Limitations of the whole cell patch clamp technique in the control of intracellular concentrations.

Recent experimental studies (Pusch and Neher, 1988) and theoretical studies (Oliva et al., 1988) have found that the pipette tip is a significant barrier to diffusion in the whole cell patch clamp configuration. In this paper, we extend the theoretical analysis of fluxes between the pipette and cell to include transmembrane fluxes. The general conclusions are: (a) within the pipette, ion fluxes are driven primarily by diffusion rather than voltage gradients. (b) At steady state there is a concentration difference between the bulk pipette and intracellular solution that is described by delta c = jRp/Dp, where delta c = 1 mM for a flux, j = 1 fmol/s, through a pipette of resistance, Rp = 1 M omega, filled with a solution of resistivity, p = 100 omega --cm, given a solute diffusion coefficient, D = 10(-5) cm2/s. (c) The time to steady state is always accelerated by membrane transport, regardless of the direction of transport. We apply our analysis to the measurement of transport by the Na/K pump and Na/Ca exchanger in cells from the ventricles of mammalian heart. We find that the binding curve for intracellular Na+ to the Na/K pump will appear significantly less steep and more linear if one does not correct for the concentration difference between intracellular and pipette Na+. Similar shifts in the binding curve for extracellular Na+ to the Na/Ca exchanger can occur due to depletion of intracellular Ca(+)+ when the exchanger is stimulated. Lastly, in Appendix we analyze the effects of mobile and fixed intracellular buffers on the movement of Ca(+)+ between the pipette and cell. Fixed buffers greatly slow the time for equilibration of pipette and intracellular Ca(+)+. Mobile buffers act like a shuttle system, as they carry Ca(+)+ from pipette to cell then diffuse back when they are empty. Vigorous transport by the Na/Ca exchanger depletes mobile buffered calcium, thus stimulating diffusion from the pipette to match the rate of Ca(+)+ transport. Moreover, we find that binding of Ca(+)+ to the exchanger can be affected by the mobile buffer.     

Endogenous buffers limit the spread of free calcium in hair cells.

Mobile Ca2+ buffers in hair cells have been postulated to play a dual role. On one hand, they carry incoming Ca2+ away from synaptic areas, allowing synapses to be rapidly reset. On the other hand, they limit the spread of free Ca2+ into the cell, preventing cross-talk between different pathways that employ Ca2+ as a second messenger. We have obtained evidence for such mobile Ca2+ buffers in hair cells by comparing the patterns of Ca2+-induced fluo-3 fluorescence under whole-cell and perforated-patch recording conditions. Fluorescent signals under perforated-patch conditions are relatively weak and are limited to the immediate vicinity of the membrane. These observations can be explained by a diffusion-reaction scheme that, in addition to Ca2+ and fluo-3, incorporates endogenous fixed and mobile Ca2+ buffers. Our experiments also suggest that the mobility of the endogenous buffer might be higher than previously thought. A high buffer mobility is expected to enhance the cell's ability to rapidly modulate transmitter release.     

Diffusion around a cardiac calcium channel and the role of surface bound calcium.

The diffusion of Ca as it converges to the external mouth of a Ca channel is examined. Diffusional limitation on Ca ions entering Ca channels during current flow, cause local extracellular Ca depletions. Such extracellular Ca depletions have been reported in cardiac muscle. The cardiac sarcolemma has a large number of low-affinity Ca binding sites that can buffer these local Ca depletions. For a hemisphere of extracellular space (of radius less than 0.33 microns) centered on the external mouth of a Ca channel the amount of Ca bound at the membrane surface exceeds that which is free within the associated hemisphere. The ratio of bound Ca/free Ca increases as r decreases, such that the [Ca] nearest the Ca channel is the most strongly buffered by sarcolemmal bound Ca. It is demonstrated that Ca ions coming from these sarcolemmal Ca binding sites contribute quantitatively to the integrated Ca current. The electric field generated by the local depletion of Ca near the channel mouth has little impact on the extent of Ca depletion, but if an additional electric field exists at the mouth of the channel, Ca depletion can be significantly altered. Other low-affinity Ca binding sites in the interstitium may also contribute to the buffering of extracellular Ca. The complex geometry of the extracellular space in cardiac muscle (e.g., transverse tubules and restrictions of extracellular space between cells) increases both the predicted Ca depletions (in the absence of binding) and the bound/free ratio. Thus, the impact of this surface Ca binding is greatly increased. By considering arrays of Ca channels in transverse tubules or in parallel planes (e.g., membranes of neighboring cells), extracellular Ca depletions are predicted which agree with those measured experimentally. Membrane Ca binding may also be expected to buffer increases in [Ca] around the inner mouth of Ca channels. It is demonstrated that in the absence of other intracellular systems most of the Ca entering the cell via Ca channels might be expected to be bound to the inner sarcolemmal surface. It is concluded that surface Ca binding may have a substantial impact on the processes of extracellular Ca depletion (and intracellular Ca accumulation).     

A theoretical study of calcium microdomains in turtle hair cells.

Confocal imaging has revealed microdomains of intracellular free Ca2+ in turtle hair cells evoked by depolarizing pulses and has delineated factors affecting the growth and dissipation of such domains. However, imaging experiments have limited spatial and temporal resolution. To extend the range of the results we have developed a three-dimensional model of Ca2+ diffusion in a cylindrical hair cell, allowing part of the Ca2+ influx to occur over a small circular region (radius 0.125-1.0 micron) representing a high-density array of voltage-dependent channels. The model incorporated experimental information about the number of channels, the fixed and mobile Ca2+ buffers, and the Ca2+ extrusion mechanism. A feature of the calculations was the use of a variable grid size depending on the proximity to the Ca2+ channel cluster. The results agreed qualitatively with experimental data on the localization of the Ca2+ transients, although the experimental responses were smaller and slower, which is most likely due to temporal and spatial averaging in the imaging. The model made predictions about 1) the optimal Ca2+ channel number and density within a cluster, 2) the conditions to ensure independence of neighboring clusters, and 3) the influence of the Ca2+ buffers on the kinetics and localization of the microdomains. We suggest that an increase in the mobile Ca2+ buffer concentration in high-frequency hair cells (which possess a larger number of release sites) would allow lower amplitude and faster Ca2+ responses and promote functional independence of the sites.     

Buffering of calcium in the vicinity of a channel pore.

The function of calcium entry or release channels is often modulated by the cytosolic free calcium concentration. When such channels are studied in isolation, calcium buffer solutions are usually used to control the free calcium at the cytosolic face of the channel. Such solutions are generally formulated on the basis of equilibrium considerations. We calculate the gradient of [Ca2+] in the vicinity of a channel pore, in the presence of such buffers. We find that the effective degree of buffering near the pore is markedly affected by kinetic considerations. Commonly used EGTA solutions are completely ineffective in buffering [Ca2+] within macromolecular distances of the pore. In order to achieve useful buffering, the fastest buffers (e.g. BAPTA derivatives) must be used, in concentrations very much higher than those conventionally employed. Because of the diffusion limit on the maximum rate of binding of calcium to the buffer ligand, it is physically impossible to achieve good control of [Ca2+] at cytosolic levels at distances of less than a few nm from a pore conducting pico-ampere calcium current.     

Buffers and oscillations in intracellular Ca2+ dynamics

I model the behavior of intracellular Ca(2+) release with high buffer concentrations. The model uses a spatially discrete array of channel clusters. The channel subunit dynamics is a stochastic representation of the DeYoung-Keizer model. The calculations show that the concentration profile of fast buffer around an open channel is more localized than that of slow buffers. Slow buffers allow for release of larger amounts of Ca(2+) from the endoplasmic reticulum and hence bind more Ca(2+) than fast buffers with the same dissociation constant and concentration. I find oscillation-like behavior for high slow buffer concentration and low Ca(2+) content of the endoplasmic reticulum. High concentration of slow buffer leads to oscillation-like behavior by repetitive wave nucleation for high Ca(2+) content of the endoplasmic reticulum. Localization of Ca(2+) release by slow buffer, as used in experiments, can be reproduced by the modeling approach.     

Development and dissipation of Ca(2+) gradients in adrenal chromaffin cells

We used pulsed laser imaging to measure the development and dissipation of Ca(2+) gradients evoked by the activation of voltage-sensitive Ca(2+) channels in adrenal chromaffin cells. Ca(2+) gradients appeared rapidly (<5 ms) upon membrane depolarization and dissipated over several hundred milliseconds after membrane repolarization. Dissipation occurred with an initial fast phase, as the steep gradient near the membrane collapsed, and a slower phase as the remaining shallow gradient dispersed. Inhibition of active Ca(2+) uptake by the endoplasmic reticulum (thapsigargin) and mitochondria (carbonylcyanide p-trifluoro-methoxyphenylhydrazone/oligomycin) had no effect on the size of Ca(2+) changes or the rate of gradient dissipation, suggesting that passive endogenous Ca(2+) buffers are responsible for the slow Ca(2+) redistribution. We used a radial diffusion model incorporating Ca(2+) diffusion and binding to intracellular Ca(2+) buffers to simulate Ca(2+) gradients. We included a 3D optical sectioning model, simulating the effects of out-of-focus light, to allow comparison with the measured gradients. Introduction of a high-capacity immobile Ca(2+) buffer, with a buffer capacity on the order of 1000 and appropriate affinity and kinetics, approximated the size of the Ca(2+) increases and rate of dissipation of the measured gradients. Finally, simulations without exogenous buffer suggest that the Ca(2+) signal due to Ca(2+) channel activation is restricted by the endogenous buffer to a space less than 1 microm from the cell membrane.     

Mobilization of intracellular calcium by extracellular ATP and by calcium ionophores in the Ehrlich ascites-tumour cell

We have studied the changes of the intracellular free calcium concentration ([Ca2+]i) effected by external ATP, which induces formation of inositol trisphosphate, and by the divalent cation ionophores ionomycin and A23187. Both, ATP (40 microM) and ionophores (1-80 mumol/l cells ionomycin; 20-400 mumol/l cells A23187), produced a transient rise of [Ca2+]i which reached its maximum within 15-30 s and declined near resting values (about 200 nM) within 1-3 min. When the [Ca2+]i peak surpassed 500 nM a transient cell shrinkage due to simultaneous activation of Ca2+-dependent K+ and Cl- channels was also observed. The cell response was similar in medium containing 1 mM Ca2+ and in Ca2+-free medium, suggesting that the Ca mobilized to the cytosol comes preferently from the intracellular stores. Treatment with low doses of ionophore (1 mumol/l cells for ionomycin; 20 mumol/l cells for A23187) depressed the response to a subsequent treatment, either with ionophore or with ATP. Treatment with ATP did also inhibit the subsequent response to ionophore, but in this case the inhibition was dependent on time, the stronger the shorter the interval between both treatments. This result suggests that the permeabilization of Ca stores by ATP is transient and that Ca can be taken up again by the intracellular stores. Refill was most efficient when Ca2+ was present in the incubation medium. Addition of either ATP or ionomycin (1-25 mumol/l cells) to cells incubated in medium containing 1 mM Ca2+ decreased drastically the total cell Ca content during the following 3 min of incubation. In the case of ATP the total cell levels of Ca returned to the initial values after 7-15 min, whereas in the case of the ionophore they remained decreased during the whole incubation period. These results indicate that Ca released from the intracellular stores by either ATP or ionophores is quickly extruded by active mechanisms located at the plasma membrane. They also suggest that, under the conditions studied here, with 1 mM Ca2+ outside, the Ca-mobilizing effect of ionophores is stronger in endomembranes than in the plasma membrane.     

Intracellular calcium changes in Balanus photoreceptor. A study with calcium ion-selective electrodes and Arsenazo III

Intracellular Ca2+ concentration (Cai) in the dark and during light stimulation, was measured in Balanus photoreceptors with Ca2+ ion-selective electrodes (Ca-ISE) and Arsenazo III absorbance changes (AIII). The average basal Cai of 17 photoreceptors in darkness was 300 +/- 160 nM determined with liquid ion-exchanger (t-HDOPP) Ca-ISE. Ca-ISE measurements indicated that light increased Cai by 700 nM (average), whereas AIII indicated an average change of 450 nM. The time course of AIII absorbance changes matched the time course of changes in the receptor potential more closely than did the Ca-ISE. Changes in Cai were graded with light intensity but the change in Cai was much greater for a decade change in intensity at high light intensity than at low intensity. The peak light induced conductance change of voltage clamped cells had a relationship to light intensity similar to that of the change in Cai. The peak Cai level measured with Ca-ISE was in good agreement with the free Ca2+ concentration of injected buffer solutions. Control Cai levels were usually restored within 5 min following injection of Ca2+ buffers. Injection of Ca2+ buffers with free Ca2+ of 0.6 microM produced a membrane depolarization. Larger increases in Cai (greater than microM) produced by injection of CaCl2 or release of Ca2+ from injected buffers by acidifying the cell, produced a pronounced membrane hyperpolarization. Increasing Cai with all of these techniques reduced the amplitude of the receptor potential. The time course of the receptor potential recovery was usually similar to that of Cai recovery.     

Properties of a model of Ca(++)-dependent vesicle pool dynamics and short term synaptic depression

We explore the properties of models of synaptic vesicle dynamics, in which synaptic depression is attributed to depletion of a pool of release-ready vesicles. Two alternative formulations of the model allow for either recruitment of vesicles from an unlimited reserve pool (vesicle state model) or for recovery of a fixed number of release sites to a release-ready state (release-site model). It is assumed that, following transmitter release, the recovery of the release-ready pool of vesicles is regulated by the intracellular free Ca(++) concentration, [Ca(++)](i). Considering the kinetics of [Ca(++)](i) after single presynaptic action potentials, we show that pool recovery can be described by two distinct kinetic components. With such a model, complex kinetic and steady-state properties of synaptic depression as found in several types of synapses can be accurately described. However, the specific assumption that enhanced recovery is proportional to [Ca(++)](i), as measured with Ca(++) indicator dyes, is not confirmed by experiments at the calyx of Held, in which [Ca(++)](i)-homeostasis was altered by adding low concentrations of the exogenous Ca(++) buffer, fura-2, to the presynaptic terminal. We conclude that synaptic depression at the calyx of Held is governed by localized, near membrane [Ca(++)](i) signals not visible to the indicator dye, or else by an altogether different mechanism. We demonstrate that, in models in which a Ca(++)-dependent process is linearly related to [Ca(++)](i), the addition of buffers has only transient but not steady-state consequences.     

Calcium dependence of bleb formation and cell death in hepatocytes

Calcium dependence of bleb formation and cell death was evaluated in rat hepatocytes following ATP depletion by metabolic inhibition with KCN and iodoacetate ('chemical hypoxia'). Cytosolic free Ca2+ was measured in single cells by ratio imaging of Fura-2 fluorescence using multiparameter digitized video microscopy. Cells formed surface blebs within 10 to 20 minutes after chemical hypoxia and most cells lost viability within an hour. An increase of cytosolic free Ca2+ was not required for bleb formation to occur. One to a few minutes prior to the onset of cell death, free Ca2+ increased rapidly in high Ca2+ buffer (1.2 mM) but not in low Ca2+ buffer (less than 1 microM). In either buffer, the rate of cell killing was the same. As the onset of cell death was approached in both high and low Ca2+ buffers, Fura-2 began to leak from the cells at an accelerating rate indicating rapidly increasing plasma membrane permeability. In high Ca2+ buffer, cytosolic free Ca2+ increased in parallel with dye leakage. No regional changes in cytosolic free Ca2+ were observed during this metastable period of increased membrane permeability. In many experiments, actual rupture of cell surface blebs could be observed which led to micron-size discontinuities of the cell surface and cell death. We conclude that a metastable period characterized by increasing plasma membrane permeability marked the onset of cell death in cultured hepatocytes which culminated in rupture of a cell surface bleb. An increase of cytosolic free Ca2+ was not required for the metastable state to develop or cell death to occur.     

Two-dimensional determination of the cellular Ca2+ binding in bovine chromaffin cells.

The spatiotemporal profile of intracellular calcium signals is determined by the flux of calcium ions across different biological membranes as well as by the diffusional mobility of calcium and different calcium buffers in the cell. To arrive at a quantitative understanding of the determinants of these signals, one needs to dissociate the flux contribution from the redistribution and buffering of calcium. Since the cytosol can be heterogeneous with respect to its calcium buffering property, it is essential to assess this property in a spatially resolved manner. In this paper we report on two different methods to estimate the cellular calcium binding of bovine adrenal chromaffin cells. In the first method, we use voltage-dependent calcium channels as a source to generate calcium gradients in the cytosol. Using imaging techniques, we monitor the dissipation of these gradients to estimate local apparent calcium diffusion coefficients and, from these, local calcium binding ratios. This approach requires a very high signal-to-noise ratio of the calcium measurement and can be used when well-defined calcium gradients can be generated throughout the cell. In the second method, we overcome these problems by using calcium-loaded DM-nitrophen as a light-dependent calcium source to homogeneously and quantitatively release calcium in the cytosol. By measuring [Ca2+] directly before and after the photorelease process and knowing the total amount of calcium being released photolytically, we get an estimate of the fraction of calcium ions which does not appear as free calcium and hence must be bound to either the indicator dye or the endogenous calcium buffer. This finally results in a two-dimensional map of the distribution of the immobile endogenous calcium buffer. We did not observe significant variations of the cellular calcium binding at a spatial resolution of approximately 2 micron. Furthermore, the calcium binding is not reduced by increasing the resting [Ca2+] to levels as high as 1.1 microM. This is indicative of a low calcium affinity of the corresponding buffers and is in agreement with a recent report on the affinity of these buffers (Xu, T., M. Naraghi, H. Kang, and E. Neher. 1997. Biophys. J. 73:532-545). In contrast to the homogeneous distribution of the calcium buffers, the apparant calcium diffusion coefficient did show inhomogeneities, which can be attributed to restricted diffusion at the nuclear envelope and to rim effects at the cell membrane.     

Bicarbonate and other buffer systems can enhance the rate of H+ diffusion through mucus in vitro.

The effect of various diffusible buffers on mucus H+ permeability, and in particular the potency of the HCO3-/CO2 buffer system relative to other selected buffers is reported here. The diffusional resistance of mucus and water was demonstrated to be dependent on buffer concentration, and the contrast between the two types of layer was most pronounced for low DH+ values near neutrality. This concentration dependence was most marked with mucus layers in the buffer systems investigated. Furthermore, the nature and pKa values of the diffusible buffer systems used in this study had a profound effect on measured DH+. The effect was particularly striking in the case of HCO3- buffer with mucus. Possible implications of these in vitro findings in mucosal protection from acid are discussed.     

Cryopreservation of dog semen: the effects of Equex STM paste on plasma membrane fluidity and the control of intracellular free calcium

Understanding cryoinjury of dog spermatozoa is crucial to preserving fertilizing ability. This study examined flow cytometric indicators of sperm function to explore the reported benefits of Equex STM paste. The motility of cryopreserved spermatozoa immediately and 1h after thawing was higher in the extender containing 0.5% Equex; no significant differences between the two extenders were observed regarding viability, acrosomal integrity and intracellular Ca(2+) concentration. The proportion of spermatozoa having high membrane fluidity increased significantly post-thawing. The interaction between time after thawing and treatment was significant for plasma membrane fluidity. Dilution in a commercial diluent for transport before processing caused a significant increase in intracellular Ca(2+), which may affect functional survival. No significant difference with or without Equex was detected in plasma membrane fluidity. However, a significant interaction between Equex and dogs was detected. A significant decrease in intracellular Ca(2+) was detected in the live cell population both after dilution in Andersen's buffer and again after cooling and equilibration. One hour post-thaw, the proportion of live spermatozoa with high calcium concentration increased to a similar proportion as that seen in diluted semen; the interaction between diluent and dog was significant. The results suggest that Equex in the diluent benefited motility after cryopreservation. Live spermatozoa with high intracellular Ca(2+) after cryopreservation seem to have a favoured survival in the first hour after thawing. Nevertheless, survival after cryopreservation was severely compromised, explaining the relatively poor fertility of cryopreserved dog semen.     

The recovery of human polymorphonuclear leucocytes from sublytic complement attack is mediated by changes in intracellular free calcium.

Using polymorphonuclear leucocyte-erythrocyte ghost hybrids entrapping the calcium-activated photoprotein obelin, we have demonstrated that sublytic amounts of the complement membrane attack complex induce a rapid but transient increase in intracellular free calcium ion concentration ([Ca2+]i). This increase in [Ca2+]i occurs prior to, and is required for, rapid removal of membrane attack complexes from the cell surface. The increase in [Ca2+]i is not only due to increased influx from outside the cell, but also results from mobilization of intracellular stores. The possible mechanism of mobilization of calcium, and the importance of an increase in [Ca2+]i as a mediator of recovery processes in nucleated cells, are discussed.     

Dynamics and calcium sensitivity of the Ca2+/myristoyl switch protein hippocalcin in living cells

Hippocalcin is a neuronal calcium sensor protein that possesses a Ca2+/myristoyl switch allowing it to translocate to membranes. Translocation of hippocalcin in response to increased cytosolic [Ca2+] was examined in HeLa cells expressing hippocalcin-enhanced yellow fluorescent protein (EYFP) to determine the dynamics and Ca2+ affinity of the Ca2+/myristoyl switch in living cells. Ca2+-free hippocalcin was freely diffusible, as shown by photobleaching and use of a photoactivable GFP construct. The translocation was dependent on binding of Ca2+ by EF-hands 2 and 3. Using photolysis of NP-EGTA, the maximal kinetics of translocation was determined (t1/2 = 0.9 s), and this was consistent with a diffusion driven process. Low intensity photolysis of NP-EGTA produced a slow [Ca2+] ramp and revealed that translocation of hippocalcin-EYFP initiated at around 180 nM and was half maximal at 290 nM. Histamine induced a reversible translocation of hippocalcin-EYFP. The data show that hippocalcin is a sensitive Ca2+ sensor capable of responding to increases in intracellular Ca2+ concentration over the narrow dynamic range of 200-800 nM free Ca2+.     

The role of electro-mechanical and reaction-diffusion systems in the intra-neuron processing of information: effect of in-flow of calcium and intracellular calcium on cAMP activity

Influx of calcium ions cannot control a generatory potential induced by the intraneuronal system because calcium ions enter the cell during impulses. These impulses are the result of problem solving and must not influence directly the generatory potential. Therefore cAMP and not calcium controls the permeability of sodium and potassium channels from the inside of the neuron. However the calcium ions and membrane potential of mitochondria affect the impact of cAMP injections. An increase in the intracellular concentration of free Ca2+ induced by the injection of Ca-EGTA buffer with 5.10(-7) M free Ca2+, electric excitation, uncouplers of oxidative phosphorylation or arsenate leads to an increase of cAMP-dependent depolarization and the inward current. The injection of Ca-EGTA buffer with 10(-5) M free Ca2+ and drop in [Ca2+]in by EGTA as well as generation of impulses after cAMP injection decrease the cAMP effect. As rise in [Ca2+]in activates phosphodiesterase and uncouples oxidative phosphorylation, and vanadate in contrast to arsenate suppresses the cAMP effect, a hypothesis is advanced that activating effect of calcium on cAMP action is associated with neuron deenergization.     

Calcium clamp of the intracellular environment

Quantitative analysis of the effects of calcium on cell function requires methods for altering intracellular free Ca in a precise and reproducible manner. Microinjection of Ca is very unreliable largely because of the powerful Ca-binding properties of cytoplasm. Much more satisfactory are microinjection of Ca-buffers - provided enough buffer is introduced - and various forms of intracellular dialysis and perfusion which permit full equilibration of the cell interior with a defined artificial intracellular environment.     

Effect of ethanol on troponin calcium binding

The Chelex resin method was found to be suitable for studying drug effects on Ca2+ binding of proteins. In comparison to conventional dialysis techniques, the Chelex method has the following advantages: Ca2+-EGTA buffer is not necessary, free Ca2+ concentration as low as 10(-9) M can be determined directly, and the reaction is complete in 30 min, thus creating fewer problems with protein denaturation at elevated temperatures. Methods to cope with problems inherent to this assay, such as the excluded volume effect of the resin and protein adsorption by the resin are described. The validity of the method was confirmed by the measurements of Ca2+ binding of troponin in the presence and absence of Mg2+. Using this method, it was demonstrated that ethanol concentration as high as 25% does not influence the Ca2+ binding of troponin.     

Analytical steady-state solution to the rapid buffering approximation near an open Ca2+ channel.

We derive an analytical steady-state solution for the Ca2+ profile near an open Ca2+ channel based on a transport equation which describes the buffered diffusion of Ca2+ in the presence of rapid stationary and mobile Ca2+ buffers (Wagner and Keizer, 1994). This steady-state rapid buffering approximation gives an upper bound on local Ca2+ elevations such as Ca2+ puffs or sparks when conditions for the validity of the rapid buffering approximation are met and is an alternative to approximations that assume that mobile buffers are unsaturable. This result also provides an analytical estimate of the cytosolic Ca2+ domain concentration ([Ca2+]d) near a channel pore and shows the dependence of [Ca2+]d on moderate concentrations of endogenous mobile buffer, Ca2+ indicator dye, and bulk cytosolic Ca2+. Assuming a simple relationship between [Ca2+]d and the lumenal depletion domain of an intracellular Ca2+ channel, lumenal and cytosolic Ca2+ profiles are matched to give an implicit analytical expression for the effect of bulk lumenal Ca2+ on [Ca2+]d.