Monitoring the total available calmodulin concentration in intact cells over the physiological range in free Ca2+

We describe the design, characterization and application of a new genetically encoded fluorescent biosensor for intracellular detection of both free Ca(2+)-calmodulin and apocalmodulin, which together comprise the available calmodulin concentration. The biosensor binds both forms of calmodulin with an apparent Kd value of 3 microM, and has kinetic properties making it suitable for monitoring dynamic changes on a subsecond time scale. It can be used in conjunction with the fluorescent Ca(2+)-indicator, indo-1, allowing the available calmodulin and free Ca2+ concentrations to be monitored concurrently. We have determined an intracellular available calmodulin concentration of 8.8 +/- 2.2 microM under resting conditions in a human kidney cell line stably expressing the biosensor. Elevation of the intracellular free Ca2+ concentration by agonist, store-operated Ca(2+)-entry or ionophore results in Ca(2+)-dependent consumption of the available calmodulin. A plot of normalized values for the available calmodulin concentration versus the free Ca2+ concentration fits a consumption curve with a cooperativity coefficient of 1.8 and a [Ca2+]50 of 850 nM. There is no detectible binding of calmodulin to the biosensor above a free Ca2+ concentration of approximately 4 microM, consistent with an available calmodulin concentration < or = 200 nM under these conditions, and an overall excess of calmodulin-binding sites.     

TRPP2 channels regulate apoptosis through the Ca2+ concentration in the endoplasmic reticulum

Ca²⁺ is an important signalling molecule that regulates multiple cellular processes, including apoptosis. Although Ca²⁺ influx through transient receptor potential (TRP) channels in the plasma membrane is known to trigger cell death, the function of intracellular TRP proteins in the regulation of Ca²⁺‐dependent signalling pathways and apoptosis has remained elusive. Here, we show that TRPP2, the ion channel mutated in autosomal dominant polycystic kidney disease (ADPKD), protects cells from apoptosis by lowering the Ca²⁺ concentration in the endoplasmic reticulum (ER). ER‐resident TRPP2 counteracts the activity of the sarcoendoplasmic Ca²⁺ ATPase by increasing the ER Ca²⁺ permeability. This results in diminished cytosolic and mitochondrial Ca²⁺ signals upon stimulation of inositol 1,4,5‐trisphosphate receptors and reduces Ca²⁺ release from the ER in response to apoptotic stimuli. Conversely, knockdown of TRPP2 in renal epithelial cells increases ER Ca²⁺ release and augments sensitivity to apoptosis. Our findings indicate an important function of ER‐resident TRPP2 in the modulation of intracellular Ca²⁺ signalling, and provide a molecular mechanism for the increased apoptosis rates in ADPKD upon loss of TRPP2 channel function.     

WFS1 protein modulates the free Ca(2+) concentration in the endoplasmic reticulum

The WFS1 gene, encoding an endoplasmic reticulum (ER) membrane glycoprotein, is mutated in Wolfram syndrome characterized by diabetes mellitus and optic atrophy. Herein, Ca(2+) dynamics were examined in WFS1-knockdown and -overexpressing HEK293 cells. Studies using ER-targeted Ca(2+)-sensitive photoprotein aequorin demonstrated WFS1 protein to positively modulate ER Ca(2+) levels by increasing the rate of Ca(2+) uptake. Furthermore, Ca(2+) imaging with Fura-2 showed the magnitude of the store-operated Ca(2+) entry to parallel WFS1 expression levels. These data indicate that WFS1 protein participates in the regulation of cellular Ca(2+) homeostasis, at least partly, by modulating the filling state of the ER Ca(2+) store.     

Mitochondrial free Ca2+ concentration in living cells

Evidence has accrued during the past two decades that mitochondrial Ca²⁺ plays an important role in the regulation of numerous cell functions such as energy metabolism. This implies that mitochondrial Ca²⁺ transport systems might be able to relay the changes of cytosolic Ca²⁺ concentration ([Ca²⁺]_c) into mitochondrial matrix for regulating biochemical activities. To substantiate this idea, measurements of intramitochondrial free Ca²⁺ concentration ([Ca²⁺]_m) become essential. In this article, we review the results from recent studies attempting to measure [Ca²⁺]_m in living cells. In addition, the significance of each study is discussed.     

The relationship between the free concentrations of Ca2+ and Ca2+-calmodulin in intact cells

Using stably expressed fluorescent indicator proteins, we have determined for the first time the relationship between the free Ca2+ and Ca2+-calmodulin concentrations in intact cells. A similar relationship is obtained when the free Ca2+ concentration is externally buffered or when it is transiently increased in response to a Ca2+-mobilizing agonist. Below a free Ca2+ concentration of 0.2 microM, no Ca2+-calmodulin is detectable. A global maximum free Ca2+-calmodulin concentration of approximately 45 nM is produced when the free Ca2+ concentration exceeds 3 microM, and a half-maximal concentration is produced at a free Ca2+ concentration of 1 microM. Data for fractional saturation of the indicators suggest that the total concentration of calmodulin-binding proteins is approximately 2-fold higher than the total calmodulin concentration. We conclude that high-affinity calmodulin targets (Kd /= 100 nM) occurs only where free Ca2+-calmodulin concentrations can be locally enhanced.     

Bcl-2 and Ca2+ homeostasis in the endoplasmic reticulum

Recent data have revealed an unexpected role of Bcl-2 in modulating the steady-state levels and agonist-dependent fluxes of Ca(2+) ions. Direct monitoring of endoplasmic reticulum (ER) Ca(2+) concentration with recombinant probes reveals a lower state of filling in Bcl-2-overexpressing cells and a higher leak rate from the organelle. The broader set of indirect data using cytosolic probes reveals a more complex scenario, as in many cases no difference was detected in the Ca(2+) content of the intracellular pools. At the same time, Ca(2+) signals have been shown to affect important checkpoints of the apoptotic process, such as mitochondria, thus tuning the sensitivity of cells to various challenges. In this contribution, we will review (i) the data on the effect of Bcl-2 on [Ca(2+)](er), (ii) the functional significance of the Ca(2+)-signalling alteration and (iii) the current insight into the possible mechanisms of this effect.     

Ca2+ refilling of the endoplasmic reticulum is largely preserved albeit reduced Ca2+ entry in endothelial cells

In this study the relationship between the efficiency of endoplasmic reticulum (ER) Ca2+ refilling and the extent of Ca2+ entry was investigated in endothelial cells. ER and mitochondrial Ca2+ concentration were measured using genetically encoded Ca2+ sensors, while the amount of entering Ca2+ was controlled by varying either the extracellular Ca2+ or the electrical driving force for Ca2+ by changing the plasma membrane potential. In the absence of an agonist, ER Ca2+ replenishment was fully accomplished even if the Ca2+ concentration applied was reduced from 2 to 0.5mM. A similar strong efficiency of ER Ca2+ refilling was obtained under condition of plasma membrane depolarization. However, in the presence of histamine, ER Ca2+ refilling depended on mitochondrial Ca2+ transport and was more susceptible to membrane depolarization. Store-operated Ca2+ entry (SOCE), was strongly reduced under low Ca2+ and depolarizing conditions but increased if ER Ca2+ uptake was blocked or if ER Ca2+ was released continuously by IP(3). A correlation of the kinetics of ER Ca2+refilling with cytosolic Ca2+ signals revealed that termination of SOCE is a rapid event that is not delayed compared to ER refilling. Our data indicate that ER refilling occurs in priority to, and independently from the cytosolic Ca2+ elevation upon Ca2+ entry and that this important process is widely achieved even under conditions of diminished Ca2+entry.     

Mitochondrial Ca2+ uptake requires sustained Ca2+ release from the endoplasmic reticulum

We analyzed the role of inositol 1,4,5-trisphosphate-induced Ca(2+) release from the endoplasmic reticulum (ER) (i) in powering mitochondrial Ca(2+) uptake and (ii) in maintaining a sustained elevation of cytosolic Ca(2+) concentration ([Ca(2+)](c)). For this purpose, we expressed in HeLa cells aequorin-based Ca(2+)-sensitive probes targeted to different intracellular compartments and studied the effect of two agonists: histamine, acting on endogenous H(1) receptors, and glutamate, acting on co-transfected metabotropic glutamate receptor (mGluR1a), which rapidly inactivates through protein kinase C-dependent phosphorylation and thus causes transient inositol 1,4,5-trisphosphate production. Glutamate induced a transient [Ca(2+)](c) rise and drop in ER luminal [Ca(2+)] ([Ca(2+)](er)), and then the ER refilled with [Ca(2+)](c) at resting values. With histamine, [Ca(2+)](c) after the initial peak stabilized at a sustained plateau, and [Ca(2+)](er) decreased to a low steady-state value. In mitochondria, histamine evoked a much larger mitochondrial Ca(2+) response than glutamate ( approximately 15 versus approximately 65 microm). Protein kinase C inhibition, partly relieving mGluR1a desensitization, reestablished both the [Ca(2+)](c) plateau and the sustained ER Ca(2+) release and markedly increased the mitochondrial Ca(2+) response. Conversely, mitochondrial Ca(2+) uptake evoked by histamine was drastically reduced by very transient ( approximately 2-s) agonist applications. These data indicate that efficient mitochondrial Ca(2+) uptake depends on the preservation of high Ca(2+) microdomains at the mouth of ER Ca(2+) release sites close to mitochondria. This in turn depends on continuous Ca(2+) release balanced by Ca(2+) reuptake into the ER and maintained by Ca(2+) influx from the extracellular space.     

Akt kinase reducing endoplasmic reticulum Ca2+ release protects cells from Ca2+-dependent apoptotic stimuli

The proto-oncogene Akt is a potent inhibitor of apoptosis, and it is activated in many human cancers. A number of recent studies have highlighted the importance of the inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) in mediating calcium (Ca²⁺) transfer from the endoplasmic reticulum (ER) to the mitochondria in several models of apoptosis. Akt is a serine-threonine kinase and recent data indicate the IP3R as a target of its phosphorylation activity.Here we show that HeLa cells, overexpressing the constitutively active myristoylated/palmitylated AKT1 (m/p-AKT1), were found to have a reduced Ca²⁺ release from ER after stimulation with agonist coupled to the generation of IP3. In turn, this affected cytosolic and mitochondria Ca²⁺ response after Ca²⁺ release from the ER induced either by agonist stimulation or by apoptotic stimuli releasing Ca²⁺ from intracellular stores.Most importantly, this alteration of ER Ca²⁺ content and release, reduces significantly cellular sensitivity to Ca²⁺ mediated proapoptotic stimulation. These results reveal a primary role of Akt in shaping intracellular Ca²⁺ homeostasis, that may underlie its protective role against some proapoptotic stimuli.     

Ca2+ shuttling between endoplasmic reticulum and mitochondria underlying Ca2+ oscillations

Although many cell functions are regulated by Ca(2+) oscillations induced by a cyclic release of Ca(2+) from intracellular Ca(2+) stores, the pacemaker mechanism of Ca(2+) oscillations remains to be explained. Using green fluorescent protein-based Ca(2+) indicators that are targeted to intracellular Ca(2+) stores, the endoplasmic reticulum (ER) and mitochondria, we found that Ca(2+) shuttles between the ER and mitochondria in phase with Ca(2+) oscillations. Following agonist stimulation, Ca(2+) release from the ER generated the first Ca(2+) oscillation and loaded mitochondria with Ca(2+). Before the second Ca(2+) oscillation, Ca(2+) release from the mitochondria by means of the Na(+)/Ca(2+) exchanger caused a gradual increase in cytoplasmic Ca(2+) concentration, inducing a regenerative ER Ca(2+) release, which generated the peak of Ca(2+) oscillation and partially reloaded the mitochondria. This sequence of events was repeated until mitochondrial Ca(2+) was depleted. Thus, Ca(2+) shuttling between the ER and mitochondria may have a pacemaker role in the generation of Ca(2+) oscillations.     

Modulation of active Ca2+ uptake by the islet-cell endoplasmic reticulum.

The possible effects of calmodulin and cyclic AMP on active Ca2+ uptake by the islet-cell endoplasmic reticulum were investigated. Neither calmodulin nor cyclic AMP affected the rate of active Ca2+ uptake, or the steady-state filling capacity of the endoplasmic reticulum when measured in the absence of oxalate. Consistent with these results, calmodulin did not activate the Ca2+-stimulated ATPase activity associated with this cell fraction. During the course of these experiments., it was unexpectedly discovered that the rate of Ca2+ uptake, as well as the steady-state Ca2+ filling capacity of the endoplasmic reticulum, were markedly increased by unidentified factor(s) in the cytosol. This effect could be demonstrated by reconstitution of the membranes in cytosol, or by direct addition of fresh or dialysed cytosol to the Ca2+ uptake assays. The degree of activation by the cytosol indicates that the endoplasmic reticulum may play a prominent role in controlling beta-cell Ca2+ concentrations and that the unidentified activator(s) present in the cytosol may be involved in regulation of this function.     

Calreticulin, Ca2+-binding chaperon of the endoplasmic reticulum

The endoplasmic reticulum (ER) plays a vital role in many cellular processes, including Ca2+ storage and release. Calreticulin is a Ca2+-binding chaperon residing in ER. The protein is a key component of the quality control pathways in ER. In the ER lumen, calreticulin performs two major functions, works as a chaperon and regulates Ca2+ homeostasis. In cardiac muscle, calreticulin plays an important role in cardiac development and pathology.     

Glucose regulation of free Ca(2+) in the endoplasmic reticulum of mouse pancreatic beta cells

Free Ca(2+) was measured in organelles of individual mouse pancreatic beta cells loaded with the low affinity indicator furaptra. After removal of cytoplasmic indicator by controlled digitonin permeabilization the organelle Ca(2+) was located essentially in the endoplasmic reticulum (ER), >90% being sensitive to inhibition of sarco(endo)plasmic reticulum Ca(2+)-ATPases. The Ca(2+) accumulation in the ER of intact beta cells depended in a hyperbolic fashion on the glucose concentration with half-maximal and maximal filling at 5.5 and >20 mM, respectively. Also elevation of cytoplasmic Ca(2+) by K(+) depolarization significantly enhanced the Ca(2+) accumulation. In permeabilized beta cells 1-3 mM ATP caused rapid Ca(2+) filling of the ER reaching almost 500 microM. At 50 nM, Ca(2+) ER became half-maximally filled at 45 microM ATP, whereas only 3.5 microM ATP was required at 200 nM Ca(2+). Inositol 1,4,5-trisphosphate induced a rapid release of about 65% of the ER Ca(2+), and its precursor phosphatidylinositol 4,5-bisphosphate was found to slowly mobilize 75% by another mechanism. It is concluded that glucose is an efficient stimulator of Ca(2+) uptake in the ER of pancreatic beta cells both by increasing ATP and cytoplasmic Ca(2+). Because physiological concentrations of cytoplasmic ATP are in the mM range, Ca(2+) sequestration can be anticipated to be modulated by factors reducing its ATP sensitivity.     

Ca2+ regulation of interactions between endoplasmic reticulum chaperones

Casade Blue (CB), a fluorescent dye, was used to investigate the dynamics of interactions between endoplasmic reticulum (ER) lumenal chaperones including calreticulin, protein disulfide isomerase (PDI), and ERp57. PDI and ERp57 were labeled with CB, and subsequently, we show that the fluorescence intensity of the CB-conjugated proteins changes upon exposure to microenvironments of a different polarity. CD analysis of the purified proteins revealed that changes in the fluorescence intensity of CB-ERp57 and CB-PDI correspond to conformational changes in the proteins. Using this technique we demonstrate that PDI interacts with calreticulin at low Ca2+ concentration (below 100 microM), whereas the protein complex dissociates at >400 microM Ca2+. These are the Ca2+ concentrations reminiscent of Ca2+ levels found in empty or full ER Ca2+ stores. The N-domain of calreticulin interacts with PDI, but Ca2+ binding to the C-domain of the protein is responsible for Ca2+ sensitivity of the interaction. ERp57 also interacts with calreticulin through the N-domain of the protein. Initial interaction between these proteins is Ca2+-independent, but it is modulated by Ca2+ binding to the C-domain of calreticulin. We conclude that changes in ER lumenal Ca2+ concentration may be responsible for the regulation of protein-protein interactions. Calreticulin may play a role of Ca2+ "sensor" for ER chaperones via regulation of Ca2+-dependent formation and maintenance of structural and functional complexes between different proteins involved in a variety of steps during protein synthesis, folding, and post-translational modification.     

Release of Ca2+ from the endoplasmic reticulum contributes to Ca2+ signaling in Dictyostelium discoideum

Ca2+ responses to two chemoattractants, folate and cyclic AMP (cAMP), were assayed in Dictyostelium D. discoideum mutants deficient in one or both of two abundant Ca2+-binding proteins of the endoplasmic reticulum (ER), calreticulin and calnexin. Mutants deficient in either or both proteins exhibited enhanced cytosolic Ca2+ responses to both attractants. Not only were the mutant responses greater in amplitude, but they also exhibited earlier onsets, faster rise rates, earlier peaks, and faster fall rates. Correlations among these kinetic parameters and the response amplitudes suggested that key events in the Ca2+ response are autoregulated by the magnitude of the response itself, i.e., by cytosolic Ca2+ levels. This autoregulation was sufficient to explain the altered kinetics of the mutant responses: larger responses are faster in both mutant and wild-type cells in response to both folate (vegetative cells) and cAMP (differentiated cells). Searches of the predicted D. discoideum proteome revealed three putative Ca2+ pumps and four putative Ca2+ channels. All but one contained sequence motifs for Ca2+- or calmodulin-binding sites, consistent with Ca2+ signals being autoregulatory. Although cytosolic Ca2+ responses in the calnexin and calreticulin mutants are enhanced, the influx of Ca2+ from the extracellular medium into the mutant cells was smaller. Compared to wild-type cells, Ca2+ release from the ER in the mutants thus contributes more to the total cytosolic Ca2+ response while influx from the extracellular medium contributes less. These results provide the first molecular genetic evidence that release of Ca2+ from the ER contributes to cytosolic Ca2+ responses in D. discoideum.     

Identification of a calmodulin-regulated Ca2+-ATPase in the endoplasmic reticulum

A unique subfamily of calmodulin-dependent Ca2+-ATPases was recently identified in plants. In contrast to the most closely related pumps in animals, plasma membrane-type Ca2+-ATPases, members of this new subfamily are distinguished by a calmodulin-regulated autoinhibitor located at the N-terminal instead of a C-terminal end. In addition, at least some isoforms appear to reside in non-plasma membrane locations. To begin delineating their functions, we investigated the subcellular localization of isoform ACA2p (Arabidopsis Ca2+-ATPase, isoform 2 protein) in Arabidopsis. Here we provide evidence that ACA2p resides in the endoplasmic reticulum (ER). In buoyant density sucrose gradients performed with and without Mg2+, ACA2p cofractionated with an ER membrane marker and a typical "ER-type" Ca2+-ATPase, ACA3p/ECA1p. To visualize its subcellular localization, ACA2p was tagged with a green fluorescence protein at its C terminus (ACA2-GFPp) and expressed in transgenic Arabidopsis. We collected fluorescence images from live root cells using confocal and computational optical-sectioning microscopy. ACA2-GFPp appeared as a fluorescent reticulum, consistent with an ER location. In addition, we observed strong fluorescence around the nuclei of mature epidermal cells, which is consistent with the hypothesis that ACA2p may also function in the nuclear envelope. An ER location makes ACA2p distinct from all other calmodulin-regulated pumps identified in plants or animals.     

Ca 2+ outflow from sarcoplasmic reticulum vesicles during changes in membrane potential, Ca2+ concentration and pH

The concentration gradient Ca2+ outflux from the vesicles of the fragmented sarcoplasmic reticulum of rabbit skeletal muscles has been studied under conditions of the induced membrane potential, the concentrations of Ca2+ and H+ in the medium washing over the vesicles being different. The Ca2+ outflux from vesicles is shown to be the same with a decrease of the membrane potential from--80 down to -10 mV and gets higher with the zero and subsequent positive values of the latter. A significant intensification of the Ca2+ outflux from vesicles under the effect of external-vesicular Ca2+ has been observed at its concentration of 10(-5) M. Against this background of external-vesicular Ca2+ and zero value of the membrane potential either exogenous AMP or the pH increase from 6.5 up to 7.8 favour a release of more than 70% of passively accumulated Ca2+. The pH effect grows with a decrease in the external-vesicular concentration of Ca2+. A conclusion is drawn on the significance of protons in the regulation of the Ca2+ release from the sarcoplasmic reticulum.     

Uptake and release of Ca2+ by the endoplasmic reticulum contribute to the oscillations of the cytosolic Ca2+ concentration triggered by Ca2+ influx in the electrically excitable pancreatic B-cell.

The role of intracellular Ca2+ pools in oscillations of the cytosolic Ca2+ concentration ([Ca2+]c) triggered by Ca2+ influx was investigated in mouse pancreatic B-cells. [Ca2+]c oscillations occurring spontaneously during glucose stimulation or repetitively induced by pulses of high K+ (in the presence of diazoxide) were characterized by a descending phase in two components. A rapid decrease in [Ca2+]c coincided with closure of voltage-dependent Ca2+ channels and was followed by a slower phase independent of Ca2+ influx. Blocking the SERCA pump with thapsigargin or cyclopiazonic acid accelerated the rising phase of [Ca2+]c oscillations and increased their amplitude, which suggests that the endoplasmic reticulum (ER) rapidly takes up Ca2+. It also suppressed the slow [Ca2+]c recovery phase, which indicates that this phase corresponds to the slow release of Ca2+ that was taken up by the ER during the upstroke of the [Ca2+]c transient. Glucose promoted the buffering capacity of the ER and amplified the slow [Ca2+]c recovery phase. The slow phase induced by high K+ pulses was not affected by modulators of Ca2+- or inositol 1,4,5-trisphosphate-induced Ca2+ release, did not involve a depolarization-induced Ca2+ release, and was also observed at the end of a rapid rise in [Ca2+]c triggered from caged Ca2+. It is attributed to passive leakage of Ca2+ from the ER. We suggest that the ER displays oscillations of the Ca2+ concentration ([Ca2+]ER) concomitant and parallel to [Ca2+]c. The observation that thapsigargin depolarizes the membrane of B-cells supports the proposal that the degree of Ca2+ filling of the ER modulates the membrane potential. Therefore, [Ca2+]ER oscillations occurring during glucose stimulation are likely to influence the bursting behavior of B-cells and eventually [Ca2+]c oscillations.     

Calreticulin, a multifunctional Ca2+ binding chaperone of the endoplasmic reticulum.

Calreticulin is a ubiquitous endoplasmic reticulum Ca2+ binding chaperone. The protein has been implicated in a variety of diverse functions. Calreticulin is a lectin-like chaperone and, together with calnexin, it plays an important role in quality control during protein synthesis, folding, and posttranslational modification. Calreticulin binds Ca2+ and affects cellular Ca2+ homeostasis. The protein increases the Ca2+ storage capacity of the endoplasmic reticulum and modulates the function of endoplasmic reticulum Ca2+-ATPase. Calreticulin also plays a role in the control of cell adhesion and steroid-sensitive gene expression. Recently, the protein has been identified and characterized in higher plants but its precise role in plant cells awaits further investigation.     

On the roles of Ca2+ diffusion, Ca2+ buffers, and the endoplasmic reticulum in IP3-induced Ca2+ waves.

We have investigated the effects of Ca2+ diffusion, mobile and stationary Ca2+ buffers in the cytosol, and Ca2+ handling by the endoplasmic reticulum on inositol 1,4,5-trisphosphate-induced Ca2+ wave propagation. Rapid equilibration of free and bound Ca2+ is used to describe Ca2+ sequestration by buffers in both the cytosol and endoplasmic reticulum (ER) lumen. Cytosolic Ca2+ regulation is based on a kinetic model of the inositol 1,4,5-trisphosphate (IP3) receptor of De Young and Keizer that includes activation and inhibition of the IP3 receptor Ca2+ channel in the ER membrane and SERCA Ca2+ pumps in the ER. Diffusion of Ca2+ in the cytosol and the ER and the breakdown and diffusion of IP3 are also included in our calculations. Although Ca2+ diffusion is severely limited because of buffering, when conditions are chosen just below the threshold for Ca2+ oscillations, a pulse of IP3 or Ca2+ results in a solitary trigger wave that requires diffusion of Ca2+ for its propagation. In the oscillatory regime repetitive wave trains are observed, but for this type of wave neither the wave shape nor the speed is strongly dependent on the diffusion of Ca2+. Local phase differences lead to waves that are predominately kinematic in nature, so that the wave speed (c) is related to the wavelength (lambda) and the period of the oscillations (tau) approximately by the formula c = lambda/tau. The period is determined by features that control the oscillations, including [IP3] and pump activity, which are related to recent experiments. Both solitary waves and wave trains are accompanied by a Ca2+ depletion wave in the ER lumen, similar to that observed in cortical preparations from sea urchin eggs. We explore the effect of endogenous and exogenous Ca2+ buffers on wave speed and wave shape, which can be explained in terms of three distinct effects of buffering, and show that exogenous buffers or Ca2+ dyes can have considerable influence on the amplitude and width of the waves.