Biol Res 37: 665-674, 2004

ARTICLE

Interplay between ER Ca²⁺ uptake and release fluxes in neurons and its impact on [Ca²⁺] dynamics

DAVID FRIEL

Dept. of Neurosciences, School of Medicine, Case Western Reserve University, Cleveland, OH, USA

Dirección para Correspondencia

ABSTRACT

In neurons, depolarizing stimuli open voltage-gated Ca²⁺ channels, leading to Ca²⁺ entry and a rise in the cytoplasmic free Ca²⁺ concentration ([Ca²⁺]_i). While such [Ca²⁺]_i elevations are initiated by Ca²⁺ entry, they are also influenced by Ca²⁺ transporting organelles such as mitochondria and the endoplasmic reticulum (ER). This review summarizes contributions from the ER to depolarization-evoked [Ca²⁺]_i responses in sympathetic neurons. As in other neurons, ER Ca²⁺ uptake depends on SERCAs, while passive Ca²⁺ release depends on ryanodine receptors (RyRs). RyRs are Ca²⁺ permeable channels that open in response to increases in [Ca²⁺]_i, thereby permitting [Ca²⁺]_i elevations to trigger Ca²⁺ release through Ca²⁺-induced Ca²⁺ release (CICR). However, whether this leads to net Ca²⁺ release from the ER critically depends upon the relative rates of Ca²⁺ uptake and release. We found that when RyRs are sensitized with caffeine, small evoked [Ca²⁺]_i elevations do trigger net Ca²⁺ release, but in the absence of caffeine, net Ca²⁺ uptake occurs, indicating that Ca²⁺ uptake is stronger than Ca²⁺ release under these conditions. Nevertheless, by increasing ER Ca²⁺ permeability, RyRs reduce the strength of Ca²⁺ buffering by the ER in a [Ca²⁺]_I-dependent manner, providing a novel mechanism for [Ca²⁺]_i response acceleration. Analysis of the underlying Ca²⁺ fluxes provides an explanation of this and two other modes of CICR that are revealed as [Ca²⁺]_i elevations become progressively larger.

Key words: Calcium dynamics, sympathetic neurons, endoplasmic reticulum, ryanodine receptors.

Abbreviations: Ca²⁺: ionized free Ca; [Ca²⁺]: Ca²⁺ concentration; [Ca²⁺]_i, cytoplasmic [Ca²⁺]; [Ca²⁺]_ER, ER [Ca²⁺]. ER: Endoplasmic reticulum; EPMA: Electron probe microanalysis; RyR: ryanodine receptors; SERCA: sarco-endoplasmic reticulum Ca ATPase; Tg: thapsigargin; t-BuBHQ, 2,5-Di-(t-butyl)-1,4-hydroquinone.

INTRODUCTION

Many physiological stimuli produce their intracellular effects by increasing the intracellular free calcium concentration ([Ca²⁺]) (Carafoli, 2004, this issue). Such [Ca²⁺] elevations can be elicited by release of Ca²⁺ from intracellular stores or by promoting Ca²⁺ entry from the extracellular medium. Ca²⁺-induced Ca²⁺ release (CICR) is an intriguing mechanism found in many excitable cells that employs both pathways. Here, Ca²⁺ entry through surface membrane Ca²⁺ channels triggers Ca²⁺ release from internal stores, principally the endoplasmic reticulum (ER). This review describes the effects of CICR on [Ca²⁺] responses induced by depolarizing stimuli in sympathetic neurons. While some of these effects are very similar to those previously described in other excitable cells, other effects are in fact quite surprising. However, after considering the underlying Ca²⁺ transport mechanisms that define the rate of net Ca²⁺ transport between the ER and cytoplasm, it becomes clear that these effects are simple consequences of the interplay between ER Ca²⁺ uptake and release pathways that are differentially regulated by Ca²⁺.

This review is not intended to be a comprehensive discussion of the role of the endoplasmic reticulum in calcium signaling, but rather a brief summary of our work on sympathetic neurons over the last few years (for a more extensive review of calcium signaling in these cells, see Friel, 2003). Space limitations make it impossible to cite all the original studies that we have drawn upon, but references to several review articles have been included to help guide interested readers to the work of other investigators on a variety of preparations.

GENERAL BACKGROUND

Previous studies in a variety of cells have shown that [Ca²⁺]_i responses evoked by depolarizing stimuli are influenced by multiple factors, including Ca²⁺ entry and extrusion across the plasma membrane, Ca²⁺ uptake and release from internal stores, and Ca²⁺ binding to endogenous and exogenous buffers. One of the properties of calcium signaling that makes it so interesting and complex is that the impact of a given Ca²⁺ transport or buffering system on stimulus-evoked calcium signals depends not only on its properties in isolation, but on the other Ca²⁺ handling systems with which it functionally interacts in vivo. Early work from my laboratory described how cytoplasmic and mitochondrial calcium dynamics depend on the interplay between Ca²⁺ transport across the plasma membrane and calcium uptake and release by mitochondria (Colegrove et al., 2000a,b; for a review see Friel, 2003). These studies showed how multiple Ca²⁺ transport systems that are regulated by Ca²⁺ in quantitatively different ways collectively give rise to evoked calcium signals with complex temporal profiles that are expected to have a strong impact on downstream processes that `read out' the Ca²⁺ signal. An important step in this work was the development of experimental approaches for dissecting the net Ca²⁺ fluxes that drive changes in calcium concentrations within particular cellular compartments into components representing individual transport pathways. This made it possible to determine how the rate of transport by each system varies with calcium concentration and use computer simulations to test if these transport systems account for evoked [Ca²⁺] signals that are observed experimentally.

This review will summarize a complementary study that was designed to elucidate how Ca²⁺ transport by the ER participates in neuronal calcium signaling. The goal of the study was to determine how CICR arises from the functional interplay between ER Ca²⁺ uptake and release pathways, and those regulating Ca²⁺ extrusion across the plasma membrane. The main conclusions can be summarized as follows. During depolarization-induced Ca²⁺ entry: (1) The ER can act as either a Ca²⁺ source or sink, depending on the size of the evoked [Ca²⁺]_i elevation, with net uptake favored at low [Ca²⁺]_i, and net release favored at high [Ca²⁺]_i; (2) The transition from sink to source that occurs as [Ca²⁺]_i increases can be explained by Ca²⁺ uptake and release pathways whose activities are regulated by Ca²⁺ in quantitatively different ways.

BASIC PROPERTIES OF ER Ca²⁺ HANDLING

The ER is an important component of Ca²⁺ signaling in most, if not all, non-muscle cells (Meldolesi and Pozzan, 1998). Ca²⁺ transport by the ER is thought to have at least three main functions. First, it permits the ER to act as a Ca²⁺ pool that can be mobilized by various IP₃-generating stimuli (e.g. Foskett and Mak, 2004). Second, ER Ca²⁺ transport modulates evoked changes in [Ca²⁺]_i, thereby influencing all cellular processes that are regulated by cytoplasmic Ca²⁺. Finally, intraluminal Ca²⁺ participates in protein processing (Verkhratsky and Petersen, 2002).

Net Ca²⁺ transport by the ER is accomplished by a set of distinctive Ca²⁺ transport systems (here broadly interpreted to include channels, pumps and exchangers). Ca²⁺ uptake is regulated by Ca²⁺ pumps termed SERCAs (sarco- and endoplasmic reticulum Ca ATPases) and passive Ca²⁺ release is regulated by channels that open in response to elevations in [Ca²⁺]_i and permit Ca²⁺ to flow passively down a steep concentration gradient into the cytoplasm (See Carafoli, 2004, this issue). Ca²⁺ release channels are particularly interesting because they render the Ca²⁺ permeability of the ER sensitive to [Ca²⁺]_i, setting the stage for CICR (Bezprozvanny et al., 1991; for reviews, see Kuba, 1994; Verkhratsky and Shmigol, 1996; and Usachev and Thayer, 1999). Two major classes of Ca²⁺ release channels have been identified: ryanodine receptors (RyRs) and inositol (1,4,5)-trisphosphate receptors, which can be distinguished based on Ca²⁺-sensitivity, pharmacology, and expression patterns that define their distinctive contributions to Ca²⁺ signaling in different cells (e.g. see Danila and Hamilton, 2004; Foskett and Mak, 2004, both in this issue).

How does the ER contribute to depolarization-evoked calcium responses in neurons?

Previous studies have described the ER as either a Ca²⁺ source or sink during depolarization-induced [Ca²⁺]_i elevations, in some cases, even within the same cell type. While these modes of signaling are expected to have very different functional effects on intraluminal and cytoplasmic Ca²⁺ binding proteins, until recently the stimulus parameters that determine whether net Ca²⁺ uptake or release occurs have been unclear. We therefore conducted a study to determine the specific conditions of stimulation that promote net Ca²⁺ uptake vs. net Ca²⁺ release. We then sought to understand what distinguishes these conditions based on the properties of the underlying Ca²⁺ transport systems.

Sympathetic neurons in primary culture were used as model cells, and cytoplasmic Ca²⁺ levels were monitored using the fluorescent Ca²⁺ indicator Fura-2. Ca²⁺ entry was stimulated by depolarizing cells from near the resting potential (~-70 mV) to various voltages ranging from _35 to _20 mV under voltage clamp (perforated patch conditions), or by exposing cells to external solutions containing high K⁺, using

Membrane depolarization opens voltage-gated Ca²⁺ channels, initiating a rise in [Ca²⁺]_i. Evidence that Ca²⁺ transport by the ER might contribute to these responses comes first from the observation that under resting conditions, rapid application of SERCA inhibitors, such as thapsigargin (Tg, 0.2-1.0 mM) or t-BuBHQ (50-100 mM), elicit a transient rise in [Ca²⁺]_i. That these [Ca²⁺]_i transients are observed in the absence of extracellular Ca²⁺ and occur without delay after applying the inhibitor provides evidence for ongoing Ca²⁺ transport at rest during which active Ca²⁺ uptake is balanced by passive Ca²⁺ release, such that sudden inhibition of uptake unmasks release, leading to a rise in [Ca²⁺]_i. Since cytoplasmic Ca²⁺ serves as a substrate and/or modulator of both the uptake and release pathways, changes in [Ca²⁺]_i initiated by Ca²⁺ entry are expected to have effects on both transport pathways. Moreover, since the rate of net ER Ca²⁺ transport that determines how intraluminal Ca concentration changes during stimulation depends on the relative rates of uptake and release, differences in the Ca²⁺ sensitivity of these transport activities can impart complex temporal properties to evoked calcium responses.

Initial evidence suggesting that ryanodine-sensitive Ca²⁺ release channels, in particular, contribute to depolarization-evoked responses came from the finding that [Ca²⁺]_i responses are amplified if cells are depolarized in the presence of caffeine (Fig. 1). Caffeine has previously been shown to increase the Ca²⁺-sensitivity of RyRs, causing them to open with high probability even at low resting [Ca²⁺]_i levels (Zucchi and Ronca-Testoni, 1997). Under control conditions, Ca²⁺ entry induced by weak depolarization from -70 to -35 mV under voltage clamp elicits a slow increase in [Ca²⁺]_i from a resting level of 50-100 nM to ~300 nM. In contrast, in the presence of caffeine, Ca²⁺ entry triggers a rapid [Ca²⁺]_i spike suggestive of net CICR, followed by a return to the same elevated steady level seen in control responses. The effects of caffeine on these evoked [Ca²⁺]_i responses cannot be explained by changes in voltage-sensitive Ca²⁺ entry and are blocked by Tg and ryanodine (1 mM), indicating that response amplification depends on the activity of a ryanodine-sensitive Ca²⁺ release pathway, presumably representing RyRs (Friel and Tsien, 1992, Albrecht et al., 2001).

Figure 1. Caffeine amplifies the effects of Ca2+ entry on [Ca2+]i. [Ca2+]i responses elicited from a representative sympathetic neuron by voltage clamp depolarizations under control conditions (Cont, light trace) and during continuous exposure to 5 mM caffeine (+Caff, dark trace). In the presence of caffeine, depolarization-induced [Ca2+]i responses exhibited an initial [Ca2+]i spike representing triggered CICR. Top trace indicates membrane potential. Exposure to caffeine at the holding potential (-70 mV) after the first (Cont) response but before the second (+Caff) response elicited a large [Ca2+]i transient caused by Ca2+ release from the ER; the small reversible reduction in basal [Ca2+]i seen in the presence of caffeine is due, at least in part, to an effect of caffeine on fura-2 fluorescence independent of changes in [Ca2+]i. Adapted from Albrecht et al., (2001).

SMALL [Ca²⁺]_I ELEVATIONS ELICITED BY WEAK DEPOLARIZATION STIMULATE ER CALCIUM ACCUMULATION

While these and other observations indicate that sympathetic neurons possess a ryanodine-sensitive Ca²⁺ release pathway, which allows the Ca²⁺ entry to trigger net CICR when modified by caffeine, they do not address the role of this pathway in regulating ER and cytoplasmic Ca²⁺ dynamics in the absence of caffeine. We found that under control conditions, weak depolarizations that raise [Ca²⁺]_i to ~300 nM stimulate net Ca²⁺ accumulation by the ER. This was suggested by the observation that [Ca²⁺]_i elevations are accelerated after cells have been treated with Tg, as if Tg disables a Ca²⁺ buffering system that normally sequesters Ca²⁺ as it enters the cytoplasm during depolarization; no effect on voltage-gated Ca²⁺ entry was detectable at the Tg concentrations employed (Fig. 2). Direct confirmation of net calcium uptake was obtained by measuring evoked changes in total ER Ca concentration, using EPMA (Hongpaisan et al., 2001) (see Fig. 4). Therefore, in sympathetic neurons, small [Ca²⁺]_i elevations (~300 nM) promote net calcium uptake, rather than net Ca²⁺ release.

Figure 2. Thapsigargin accelerates depolarization-evoked [Ca2+]i elevations. (Top) [Ca2+]i responses elicited by step depolarizations from -70 to -35 mV before (light trace) and after exposure to Tg (200 nM) (dark trace) measured under voltage clamp. Top trace indicates membrane potential. In each case, the depolarization elicited similar voltage-sensitive Ca2+ currents (bottom), but [Ca2+]i increased more rapidly and reached its peak earlier after Tg treatment, supporting the conclusion that the Tg-sensitive ER pool accumulates Ca2+ during these stimuli. Tg elicited a transient [Ca2+]i elevation between these responses (not shown), caused by passive Ca2+ release from the ER. From Albrecht et al., (2001).

EFFECTS OF RYR ACTIVITY ON THE RATE OF Ca²⁺ ACCUMULATION DURING WEAK DEPOLARIZATION

How can net Ca²⁺ uptake by the ER during weak stimulation be reconciled with the presence of a ryanodine-sensitive CICR pathway that might be expected to favor triggered Ca²⁺ release from the ER? A key piece of information was provided by the following observation: after exposing cells to ryanodine (1 mM), depolarization-evoked [Ca²⁺]_i elevations show a significantly slower onset and recovery than control responses (Fig. 3). As with the effect of Tg, this effect could not be explained by ryanodine-induced changes in voltage-sensitive Ca²⁺ entry. Since the direction of net Ca²⁺ transport by the ER depends on the relative rates of Ca²⁺ uptake and release via distinct pathways, the finding that small [Ca²⁺]_i elevations stimulate net calcium uptake means that under these conditions, the average rate of Ca²⁺ uptake exceeds the average rate of Ca²⁺ release. This raises the following question: how would Ca²⁺-dependent activation of RyRs contribute to net Ca²⁺ transport by the ER when it acts as a Ca²⁺ buffer? During stimulation, RyR activation would be expected to increase the Ca²⁺ permeability of the ER. For a given electrochemical driving force that favors passive Ca²⁺ release, this would increase the rate of passive release. If uptake is faster than release, the overall consequence of a small increase in release rate would be a reduced imbalance between uptake and release rates, leading to slower net Ca²⁺ uptake. During weak stimulation, RyR activation would therefore accelerate evoked [Ca²⁺]_i elevations not by promoting net Ca²⁺ release from the ER, but by reducing the rate of net uptake. This provides a potential explanation of the observed effects of ryanodine. If ryanodine prevents a Ca²⁺-induced increase in RyR activity during stimulation, it would lower the rate of passive Ca²⁺ release, increase the imbalance between uptake and release rates, and thereby enhance net Ca²⁺ uptake. Thus, in the presence of ryanodine, small depolarizations that normally stimulate weak Ca²⁺ accumulation would cause more powerful accumulation, simply because the uptake pathway now operates in parallel with a smaller leak. Overall, ryanodine would cause the ER to become a more powerful Ca²⁺ buffer during periods of stimulated Ca²⁺ entry.

We carried out two series of experiments to test this idea. First, if the interpretation presented above is correct, then Tg should reverse the effects of ryanodine. We found that this is indeed the case (Albrecht et al., 2001). Second, direct measurement showed that following treatment with ryanodine, elevations in intraluminal total Ca concentration induced by weak depolarization are larger than controls, confirming the idea that ryanodine increases the average rate of net Ca²⁺ uptake during stimulation. Interestingly, ryanodine also reduced the basal ER total calcium concentration by ~60%, which can be explained, at least in part, by a parallel increase in basal ER Ca²⁺ permeability. This increase was presumably the result of an increase in steady-state RyR open probability like that observed when purified RyRs in planar lipid bilayers are exposed to ryanodine at concentrations that are below those causing the channel blockade (Sutko et al. 1997).

LARGE [Ca²⁺]_i ELEVATIONS STIMULATE NET Ca²⁺ RELEASE

We found that with stronger depolarizations that raise [Ca²⁺]_i to higher levels, the ER undergoes a transition from net Ca²⁺ uptake to net Ca²⁺ release. While weak depolarizing stimuli (V_m~-35 mV) that raise [Ca²⁺]_i from a resting level of 50-100 nM to ~300 nM lead to net Ca²⁺ uptake, stronger depolarizations (V_m~-20 mV) that produced intermediate [Ca²⁺]_i elevations (400-600 nM) lead to little or no net ER Ca²⁺ transport. In contrast, stimuli that raise [Ca²⁺]_i to levels in excess of ~1 mM cause net Ca²⁺ release (Fig. 4) (see Hongpaisan et al., 2001).

Figure 3. Depolarization-evoked [Ca2+]i elevations are slowed after treatment with ryanodine. Comparison between [Ca2+]i responses elicited by 30 mM K+ depolarization before (light trace) and after (dark trace) treatment with ryanodine (1 mM), which abolished the ability of caffeine to discharge the ER. Top trace indicates estimated membrane potential at rest and during exposure to 30 mM K+ based on microelectrode measurements from sympathetic neurons (Friel and Tsien, 1992). Adapted from Albrecht et al., (2001).

Figure 4. Biphasic [Ca2+]i-dependence of evoked changes in total ER Ca concentration. Plot of [Ca]ER vs. [Ca2+]i under six different experimental conditions. As the stimulus-evoked [Ca2+]i elevation become larger, [Ca]ER increases from a high resting level, then declines to basal levels when [Ca2+]i nears ~500 nM. When the same stimulus is applied to cells after inhibiting mitochondrial Ca2+ transport with FCCP (1 mM), the evoked [Ca2+]i increase is larger, and [Ca]ER falls below resting levels, indicative of net calcium release. Net Ca2+ release in response to this stimulus does not depend upon the presence of FCCP, since a reduction in [Ca]ER is also observed in the absence of FCCP in regions near the plasma membrane where [Ca2+]i levels are especially high during depolarization, owing to their proximity to surface membrane Ca2+ channels. Dotted lines indicate [Ca]ER measured in resting cells after treatment with ryanodine. Error bars represent ±SEM. (Adapted from Honpaisan et al. 2001).

ANALYSIS OF THE UNDERLYING Ca²⁺ FLUXES

We investigated the basis for the observed biphasic [Ca⁺²]_i-dependence of net ER Ca²⁺ transport. Since the intraluminal calcium concentration changes under the influence of a net Ca²⁺ flux whose direction depends on the relative rates of Ca²⁺ uptake and release via different pathways, we predicted that the biphasic [Ca²⁺]_i-dependence of evoked changes in [Ca]_ER results from different [Ca²⁺]_i-sensitivities of the Ca²⁺ uptake and release rates. To test this idea, we examined the [Ca²⁺]_i-dependence of the rates of Ca²⁺ uptake via SERCAs (J_SERCA) and of passive Ca²⁺ release by the ER (J_Release), which depends on both the driving force for Ca²⁺ release ([Ca²⁺]_i-[Ca²⁺] _ER) and the Ca²⁺ permeability of the ER (P_ER) (Albrecht et al., 2002). Figure 5B shows smooth curves based on measurements of the fluxes that together define the net ER Ca²⁺ flux (J_ER= J_SERCA + J_Release). The quantitative properties of these fluxes provide an explanation for the biphasic [Ca²⁺]_i-dependence of evoked changes in [Ca]_ER. At low [Ca²⁺]_i, J_SERCA changes more steeply with [Ca²⁺]_i than does J_Release, leading to net Ca²⁺ uptake as [Ca²⁺]_i rises, while at high [Ca²⁺]_i the reverse is true, accounting for net Ca²⁺ release. The increase in the magnitude of J_Release reflects the [Ca²⁺]_i-dependence of P_ER, which because it is highly sensitive to caffeine and ryanodine, appears to represent the contribution to ER Ca²⁺ permeability from ryanodine sensitive Ca²⁺ release channels (Albrecht et al, 2002).

Figure 5C shows the fluxes that together define the net cytoplasmic Ca²⁺ flux (J_i), which determines how [Ca²⁺]_i changes with time. The interplay between J_ER, which increases biphasically with [Ca²⁺]_i, and the rate of Ca²⁺ extrusion across the surface membrane (J_Extru), which in turn increases monotonically with [Ca²⁺]_i, leads to the separation of the [Ca²⁺]_i range into three domains displaying distinct modes of CICR. When [Ca²⁺]_i is above the resting level but low enough so that RyR activity is weak, J_i and J_ER are both outward (positive) fluxes (Mode 1 CICR). J_ER is positive because passive Ca²⁺ release is slower than Ca²⁺ uptake, causing the ER to act as a buffer. In this mode, activation of the [Ca²⁺]_i-sensitive permeability during stimulation reduces the rate of Ca²⁺ accumulation and accelerates the rise in [Ca²⁺]_i as compared to the case where P_ER is constant at its resting level. Over the intermediate [Ca²⁺]_i range, J_i is positive but J_ER is negative (Mode 2 CICR) because passive Ca²⁺ release is faster than uptake. In this [Ca²⁺]_i domain, net Ca²⁺ release amplifies the effects of stimulated Ca²⁺ entry on [Ca²⁺]_i, causing [Ca²⁺]_ER to decline to an extent that is graded with [Ca²⁺]_i. Finally, at higher [Ca²⁺]_i levels, both J_i and J_ER are inward fluxes (Mode 3 CICR). A brief stimulus that brings [Ca²⁺]_i within this range would stimulate Ca²⁺ release at such a high rate that it would overwhelm Ca²⁺ extrusion, leading to a regenerative rise in [Ca²⁺]_i and fall in [Ca²⁺]_ER. The zero crossing in this case represents the threshold for regenerative net CICR. It should be noted that while this discussion has focused on CICR under the control of RyRs, the general ideas apply equally well to other types of Ca²⁺ release channels, e.g. InsP₃Rs.

Figure 5. Quantitative properties of ER Ca2+ uptake and release rates account for multiple modes of CICR. (A) Schematic illustrating three modes of CICR and the relationship between the magnitudes of the underlying Ca2+ fluxes (arrows) in each mode during periods of stimulated Ca2+ entry; gray arrows represent Ca2+ entry. (B,C) Instantaneous flux/[Ca2+]i,[Ca2+ ]ER relations at constant (resting) [Ca2+]ER for JER (B) and the net cytoplasmic Ca2+ flux Ji in the absence of Ca2+ entry (C) showing how each flux is the sum of two component fluxes: JER=JSERCA + JRelease and Ji=JER + JExtru, where JExtru, is the rate of Ca2+ extrusion across the surface membrane. Fluxes directed into the cytosol are negative while outward fluxes are positive. Panel B also shows the [Ca2+]i-dependence of PER (dotted curve), the Ca2+ permeability of the ER. Note that in C, JER intersects the zero net flux axis at two points (see up arrows) corresponding to the resting state and the threshold for net CICR. There are also two zero-crossing for Ji, one at resting [Ca2+]i and another at the threshold for regenerative net CICR. For illustration purposes, calculated values of JRelease assume that [Ca2+]ER is constant at its resting level, so the flux/[Ca2+]i,[Ca2+ ]ER curves represent the net fluxes that would be predicted if [Ca2+]i were suddenly increased from its resting level to various levels indicated on the abscissa. Adapted from Albrecht et al. (2002).

Our results underscore a basic ambiguity in the phrase "Ca²⁺-induced Ca²⁺ release," which arises because there is a distinction between passive Ca²⁺ transport via a CICR pathway and net Ca²⁺ transport by the ER. CICR is usually used to refer to net Ca²⁺ release, but our results indicate that activation of a Ca²⁺-sensitive release pathway can have the alternate effect of attenuating net ER Ca²⁺ accumulation. The term CICR could in principle refer to either passive release via a Ca²⁺-sensitive permeability without reference to the direction of net organellar Ca²⁺ movement, or to Ca²⁺-induced net Ca²⁺ release. Since the second definition necessarily involves the relationship between a CICR pathway and other transport systems, we prefer the first definition and use `net CICR' to describe the case in which CICR leads to a reduction in intraluminal Ca²⁺ levels. According to this usage, CICR refers to a Ca²⁺-induced increase in a passive Ca²⁺ release whose impact on intraluminal Ca²⁺ levels is context-dependent.

SUMMARY AND CONCLUSIONS

Ca²⁺ is an important signaling intermediate in virtually all cells that produces its effects by interacting with Ca²⁺ sensors in a manner that depends critically on Ca²⁺ concentration. Therefore, there is a strong rationale for understanding the physiological determinants of [Ca²⁺] dynamics. The free Ca²⁺ concentration is tightly controlled in all cellular compartments by specific Ca²⁺ transport and buffering systems that have been investigated in isolation, but largely because of technical difficulties in measuring rates of transport in situ have received little attention in intact cells. This review provides an overview of work that we have done to elucidate the functional properties of ER Ca²⁺ transport systems expressed in sympathetic neurons and how they operate together to orchestrate stimulus-evoked Ca²⁺ signals. Our results show how multiple Ca²⁺ transporters operating in parallel can impart to Ca²⁺ dynamics a complicated dependence on Ca²⁺ concentration, illustrating the importance of obtaining quantitative functional descriptions of Ca²⁺ transport systems in intact cells. Our results also demonstrate that in addition to exhibiting different spatial signaling domains, cells also show distinctive Ca²⁺ concentration `domains' in which there are unique patterns in the way [Ca²⁺] changes in different cellular compartments, patterns that are likely to have unique effects on cell function.

ACKNOWLEDGEMENTS

The author would like to thank Meredith Albrecht, S. Brian Andrews, Stephen Colegrove, Jarin Hongpaisan and Natalia Pivovarova for their contributions to this work.

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Corresponding author: David Friel, Dept. of Neurosciences, School of Medicine, E610 Case Western Reserve University, 10900 Euclid Ave., Cleveland, OH 44106, USA. Phone: (216)368 4930, Fax: (216)368 4650, E-mail: ddf2@po.cwru.edu

Received: January 21, 2004. Accepted: March 22, 2004.