Using total fluorescence increase (signal mass) to determine the Ca2+ current underlying localized Ca2+ events

The feasibility of determining localized Ca(2+) influx using only wide-field fluorescence images was explored by imaging (using fluo-3) single channel Ca(2+) fluorescence transients (SCCaFTs), due to Ca(2+) entry through single openings of Ca(2+)-permeable ion channels, while recording unitary channel currents. Since the image obtained with wide-field optics is an integration of both in-focus and out-of-focus light, the total fluorescence increase (DeltaF(total) or "signal mass") associated with a SCCaFT can be measured directly from the image by adding together the fluorescence increase due to Ca(2+) influx in all of the pixels. The assumptions necessary for obtaining the signal mass from confocal linescan images are not required. Two- and three-dimensional imaging was used to show that DeltaF(total) is essentially independent of the position of the channel with respect to the focal plane of the microscope. The relationship between Ca(2+) influx and DeltaF(total) was obtained using SCCaFTs from plasma membrane caffeine-activated cation channels when Ca(2+) was the only charge carrier of the inward current. This relationship was found to be linear, with the value of the slope (or converting factor) affected by the particular imaging system set-up, the experimental conditions, and the properties of the fluorescent indicator, including its binding capacity with respect to other cellular buffers. The converting factor was used to estimate the Ca(2+) current passing through caffeine-activated channels in near physiological saline and to estimate the endogenous buffer binding capacity. In addition, it allowed a more accurate estimate of the Ca(2+) current underlying Ca(2+) sparks resulting from Ca(2+) release from intracellular stores via ryanodine receptors in the same preparation.     

Allosteric activation of Na+-Ca2+ exchange by L-type Ca2+ current augments the trigger flux for SR Ca2+ release in ventricular myocytes

The possible contribution of Na(+)-Ca(2+) exchange to the triggering of Ca(2+) release from the sarcoplasmic reticulum in ventricular cells remains unresolved. To gain insight into this issue, we measured the "trigger flux" of Ca(2+) crossing the cell membrane in rabbit ventricular myocytes with Ca(2+) release disabled pharmacologically. Under conditions that promote Ca(2+) entry via Na(+)-Ca(2+) exchange, internal [Na(+)] (10 mM), and positive membrane potential, the Ca(2+) trigger flux (measured using a fluorescent Ca(2+) indicator) was much greater than the Ca(2+) flux through the L-type Ca(2+) channel, indicating a significant contribution from Na(+)-Ca(2+) exchange to the trigger flux. The difference between total trigger flux and flux through L-type Ca(2+) channels was assessed by whole-cell patch-clamp recordings of Ca(2+) current and complementary experiments in which internal [Na(+)] was reduced. However, Ca(2+) entry via Na(+)-Ca(2+) exchange measured in the absence of L-type Ca(2+) current was considerably smaller than the amount inferred from the trigger flux measurements. From these results, we surmise that openings of L-type Ca(2+) channels increase [Ca(2+)] near Na(+)-Ca(2+) exchanger molecules and activate this protein. These results help to resolve seemingly contradictory results obtained previously and have implications for our understanding of the triggering of Ca(2+) release in heart cells under various conditions.     

Properties of Ca2+ sparks revealed by four-dimensional confocal imaging of cardiac muscle

Parameters (amplitude, width, kinetics) of Ca(2+) sparks imaged confocally are affected by errors when the spark source is not in focus. To identify sparks that were in focus, we used fast scanning (LSM 5 LIVE; Carl Zeiss) combined with fast piezoelectric focusing to acquire x-y images in three planes at 1-μm separation (x-y-z-t mode). In 3,000 x-y scans in each of 34 membrane-permeabilized cat atrial cardiomyocytes, 6,906 sparks were detected. 767 sparks were in focus. They had greater amplitude, but their spatial width and rise time were similar compared with all sparks recorded. Their distribution of amplitudes had a mode at ΔF/F(0) = 0.7. The Ca(2+) release current underlying in-focus sparks was 11 pA, requiring 20 to 30 open channels, a number at the high end of earlier estimates. Spark frequency was greater than in earlier imaging studies of permeabilized ventricular cells, suggesting a greater susceptibility to excitation, which could have functional relevance for atrial cells. Ca(2+) release flux peaked earlier than the time of peak fluorescence and then decayed, consistent with significant sarcoplasmic reticulum (SR) depletion. The evolution of fluorescence and release flux were strikingly similar for in-focus sparks of different rise time (T). Spark termination involves both depletion of Ca(2+) in the SR and channel closure, which may be synchronized by depletion. The observation of similar flux in sparks of different T requires either that channel closure and other termination processes be independent of the determinants of flux (including [Ca(2+)](SR)) or that different channel clusters respond to [Ca(2+)](SR) with different sensitivity.     

Optical single-channel recording by imaging Ca2+ flux through individual ion channels: theoretical considerations and limits to resolution

Recent developments in microscopy and fluorescent indicators now make it possible to monitor the activity and localization of membrane ion channels by imaging Ca(2+) flux through individual channels. Such optical approaches have advantages over electrophysiological single-channel techniques in that they are less invasive, provide spatial information and can simultaneously and independently monitor hundreds of channels. However, their kinetic resolution does not yet approach that of patch-clamp recordings. To help understand the processes that determine the temporal resolution and noise level of single-channel Ca(2+) fluorescence signals (SCCaFTs), we simulated the microdomains of Ca(2+) ions and Ca(2+)-bound indicator dye that exist around the mouth of an open channel. Further, as an aid to development of improved optical techniques, we modeled the dependence of the amplitude and kinetics of SCCaFTs on parameters such as the imaging volume, the indicator concentration, affinity and mobility, and the presence of endogenous and exogenous Ca(2+) buffers. The results indicate that under optimal conditions, including the use of confocal or total-internal reflection microscopy to image from sub-femtolitre volumes, SCCaFTs should resolve channel openings as brief as 1ms with a signal-to-noise ratio >10.     

Ca2+ -induced Ca2+ release through localized Ca2+ uncaging in smooth muscle

Ca(2+)-induced Ca(2+) release (CICR) from the sarcoplasmic reticulum (SR) occurs in smooth muscle as spontaneous SR Ca(2+) release or Ca(2+) sparks and, in some spiking tissues, as Ca(2+) release that is triggered by the activation of sarcolemmal Ca(2+) channels. Both processes display spatial localization in that release occurs at a higher frequency at specific subcellular regions. We have used two-photon flash photolysis (TPFP) of caged Ca(2+) (DMNP-EDTA) in Fluo-4-loaded urinary bladder smooth muscle cells to determine the extent to which spatially localized increases in Ca(2+) activate SR release and to further understand the molecular and biophysical processes underlying CICR. TPFP resulted in localized Ca(2+) release in the form of Ca(2+) sparks and Ca(2+) waves that were distinguishable from increases in Ca(2+) associated with Ca(2+) uncaging, unequivocally demonstrating that Ca(2+) release occurs subsequent to a localized rise in [Ca(2+)](i). TPFP-triggered Ca(2+) release was not constrained to a few discharge regions but could be activated at all areas of the cell, with release usually occurring at or within several microns of the site of photolysis. As expected, the process of CICR was dominated by ryanodine receptor (RYR) activity, as ryanodine abolished individual Ca(2+) sparks and evoked release with different threshold and kinetics in FKBP12.6-null cells. However, TPFP CICR was not completely inhibited by ryanodine; Ca(2+) release with distinct kinetic features occurred with a higher TPFP threshold in the presence of ryanodine. This high threshold release was blocked by xestospongin C, and the pharmacological sensitivity and kinetics were consistent with CICR release at high local [Ca(2+)](i) through inositol trisphosphate (InsP(3)) receptors (InsP(3)Rs). We conclude that CICR activated by localized Ca(2+) release bears essential similarities to those observed by the activation of I(Ca) (i.e., major dependence on the type 2 RYR), that the release is not spatially constrained to a few specific subcellular regions, and that Ca(2+) release through InsP(3)R can occur at high local [Ca(2+)](i).     

Local Ca2+ rise near store operated Ca2+ channels inhibits cell coupling during capacitative Ca2+ influx

Using a new fluorescence imaging technique, LAMP, we recently reported that Ca(2+) influx through store operated Ca(2+) channels (SOCs) strongly inhibits cell coupling in primary human fibroblasts (HF) expressing Cx43. To understand the mechanism of inhibition, we studied the involvement of cytosolic pH (pH(i)) and Ca(2+)([Ca(2+)](i)) in the process by using fluorescence imaging and ion clamping techniques. During the capacitative Ca(2+) influx, there was a modest decline of pH(i) measured by BCECF. Decreasing pH(i) below neutral using thioacetate had little effect by itself on cell coupling, and concomitant pH(i) drop with thioacetate and bulk [Ca(2+)(i) rise with ionomycin was much less effective in inhibiting cell coupling than Ca(2+) influx. Moreover, clamping pH(i) with a weak acid and a weak base during Ca(2+) influx largely suppressed bulk pH(i) drop, yet the inhibition of cell coupling was not affected. In contrast, buffering [Ca(2+)(i) with BAPTA, but not EGTA, efficiently prevented cell uncoupling by Ca(2+) influx. We concluded that local Ca(2+) elevation subjacent to the plasma membrane is the primary cause for closing Cx43 channels during capacitative Ca(2+) influx. To assess how Ca(2+) influx affects junctional coupling mediated by other types of connexins, we applied the LAMP assay to Hela cells expressing Cx26. Capacitative Ca(2+) influx also caused a strong reduction of cell coupling, suggesting that the inhibitory effect by Ca(2+) influx may be a more general phenomenon.     

Calcium release flux underlying Ca2+ sparks of frog skeletal muscle.

An algorithm for the calculation of Ca2+ release flux underlying Ca2+ sparks (Blatter, L.A., J. Hüser, and E. Ríos. 1997. Proc. Natl. Acad. Sci. USA. 94:4176-4181) was modified and applied to sparks obtained by confocal microscopy in single frog skeletal muscle fibers, which were voltage clamped in a two-Vaseline gap chamber or permeabilized and immersed in fluo-3-containing internal solution. The performance of the algorithm was characterized on sparks obtained by simulation of fluorescence due to release of Ca2+ from a spherical source, in a homogeneous three-dimensional space that contained components representing cytoplasmic molecules and Ca2+ removal processes. Total release current, as well as source diameter and noise level, was varied in the simulations. Derived release flux or current, calculated by volume integration of the derived flux density, estimated quite closely the current used in the simulation, while full width at half magnitude of the derived release flux was a good monitor of source size only at diameters >0. 7 micrometers. On an average of 157 sparks of amplitude >2 U resting fluorescence, located automatically in a representative voltage clamp experiment, the algorithm reported a release current of 16.9 pA, coming from a source of 0.5 micrometer, with an open time of 6.3 ms. Fewer sparks were obtained in permeabilized fibers, so that the algorithm had to be applied to individual sparks or averages of few events, which degraded its performance in comparable tests. The average current reported for 19 large sparks obtained in permeabilized fibers was 14.4 pA. A minimum estimate, derived from the rate of change of dye-bound Ca2+ concentration, was 8 pA. Such a current would require simultaneous opening of between 8 and 60 release channels with unitary Ca2+ currents of the level recorded in bilayer experiments. Real sparks differ from simulated ones mainly in having greater width. Correspondingly, the algorithm reported greater spatial extent of the source for real sparks. This may again indicate a multichannel origin of sparks, or could reflect limitations in spatial resolution.     

Ca2+ signaling in the regulation of dendritic cell functions

Dendritic cells (DCs) are highly versatile antigen-presenting cells critically involved in both innate and adaptive immunity as well as maintenance of self-tolerance. DC function is governed by Ca(2+) signaling, which directs the DC responses to diverse antigens, including Toll-like receptor ligands, intact bacteria, and microbial toxins. Ca(2+)-sensitive DC functions include DC activation, maturation, migration, and formation of immunological synapses with T cells. Moreover, alterations of cytosolic Ca(2+) trigger immune suppression or switch off DC activity. Ca(2+) signals are generated by the orchestration of Ca(2+) transport processes across plasma, endoplasmic reticulum, and inner mitochondrial membrane. These processes include active pumping of Ca(2+), Ca(2+)/Na(+) antiport, and electrodiffusion through Ca(2+)-permeable channels or uniporters. Ca(2+) channels in the plasma membrane such as Ca(2+) release-activated Ca(2+) or L-type Ca(2+) channels are tightly regulated by the membrane potential which in turn depends on the activity of voltage-gated K(+) or Ca(2+)-activated nonselective cation channels. The rapidly growing knowledge on the function and regulation of these membrane transport proteins provides novel insight into pathophysiological mechanisms underlying dysfunction of the immune system and opens novel therapeutic opportunity to favorably influence the function of the immune system.     

Endothelial Ca2+ wavelets and the induction of myoendothelial feedback

When arteries constrict to agonists, the endothelium inversely responds, attenuating the initial vasomotor response. The basis of this feedback mechanism remains uncertain, although past studies suggest a key role for myoendothelial communication in the signaling process. The present study examined whether second messenger flux through myoendothelial gap junctions initiates a negative-feedback response in hamster retractor muscle feed arteries. We specifically hypothesized that when agonists elicit depolarization and a rise in second messenger concentration, inositol trisphosphate (IP(3)) flux activates a discrete pool of IP(3) receptors (IP(3)Rs), elicits localized endothelial Ca(2+) transients, and activates downstream effectors to moderate constriction. With use of integrated experimental techniques, this study provided three sets of supporting observations. Beginning at the functional level, we showed that blocking intermediate-conductance Ca(2+)-activated K(+) channels (IK) and Ca(2+) mobilization from the endoplasmic reticulum (ER) enhanced the contractile/electrical responsiveness of feed arteries to phenylephrine. Next, structural analysis confirmed that endothelial projections make contact with the overlying smooth muscle. These projections retained membranous ER networks, and IP(3)Rs and IK channels localized in or near this structure. Finally, Ca(2+) imaging revealed that phenylephrine induced discrete endothelial Ca(2+) events through IP(3)R activation. These events were termed recruitable Ca(2+) wavelets on the basis of their spatiotemporal characteristics. From these findings, we conclude that IP(3) flux across myoendothelial gap junctions is sufficient to induce focal Ca(2+) release from IP(3)Rs and activate a discrete pool of IK channels within or near endothelial projections. The resulting hyperpolarization feeds back on smooth muscle to moderate agonist-induced depolarization and constriction.     

Dynamics of signaling between Ca(2+) sparks and Ca(2+)- activated K(+) channels studied with a novel image-based method for direct intracellular measurement of ryanodine receptor Ca(2+) current

Ca(2+) sparks are highly localized cytosolic Ca(2+) transients caused by a release of Ca(2+) from the sarcoplasmic reticulum via ryanodine receptors (RyRs); they are the elementary events underlying global changes in Ca(2+) in skeletal and cardiac muscle. In smooth muscle and some neurons, Ca(2+) sparks activate large conductance Ca(2+)-activated K(+) channels (BK channels) in the spark microdomain, causing spontaneous transient outward currents (STOCs) that regulate membrane potential and, hence, voltage-gated channels. Using the fluorescent Ca(2+) indicator fluo-3 and a high speed widefield digital imaging system, it was possible to capture the total increase in fluorescence (i.e., the signal mass) during a spark in smooth muscle cells, which is the first time such a direct approach has been used in any system. The signal mass is proportional to the total quantity of Ca(2+) released into the cytosol, and its rate of rise is proportional to the Ca(2+) current flowing through the RyRs during a spark (I(Ca(spark))). Thus, Ca(2+) currents through RyRs can be monitored inside the cell under physiological conditions. Since the magnitude of I(Ca(spark)) in different sparks varies more than fivefold, Ca(2+) sparks appear to be caused by the concerted opening of a number of RyRs. Sparks with the same underlying Ca(2+) current cause STOCs, whose amplitudes vary more than threefold, a finding that is best explained by variability in coupling ratio (i.e., the ratio of RyRs to BK channels in the spark microdomain). The time course of STOC decay is approximated by a single exponential that is independent of the magnitude of signal mass and has a time constant close to the value of the mean open time of the BK channels, suggesting that STOC decay reflects BK channel kinetics, rather than the time course of [Ca(2+)] decline at the membrane. Computer simulations were carried out to determine the spatiotemporal distribution of the Ca(2+) concentration resulting from the measured range of I(Ca(spark)). At the onset of a spark, the Ca(2+) concentration within 200 nm of the release site reaches a plateau or exceeds the [Ca(2+)](EC50) for the BK channels rapidly in comparison to the rate of rise of STOCs. These findings suggest a model in which the BK channels lie close to the release site and are exposed to a saturating [Ca(2+)] with the rise and fall of the STOCs determined by BK channel kinetics. The mechanism of signaling between RyRs and BK channels may provide a model for Ca(2+) action on a variety of molecular targets within cellular microdomains.     

ECaC: the gatekeeper of transepithelial Ca2+ transport

The epithelial Ca(2+) channels (ECaCs) are primarily expressed in Ca(2+) transporting epithelia and represent a new family of Ca(2+) channels that belong to the superfamily of transient receptor potential (TRP) channels. Two members, namely ECaC1 and ECaC2, have been identified from kidney and intestine, respectively. These channels are the prime target for hormonal control of active Ca(2+) flux from the urine space or intestinal lumen to the blood compartment. This review covers the distinctive properties of these highly Ca(2+)-selective channels and highlights the implications for our understanding of the process of transepithelial Ca(2+) transport.     

The effect of residual Ca2+ on the stochastic gating of Ca2+-regulated Ca2+ channel models

Single-channel models of intracellular Ca(2+) channels such as the inositol 1,4,5-trisphosphate receptor and ryanodine receptor often assume that Ca(2+)-dependent transitions are mediated by a constant background [Ca(2+)] as opposed to a dynamic [Ca(2+)] representing the formation and collapse of a localized Ca(2+) domain. This assumption neglects the fact that Ca(2+) released by open intracellular Ca(2+) channels may influence subsequent gating through the processes of Ca(2+)-activation or -inactivation. We study the effect of such "residual Ca(2+)" from previous channel opening on the stochastic gating of minimal and realistic single-channel models coupled to a restricted cytoplasmic compartment. Using Monte Carlo simulation as well as analytical and numerical solution of a system of advection-reaction equations for the probability density of the domain [Ca(2+)] conditioned on the state of the channel, we determine how the steady-state open probability (p(open)) of single-channel models of Ca(2+)-regulated Ca(2+) channels depends on the time constant for Ca(2+) domain formation and collapse. As expected, p(open) for a minimal model including Ca(2+) activation increases as the domain time constant becomes large compared to the open and closed dwell times of the channel, that is, on average the channel is activated by residual Ca(2+) from previous openings. Interestingly, p(open) for a channel model that is inactivated by Ca(2+) also increases as a function of the domain time constant when the maximum domain [Ca(2+)] is fixed, because slow formation of the Ca(2+) domain attenuates Ca(2+)-mediated inactivation. Conversely, when the source amplitude of the channel is fixed, increasing the domain time constant leads to elevated domain [Ca(2+)] and decreased open probability. Consistent with these observations, a realistic De Young-Keizer-like IP(3)R model responds to residual Ca(2+) with a steady-state open probability that is a monotonic function of the domain time constant, though minimal models that include both Ca(2+)-activation and -inactivation show more complex behavior. We show how the probability density approach described here can be generalized for arbitrarily complex channel models and for any value of the domain time constant. In addition, we present a comparatively simple numerical procedure for estimating p(open) for models of Ca(2+)-regulated Ca(2+) channels in the limit of a very fast or very slow Ca(2+) domain. When the ordinary differential equation for the [Ca(2+)] in a restricted cytoplasmic compartment is replaced by a partial differential equation for the buffered diffusion of intracellular Ca(2+) in a homogeneous isotropic cytosol, we find the dependence of p(open) on the buffer time constant is qualitatively similar to the above-mentioned results.     

Mechanisms underlying heterogeneous Ca2+ sparklet activity in arterial smooth muscle

In arterial smooth muscle, single or small clusters of Ca(2+) channels operate in a high probability mode, creating sites of nearly continual Ca(2+) influx (called "persistent Ca(2+) sparklet" sites). Persistent Ca(2+) sparklet activity varies regionally within any given cell. At present, the molecular identity of the Ca(2+) channels underlying Ca(2+) sparklets and the mechanisms that give rise to their spatial heterogeneity remain unclear. Here, we used total internal reflection fluorescence (TIRF) microscopy to directly investigate these issues. We found that tsA-201 cells expressing L-type Cavalpha1.2 channels recapitulated the general features of Ca(2+) sparklets in cerebral arterial myocytes, including amplitude of quantal event, voltage dependencies, gating modalities, and pharmacology. Furthermore, PKCalpha activity was required for basal persistent Ca(2+) sparklet activity in arterial myocytes and tsA-201 cells. In arterial myocytes, inhibition of protein phosphatase 2A (PP2A) and 2B (PP2B; calcineurin) increased Ca(2+) influx by evoking new persistent Ca(2+) sparklet sites and by increasing the activity of previously active sites. The actions of PP2A and PP2B inhibition on Ca(2+) sparklets required PKC activity, indicating that these phosphatases opposed PKC-mediated phosphorylation. Together, these data unequivocally demonstrate that persistent Ca(2+) sparklet activity is a fundamental property of L-type Ca(2+) channels when associated with PKC. Our findings support a novel model in which the gating modality of L-type Ca(2+) channels vary regionally within a cell depending on the relative activities of nearby PKCalpha, PP2A, and PP2B.     

Reversed Na+/Ca2+ exchange contributes to Ca2+ influx and respiratory burst in microglia

Phagocytosis and the ensuing NADPH-mediated respiratory burst are important aspects of microglial activation that require calcium ion (Ca(2+)) influx. However, the specific Ca(2+) entry pathway(s) that regulates this mechanism remains unclear, with the best candidates being surface membrane Ca(2+)-permeable ion channels or Na(+)/Ca(2+) exchangers. In order to address this issue, we used quantitative real-time RT-PCR to assess mRNA expression of the Na(+)/Ca(2+) exchangers, Slc8a1-3/NCX1-3, before and after phagocytosis by rat microglia. All three Na(+)/Ca(2+) exchangers were expressed, with mRNA levels of NCX1 > NCX3 > NCX2, and were unaltered during the one hour phagocytosis period. We then carried out a biophysical characterization of Na(+)/Ca(2+) exchanger activity in these cells. To investigate conditions under which Na(+)/Ca(2+) exchange was functional, we used a combination of perforated patch-clamp analysis, fluorescence imaging of a Ca(2+) indicator (Fura-2) and a Na(+) indicator (SBFI), and manipulations of membrane potential and intracellular and extracellular ions. Then, we used a pharmacological toolbox to compare the contribution of Na(+)/Ca(2+) exchange with candidate Ca(2+)-permeable channels, to the NADPH-mediated respiratory burst that was triggered by phagocytosis. We find that inhibiting the reversed mode of the Na(+)/Ca(2+) exchanger with KB-R7943, dose dependently reduced the phagocytosis-stimulated respiratory burst; whereas, blockers of store-operated Ca(2+) channels or L-type voltage-gated Ca(2+) channels had no effect. These results provide evidence that Na(+)/Ca(2+) exchangers are potential therapeutic targets for reducing the bystander damage that often results from microglia activation in the damaged CNS.     

Ca2+-induced Ca2+ release from the endoplasmic reticulum amplifies the Ca2+ signal mediated by activation of voltage-gated L-type Ca2+ channels in pancreatic beta-cells

Stimulus-secretion coupling in pancreatic beta-cells involves membrane depolarization and Ca(2+) entry through voltage-gated L-type Ca(2+) channels, which is one determinant of increases in the cytoplasmic free Ca(2+) concentration ([Ca(2+)](i)). We investigated how the endoplasmic reticulum (ER)-associated Ca(2+) apparatus further modifies this Ca(2+) signal. When fura-2-loaded mouse beta-cells were depolarized by KCl in the presence of 3 mm glucose, [Ca(2+)](i) increased to a peak in two phases. The second phase of the [Ca(2+)](i) increase was abolished when ER Ca(2+) stores were depleted by thapsigargin. The steady-state [Ca(2+)](i) measured at 300 s of depolarization was higher in control cells compared with cells in which the ER Ca(2+) pools were depleted. The amount of Ca(2+) presented to the cytoplasm during depolarization as estimated from the integral of the increment in [Ca(2+)](i) over time (integralDelta[Ca(2+)](i).dt) was approximately 30% higher compared with that in the Ca(2+) pool-depleted cells. neo-thapsigargin, an inactive analog, did not affect [Ca(2+)](i) response. Using Sr(2+) in the extracellular medium and exploiting the differences in the fluorescence properties of Ca(2+)- and Sr(2+)-bound fluo-3, we found that the incoming Sr(2+) triggered Ca(2+) release from the ER. Depolarization-induced [Ca(2+)](i) response was not altered by, an inhibitor of phosphatidylinositol-specific phospholipase C, suggesting that stimulation of the enzyme by Ca(2+) is not essential for amplification of Ca(2+) signaling. [Ca(2+)](i) response was enhanced when cells were depolarized in the presence of 3 mm glucose, forskolin, and caffeine, suggesting involvement of ryanodine receptors in the amplification process. Pretreatment with ryanodine (100 microm) diminished the second phase of the depolarization-induced increase in [Ca(2+)](i). We conclude that Ca(2+) entry through L-type voltage-gated Ca(2+) channels triggers Ca(2+) release from the ER and that such a process amplifies depolarization-induced Ca(2+) signaling in beta-cells.     

A constitutive, transient receptor potential-like Ca2+ influx pathway in presynaptic nerve endings independent of voltage-gated Ca2+ channels and Na+/Ca2+ exchange

Calcium levels in the presynaptic nerve terminal are altered by several pathways, including voltage-gated Ca(2+) channels, the Na(+)/Ca(2+) exchanger, Ca(2+)-ATPase, and the mitochondria. The influx pathway for homeostatic control of [Ca(2+)](i) in the nerve terminal has been unclear. One approach to detecting the pathway that maintains internal Ca(2+) is to test for activation of Ca(2+) influx following Ca(2+) depletion. Here, we demonstrate that a constitutive influx pathway for Ca(2+) exists in presynaptic terminals to maintain internal Ca(2+) independent of voltage-gated Ca(2+) channels and Na(+)/Ca(2+) exchange, as measured in intact isolated nerve endings from mouse cortex and in intact varicosities in a neuronal cell line using fluorescence spectroscopy and confocal imaging. The Mg(2+) and lanthanide sensitivity of the influx pathway, in addition to its pharmacological and short hairpin RNA sensitivity, and the results of immunostaining for transient receptor potential (TRP) channels indicate the involvement of TRPC channels, possibly TRPC5 and TRPC1. This constitutive Ca(2+) influx pathway likely serves to maintain synaptic function under widely varying levels of synaptic activity.     

Measurement and modeling of Ca2+ waves in isolated rabbit ventricular cardiomyocytes

The time course and magnitude of the Ca(2+) fluxes underlying spontaneous Ca(2+) waves in single permeabilized ventricular cardiomyocytes were derived from confocal Fluo-5F fluorescence signals. Peak flux rates via the sarcoplasmic reticulum (SR) release channel (RyR2) and the SR Ca(2+) ATPase (SERCA) were not constant across a range of cellular [Ca(2+)] values. The Ca(2+) affinity (K(mf)) and maximum turnover rate (V(max)) of SERCA and the peak permeability of the RyR2-mediated Ca(2+) release pathway increased at higher cellular [Ca(2+)] loads. This information was used to create a computational model of the Ca(2+) wave, which predicted the time course and frequency dependence of Ca(2+) waves over a range of cellular Ca(2+) loads. Incubation of cardiomyocytes with the Ca(2+) calmodulin (CaM) kinase inhibitor autocamtide-2-related inhibitory peptide (300 nM, 30 mins) significantly reduced the frequency of the Ca(2+) waves at high Ca(2+) loads. Analysis of the Ca(2+) fluxes suggests that inhibition of CaM kinase prevented the increases in SERCA V(max) and peak RyR2 release flux observed at high cellular [Ca(2+)]. These data support the view that modification of activity of SERCA and RyR2 via a CaM kinase sensitive process occurs at higher cellular Ca(2+) loads to increase the maximum frequency of spontaneous Ca(2+) waves.     

Lack of evidence for presenilins as endoplasmic reticulum Ca2+ leak channels

Familial Alzheimer disease (FAD) is linked to mutations in the presenilin (PS) homologs. FAD mutant PS expression has several cellular consequences, including exaggerated intracellular Ca(2+) ([Ca(2+)](i)) signaling due to enhanced agonist sensitivity and increased magnitude of [Ca(2+)](i) signals. The mechanisms underlying these phenomena remain controversial. It has been proposed that PSs are constitutively active, passive endoplasmic reticulum (ER) Ca(2+) leak channels and that FAD PS mutations disrupt this function resulting in ER store overfilling that increases the driving force for release upon ER Ca(2+) release channel opening. To investigate this hypothesis, we employed multiple Ca(2+) imaging protocols and indicators to directly measure ER Ca(2+) dynamics in several cell systems. However, we did not observe consistent evidence that PSs act as ER Ca(2+) leak channels. Nevertheless, we confirmed observations made using indirect measurements employed in previous reports that proposed this hypothesis. Specifically, cells lacking PS or expressing a FAD-linked PS mutation displayed increased area under the ionomycin-induced [Ca(2+)](i) versus time curve (AI) compared with cells expressing WT PS. However, an ER-targeted Ca(2+) indicator revealed that this did not reflect overloaded ER stores. Monensin pretreatment selectively attenuated the AI in cells lacking PS or expressing a FAD PS allele. These findings contradict the hypothesis that PSs form ER Ca(2+) leak channels and highlight the need to use ER-targeted Ca(2+) indicators when studying ER Ca(2+) dynamics.     

Ca2+ modulation of Ca2+ release-activated Ca2+ channels is responsible for the inactivation of its monovalent cation current

The Ca(2+) release-activated Ca(2+) (CRAC) channel is the most well documented of the store-operated ion channels that are widely expressed and are involved in many important biological processes. However, the regulation of the CRAC channel by intracellular or extracellular messengers as well as its molecular identity is largely unknown. Specifically, in the absence of extracellular divalent cations it becomes permeable to monovalent cations with a larger conductance, however this monovalent cation current inactivates rapidly by an unknown mechanism. Here we found that Ca(2+) dissociation from a site on the extracellular side of the CRAC channel is responsible for the inactivation of its Na(+) current, and Ca(2+) occupancy of this site otherwise potentiates its Ca(2+) as well as Na(+) currents. This Ca(2+)-dependent potentiation is required for the normal functioning of CRAC channels.     

Metabotropic Ca2+ channel-induced calcium release in vascular smooth muscle

Contraction of vascular smooth muscle cells (VSMCs) depends on the rise of cytosolic [Ca(2+)] owing to either Ca(2+) influx through voltage-gated Ca(2+) channels of the plasmalemma or to receptor-mediated Ca(2+) release from the sarcoplasmic reticulum (SR). Although the ionotropic role of L-type Ca(2+) channels is well known, we review here data suggesting a new role of these channels in arterial myocytes. After sensing membrane depolarization Ca(2+) channels activate G proteins and the phospholipase C/inositol 1,4,5-trisphosphate (InsP(3)) pathway. Ca(2+) released through InsP(3)-dependent channels of the SR activates ryanodine receptors to amplify the cytosolic Ca(2+) signal, thus triggering arterial cerebral vasoconstriction in the absence of extracellular calcium influx. This metabotropic action of L-type Ca(2+) channels, denoted as calcium channel-induced Ca(2+) release, could have implications in cerebral vascular pharmacology and pathophysiology, because it can be suppressed by Ca(2+) channel antagonists and potentiated with small concentrations of extracellular vasoactive agents as ATP.