells from undergoing accurate differentiation. Together, these findings demonstrate unexpected roles of calreticulin and ER Ca(2+) homeostasis/signaling in the canonical WNT signaling pathway.

Cytosolic Ca Buffering of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes and Adult Rabbit Ventricular Cardiomyocytes

A major concern about using cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CM) for human disease modeling is their immature phenotype which may result in substantial differences in their physiology when compared to native cardiomyocytes. Here we compare intracellular Ca buffering of hiPSC-CM derived from healthy humans to that of acutely-isolated adult rabbit ventricular CM. hiPSC-CM (21 days post cardiac induction) were plated at low density on matrigel-coated dishes and studied 3-5 days after plating. While hiPSC were significantly smaller than rabbit CM (cell capacity 19±2 pF vs 101±12 pF, p<0.01) and had disorganized contractile filaments on electron microscopy, hiPSC-CM nevertheless exhibited robust troponin C expression, the major cytosolic Ca binding site. To measure cytosolic Ca buffering (Trafford method), caffeine (10 mM) was rapidly applied to release Ca from the SR, which then extruded via the NaCa exchanger (NCX), generating an inward NCX current. Integration of NCX current yielded the total amount of Ca released from the SR. Then Δ[Ca]total was plotted as a function of Δ[Ca]free and maximal cytosolic buffering capacity (Bmax) and Kd calculated. Cytosolic Ca buffering parameters were not significantly different between hiPSC-CM (Bmax=100±14μmol/Lcytosol, Kd=0.61±0.09 μM, n=25) and rabbit-CM (Bmax=117±15 μmol/Lcytosol, Kd=0.61±0.11 μM, n=9). EMD57033 (3 μM), which binds to troponin C and increases cytosolic Ca binding in murine CM, also increased cytosolic Ca binding affinity in hiPSC-CM (Kd=0.39±0.05 μM; p<0.05) without affecting Bmax (96±13 μmol/Lcytosol). Importantly, rates of non-NCX mediated Ca removal were not different between hiPSC-CM and adult rabbit CM. These results demonstrate that hiPSC-CM have cytosolic Ca buffering parameters similar to those of acutely-isolated native CM and therefore could be used for modeling cardiac diseases with altered myofilament Ca binding. Supported by AHA-12POST12080080(DK), NIH-RO1-HL71670(BCK).

The Potential for Another Calcium Uptake Mode in Cardiac Mitochondria

Cytosolic Ca2+ levels are tightly regulated in cardiomyoctes. In many instances mitochondria play a direct role in this regulation. They take up Ca2+ via the Ca2+ uniporter, buffer Ca2+ by calcium-phosphate sequestration, and release Ca2+ primarily through the electrogenic Na+/Ca2+ exchanger. However, the manner in which Ca2+ is taken up is still in dispute. In this study we used energized mitochondria isolated from guinea pig hearts to explore the potential for two modes of Ca2+ uptake. In addition, we sought to demonstrate the manner in which mitochondria take up, sequester, and release Ca2+, and how Ca2+ transport is differentially modulated by Mg2+. To carry out this study, we monitored extra-matrix and matrix [Ca2+] during Ca2+ loading and unloading experiments using Fura-4F PP and Fura-4F AM fluorescence to quantify extra-matrix and matrix [Ca2+], respectively. Two loading protocols were used: 1) a bolus of CaCl2 was added to a suspension of mitochondria in respiration buffer and 2) mitochondria were added to respiration buffer already containing CaCl2. In all experiments, ruthenium red was later added to stop Ca2+ uptake and NaCl was added to initiate Ca2+ efflux. Also, each protocol was executed in the presence and absence of extra-matrix MgCl2. Depending on the protocol, two distinct profiles of Ca2+ uptake were observed, whereby using protocol 1 resulted in a faster mode of Ca2+ uptake and protocol 2 in a slower mode. Furthermore, Ca2+ uptake and efflux were inhibited by MgCl2. We found that Mg2+ reduced the ability of mitochondria to sequester Ca2+ independent of the protocol. In summary, these observations derived from our experiments show the potential for at least two modes of Ca2+ uptake and provide us with a better understanding of how matrix Ca2+ dynamics change under physiological and pathophysiological conditions.

Selective vulnerability of motoneuron and perturbed mitochondrial calcium homeostasis in amyotrophic lateral sclerosis: implications for motoneurons specific calcium dysregulation.

Amyotrophic lateral sclerosis (ALS) is a lethal neurodegenerative disorder characterized by the selective degeneration of defined subgroups of motoneuron in the brainstem, spinal cord and motor cortex with signature hallmarks of mitochondrial Ca2+ overload, free radical damage, excitotoxicity and impaired axonal transport. Although intracellular disruptions of cytosolic and mitochondrial calcium, and in particular low cytosolic calcium ([Ca2+]c) buffering and a strong interaction between metabolic mechanisms and [Ca2+]i have been identified predominantly in motoneuron impairment, the causes of these disruptions are unknown. The existing evidence suggests that the mutant superoxide dismutase1 (mtSOD1)-mediated toxicity in ALS acts through mitochondria, and that alteration in cytosolic and mitochondria-ER microdomain calcium accumulation are critical to the neurodegenerative process. Furthermore, chronic excitotoxcity mediated by Ca2+-permeable AMPA and NMDA receptors seems to initiate vicious cycle of intracellular calcium dysregulation which leads to toxic Ca2+ overload and thereby selective neurodegeneration. Recent advancement in the experimental analysis of calcium signals with high spatiotemporal precision has allowed investigations of calcium regulation in-vivo and in-vitro in different cell types, in particular selectively vulnerable/resistant cell types in different animal models of this motoneuron disease. This review provides an overview of latest advances in this field, and focuses on details of what has been learned about disrupted Ca2+ homeostasis and mitochondrial degeneration. It further emphasizes the critical role of mitochondria in preventing apoptosis by acting as a Ca2+ buffers, especially in motoneurons, in pathophysiological conditions such as ALS.

Calretinin: from a "simple" Ca(2+) buffer to a multifunctional protein implicated in many biological processes.

The hexa-EF-hand Ca2+-binding protein calretinin (CR) is predominantly expressed in specific neurons of the central and peripheral nervous system. However, CR expression is also observed in non-neuronal cells, e.g., during embryonic development and in mesothelioma cells. Of the 6 EF-hand domains, 5 are functional; the first 4 domains form 2 pairs showing high cooperativity within a pair that results in non-linear modulation of intracellular Ca2+ signals by CR. EF-hand domain 5 has a low affinity and represents the identified interaction site with CR-binding partners present in mouse cerebellar granule cells. CR binding to other targets including the pore-forming α1 subunit of the Ca2+ channel CaV2.1, as well as to huntingtin indicates additional Ca2+ sensor functions besides the well-known Ca2+-buffering functions. The absence of CR in cerebellar granule cells of CR−/− mice results in increased excitability and altered firing of Purkinje cells and promotes cerebellar 160-Hz oscillations impairing motor coordination. The putative role of CR in neuroprotection is still highly discussed. Altogether, CR emerges as a multi-functional protein also associated with development, i.e., cell proliferation, differentiation, and cell death.

Chapter 5 - Effects of calcium (Ca2 +) extrusion mechanisms on electrophysiological properties in a hypoglossal motoneuron: Insight from a mathematical model

Spike-frequency dynamics and spike shape can provide insight into the types of ion channels present in any given neuron and give a sense for the precise response any neuron may have to a given input stimulus. Motoneuron firing frequency over time is especially important due to its direct effect on motor output. Of particular interest is intracellular Ca(2+), which exerts a powerful influence on both firing properties over time and spike shape. In order to better understand the cellular mechanisms for the regulation of intracellular Ca(2+) and their effect on spiking behavior, we have modified a computational model of an HM to include a variety of Ca(2+) handling processes. For the current study, a series of HM models that include Ca(2+) pumps, Na(+)/Ca(2+) exchangers, and a generic exponential decay of excess Ca(2+) were generated. Simulations from these models indicate that although each extrusion mechanism exerts a similar effect on voltage, the firing properties change distinctly with the inclusion of additional Ca(2+)-related mechanisms: BK channels, Ca(2+) buffering, and diffusion of [Ca(2+)]i modeled via a linear diffusion partial differential equation. While an exponential decay of Ca(2+) seems to adequately capture short-term changes in firing frequency seen in biological data, internal diffusion of Ca(2+) appears to be necessary for capturing longer term frequency changes.

IP3-mediated STIM1 oligomerization requires intact mitochondrial Ca2+ uptake.

ABSTRACTMitochondria contribute to cell signaling by controlling store-operated Ca2+ entry (SOCE). SOCE is activated by Ca2+ release from the endoplasmic reticulum (ER), whereupon stromal interacting molecule 1 (STIM1) forms oligomers, redistributes to ER–plasma-membrane junctions and opens plasma membrane Ca2+ channels. The mechanisms by which mitochondria interfere with the complex process of SOCE are insufficiently clarified. In this study, we used an shRNA approach to investigate the direct involvement of mitochondrial Ca2+ buffering in SOCE. We demonstrate that knockdown of either of two proteins that are essential for mitochondrial Ca2+ uptake, the mitochondrial calcium uniporter (MCU) or uncoupling protein 2 (UCP2), results in decelerated STIM1 oligomerization and impaired SOCE following cell stimulation with an inositol-1,4,5-trisphosphate (IP3)-generating agonist. Upon artificially augmented cytosolic Ca2+ buffering or ER Ca2+ depletion by sarcoplasmic or endoplasmic reticulum Ca2+-ATPase (SERCA) inhibitors, STIM1 oligomerization did not rely on intact mitochondrial Ca2+ uptake. However, MCU-dependent mitochondrial sequestration of Ca2+ entering through the SOCE pathway was essential to prevent slow deactivation of SOCE. Our findings show a stimulus-specific contribution of mitochondrial Ca2+ uptake to the SOCE machinery, likely through a role in shaping cytosolic Ca2+ micro-domains.

Effect of Ca2+ efflux pathway distribution and exogenous Ca2+ buffers on intracellular Ca2+ dynamics in the rat ventricular myocyte: a simulation study.

We have used a previously published computer model of the rat cardiac ventricular myocyte to investigate the effect of changing the distribution of Ca2+ efflux pathways (SERCA, Na+/Ca2+ exchange, and sarcolemmal Ca2+ ATPase) between the dyad and bulk cytoplasm and the effect of adding exogenous Ca2+ buffers (BAPTA or EGTA), which are used experimentally to differentially buffer Ca2+ in the dyad and bulk cytoplasm, on cellular Ca2+ cycling. Increasing the dyadic fraction of a particular Ca2+ efflux pathway increases the amount of Ca2+ removed by that pathway, with corresponding changes in Ca2+ efflux from the bulk cytoplasm. The magnitude of these effects varies with the proportion of the total Ca2+ removed from the cytoplasm by that pathway. Differences in the response to EGTA and BAPTA, including changes in Ca2+-dependent inactivation of the L-type Ca2+ current, resulted from the buffers acting as slow and fast “shuttles,” respectively, removing Ca2+ from the dyadic space. The data suggest that complex changes in dyadic Ca2+ and cellular Ca2+ cycling occur as a result of changes in the location of Ca2+ removal pathways or the presence of exogenous Ca2+ buffers, although changing the distribution of Ca2+ efflux pathways has relatively small effects on the systolic Ca2+ transient.

Novel features on the regulation by mitochondria of calcium and secretion transients in chromaffin cells challenged with acetylcholine at 37°C.

From experiments performed at room temperature, we know that the buffering of Ca2+ by mitochondria contributes to the shaping of the bulk cytosolic calcium transient ([Ca2+]c) and secretion transients of chromaffin cells stimulated with depolarizing pulses. We also know that the mitochondrial Ca2+ transporters and the release of catecholamine are faster at 37°C with respect to room temperature. Therefore, we planned this investigation to gain further insight into the contribution of mitochondrial Ca2+ buffering to the shaping of [Ca2+]c and catecholamine release transients, using some novel experimental conditions that have not been yet explored namely: (1) perifusion of bovine chromaffin cells (BCCs) with saline at 37°C and their repeated challenging with the physiological neurotransmitter acetylcholine (ACh); (2) separate blockade of mitochondrial Ca2+ uniporter (mCUP) with Ru360 or the mitochondrial Na+/Ca2+ exchanger (mNCX) with CGP37157; (3) full blockade of the mitochondrial Ca2+ cycling (mCC) by the simultaneous inhibition of the mCUP and the mNCX. Ru360 caused a pronounced delay of [Ca2+]c clearance and augmented secretion. In contrast, CGP37157 only caused a tiny delay of [Ca2+]c clearance and a mild decrease in secretion. The mCC resulting in continued Ca2+ uptake and its release back into the cytosol was interrupted by combined Ru360 + CGP37157 (Ru/CGP), the protonophore carbonyl cyanide‐p‐trifluoromethoxyphenylhydrazone, or combined oligomycin + rotenone (O/R); these three treatments caused a mild but sustained elevation of basal [Ca2+]c that, however, was not accompanied by a parallel increase in basal secretion. Nevertheless, all treatments caused a pronounced augmentation of ACh‐induced secretion, with minor changes of the ACh‐induced [Ca2+]c transients. Combined Ru/CGP did not alter the resting membrane potential in current‐clamped cells. Additionally, Ru/CGP did not increase basal [Ca2+]c near subplasmalemmal sites and caused a mild decrease in the size of the readily releasable vesicle pool. Our results provide new functional features in support of the view that in BCCs there are two subpopulations of mitochondria, M1 underneath the plasmalemma nearby exocytotic sites and M2 at the core cell nearby vesicle transport sites. While M1 serves to shape the ACh‐elicited exocytotic response through its efficient Ca2+ removal by the mCUP, M2 shapes the lower [Ca2+]c elevations required for new vesicle supply to the exocytotic machinery, from the large reserve vesicle pool at the cell core. The mCUP of the M1 pool seems to play a more prominent role in controlling the ACh responses, in comparison with the mNCX.

Adaptation of Mammalian Auditory Hair Cell Mechanotransduction Is Independent of Calcium Entry

Adaptation is a hallmark of hair cell mechanotransduction, extending the sensory hair bundle dynamic range while providing mechanical filtering of incoming sound. In hair cells responsive to low frequencies, two distinct adaptation mechanisms exist, a fast component of debatable origin and a slow myosin-based component. It is generally believed that Ca(2+) entry through mechano-electric transducer channels is required for both forms of adaptation. This study investigates the calcium dependence of adaptation in the mammalian auditory system. Recordings from rat cochlear hair cells demonstrate that altering Ca(2+) entry or internal Ca(2+) buffering has little effect on either adaptation kinetics or steady-state adaptation responses. Two additional findings include a voltage-dependent process and an extracellular Ca(2+) binding site, both modulating the resting open probability independent of adaptation. These data suggest that slow motor adaptation is negligible in mammalian auditory cells and that the remaining adaptation process is independent of calcium entry.

Quantitative Aspects of Calcium Fluorimetry

Ca(2+) indicator dyes by necessity are Ca(2+) chelators, because it is the binding of Ca(2+) to dye molecules that induces the change in fluorescence on which the Ca(2+) signal is based. As chelators, once introduced into a cell, they contribute to cellular Ca(2+) buffering. It has been a question of much debate to what extent this added Ca(2+) buffer (exogenous Ca(2+) buffer) changes Ca(2+) homeostasis and the signals of interest. I discuss this problem here, emphasizing the distinction between the influence of the dyes on amplitudes (which may be not so severe) and on the dynamics of Ca(2+) signals (which may be drastic). Once the Ca(2+)-buffering action of dyes relative to intrinsic Ca(2+) buffers is understood for a given preparation, Ca(2+) dyes can be used as very versatile tools for studying both Ca(2+) concentrations and Ca(2+) fluxes. I describe in detail some of my own experiences in calibrating the indicator dye Fura-2. These refer exclusively to experiments in which the dye is loaded into the cell via a patch pipette because acetoxymethyl ester loading introduces problems that very often prohibit precise quantitative conclusions.

Isolation of Human Atrial Myocytes for Simultaneous Measurements of Ca&lt;sup&gt;2+&lt;/sup&gt; Transients and Membrane Currents

The study of electrophysiological properties of cardiac ion channels with the patch-clamp technique and the exploration of cardiac cellular Ca(2+) handling abnormalities requires isolated cardiomyocytes. In addition, the possibility to investigate myocytes from patients using these techniques is an invaluable requirement to elucidate the molecular basis of cardiac diseases such as atrial fibrillation (AF).(1) Here we describe a method for isolation of human atrial myocytes which are suitable for both patch-clamp studies and simultaneous measurements of intracellular Ca(2+) concentrations. First, right atrial appendages obtained from patients undergoing open heart surgery are chopped into small tissue chunks ("chunk method") and washed in Ca(2+)-free solution. Then the tissue chunks are digested in collagenase and protease containing solutions with 20 μM Ca(2+). Thereafter, the isolated myocytes are harvested by filtration and centrifugation of the tissue suspension. Finally, the Ca(2+) concentration in the cell storage solution is adjusted stepwise to 0.2 mM. We briefly discuss the meaning of Ca(2+) and Ca(2+) buffering during the isolation process and also provide representative recordings of action potentials and membrane currents, both together with simultaneous Ca(2+) transient measurements, performed in these isolated myocytes.

Ca2+buffering as a mechanism of short-term synaptic plasticity

Spatio-temporal compartmentalization allows Ca signals to simultaneously regulate multiple vital cell processes and relies in part on Ca buffers that absorb at least 98% of Ca ions entering the cytoplasm. Computational modeling has played a central role in the understanding of localized Ca signals in neurons and other cell types. Although many models consider only simple, one-to-one Ca buffering stoichiometry, practically all buffers have multiple Ca binding sites which often display cooperative binding (e.g., calretinin [1-3], calmodulin [4,5] ). Given the simplest case of two binding sites, cooperativity manifests itself in an order of magnitude difference in the binding and/or unbinding rates of the two consecutive Ca binding steps in the following buffer reaction: Here we extend recent modeling studies of cooperative buffering [1-5], and find that it can lead to spatiotemporal Ca signals that cannot be achieved by any combination of non-cooperative buffers, in particular during a sequence of action potentials or synaptic inputs. Namely, Figure 1B shows that cooperative Ca buffering can potentially serve as a mechanism of shortterm synaptic depression, in contrast to the case of noncooperative buffers (Figure 1A), which are believed to underlie short-term synaptic facilitation in certain types of mammalian synapses [6,7]. We explore this phenomenon in detail, demonstrating the dependence of such buffer-induced short-term synaptic plasticity on all relevant buffering parameters. These results may lead to better understanding of post-synaptic Ca dynamics as well, yielding a deeper insight into synaptic transmission and its dynamic regulation, and may also have relevance for Ca-dependent processes in other cells such as endocrine cells, myocytes and immune system cells.

Paired‐pulse facilitation at recurrent Purkinje neuron synapses is independent of calbindin and parvalbumin during high‐frequency activation

Paired-pulse facilitation (PPF) is a dynamic enhancement of transmitter release considered crucial in CNS information processing. The mechanisms of PPF remain controversial and may differ between synapses. Endogenous Ca(2+) buffers such as parvalbumin (PV) and calbindin-D28k (CB) are regarded as important modulators of PPF, with PV acting as an anti-facilitating buffer while saturation of CB can promote PPF. We analysed transmitter release and PPF at intracortical, recurrent Purkinje neuron (PN) to PN synapses, which show PPF during high-frequency activation (200 Hz) and strongly express both PV and CB. We quantified presynaptic Ca(2+) dynamics and quantal release parameters in wild-type (WT), and CB and PV deficient mice. Lack of CB resulted in increased volume averaged presynaptic Ca(2+) amplitudes and in increased release probability, while loss of PV had no significant effect on these parameters. Unexpectedly, none of the buffers significantly influenced PPF, indicating that neither CB saturation nor residual free Ca(2+) ([Ca(2+)]res) was the main determinant of PPF. Experimentally constrained, numerical simulations of Ca(2+)-dependent release were used to estimate the contributions of [Ca(2+)]res, CB, PV, calmodulin (CaM), immobile buffer fractions and Ca(2+) remaining bound to the release sensor after the first of two action potentials ('active Ca(2+)') to PPF. This analysis indicates that PPF at PN-PN synapses does not result from either buffer saturation or [Ca(2+)]res but rather from slow Ca(2+) unbinding from the release sensor.