Correspondence to: David D. Friel, Department of Neuroscience, Case Western Reserve University, 10900 Euclid Ave. Cleveland, OH 44106. Fax:216-368-4650 E-mail:ddf2{at}po.cwru.edu.
Released online: 28 February 2000
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Abstract |
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We studied how mitochondrial Ca2+ transport influences [Ca2+]i dynamics in sympathetic neurons. Cells were treated with thapsigargin to inhibit Ca2+ accumulation by SERCA pumps and depolarized to elevate [Ca2+]i; the recovery that followed repolarization was then examined. The total Ca2+ flux responsible for the [Ca2+]i recovery was separated into mitochondrial and nonmitochondrial components based on sensitivity to the proton ionophore FCCP, a selective inhibitor of mitochondrial Ca2+ transport in these cells. The nonmitochondrial flux, representing net Ca2+ extrusion across the plasma membrane, has a simple dependence on [Ca2+]i, while the net mitochondrial flux (Jmito) is biphasic, indicative of Ca2+ accumulation during the initial phase of recovery when [Ca2+]i is high, and net Ca2+ release during later phases of recovery. During each phase, mitochondrial Ca2+ transport has distinct effects on recovery kinetics. Jmito was separated into components representing mitochondrial Ca2+ uptake and release based on sensitivity to the specific mitochondrial Na+/Ca2+ exchange inhibitor, CGP 37157 (CGP). The CGP-resistant (uptake) component of Jmito increases steeply with [Ca2+]i, as expected for transport by the mitochondrial uniporter. The CGP-sensitive (release) component is inhibited by lowering the intracellular Na+ concentration and depends on both intra- and extramitochondrial Ca2+ concentration, as expected for the Na+/Ca2+ exchanger. Above ~400 nM [Ca2+]i, net mitochondrial Ca2+ transport is dominated by uptake and is largely insensitive to CGP. When [Ca2+]i is ~200300 nM, the net mitochondrial flux is small but represents the sum of much larger uptake and release fluxes that largely cancel. Thus, mitochondrial Ca2+ transport occurs in situ at much lower concentrations than previously thought, and may provide a mechanism for quantitative control of ATP production after brief or low frequency stimuli that raise [Ca2+]i to levels below ~500 nM.
Key Words: mitochondria, calcium, calcium signaling, neurons, CGP 37157
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INTRODUCTION |
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There is a growing interest in mitochondrial Ca2+ transport. Ca2+ uptake and release by these organelles is thought to influence the dynamics of cytosolic free Ca2+ concentration ([Ca2+]i) in a variety of cell types after stimuli that promote either Ca2+ entry from the extracellular medium or release from intracellular stores (for reviews see
Mitochondrial Ca2+ transport has been studied extensively in isolated mitochondria. Ca2+ uptake is controlled by a Ca2+-sensitive uniporter (EC50 ~1020 µM;
Despite the importance of mitochondrial Ca2+ uptake and release pathways in defining the rate of net mitochondrial Ca2+ transport, their individual contributions to [Ca2+]i dynamics in situ have not been determined, in part because they operate within an intracellular network of coupled transporters that makes contributions from individual transport systems difficult to resolve. Pharmacological agents have been useful in identifying mitochondrial contributions to depolarization-evoked [Ca2+]i responses in intact cells. Proton ionophores, such as FCCP, depolarize the inner membrane and reduce the electrochemical driving force for Ca2+ uptake, suppressing mitochondrial Ca2+ accumulation. Inhibitors of the uniporter, such as ruthenium red or its active component Ru360 (
We sought to characterize the Ca2+ transport systems that restore resting [Ca2+]i after depolarization-induced [Ca2+]i elevations in sympathetic neurons. These cells respond to depolarization with a rise in [Ca2+]i that is initiated by Ca2+ entry through voltage-gated Ca2+ channels but is strongly influenced by mitochondrial Ca2+ transport (
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MATERIALS AND METHODS |
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Cell Dissociation and Culture
All procedures conform with guidelines established by our Institutional Animal Care and Use Committee. Sympathetic neurons were obtained as described previously (
Cytosolic Calcium Measurements
Cells were incubated with 3 µM fura-2 AM (Molecular Probes) for 40 min at room temperature with gentle agitation. Fura-2 AM was dispensed from a 1-mM stock solution in DMSO containing 25% (wt/wt) pluronic F127 (BASF Corporation) that was stored at -20°C. Cells were rinsed and recordings began after ~20 min to facilitate de-esterification of the Ca2+ indicator. Cells were placed on the stage of an inverted microscope (Nikon Diaphot TMD) and superfused continuously (~5 ml/min) with normal Ringer's. Solution changes (~200 ms) were made using a system of microcapillaries (Drummond microcaps, 20 µl) mounted on a micromanipulator as described in
Cells were illuminated by light from a 150 W Xenon lamp that passed through excitation filters (350 ± 5 nm, 380 ± 5 nm) mounted on a filter wheel rotating at 40100 Hz and was focused with a 40xobjective (NA 1.3; Nikon, Fluor). Emitted light passed through a long-pass dichroic mirror (400 nm) and an emission filter (510 ± 10 nm) and was detected by a photomultiplier tube (Thorn EMI 9124). A spectrophotometer (Cairn Research Limited) was used to control the filter wheel and measure fluorescence intensity at the two excitation wavelengths. Fluorescence measurements were made at 45 Hz and saved on a laboratory computer. [Ca2+]i was calculated according to the method of
Voltage Clamp
Simultaneous measurements of depolarization-evoked [Ca2+]i elevations and voltage-sensitive Ca2+ currents (ICa) were made under voltage clamp in fura-2 AM loaded cells using the perforated patch technique. Patch electrodes (12 M) were pulled (Sutter Instruments P-97), and tips were filled with a solution containing (in mM): 125 CsCl, 5 MgCl2, 10 HEPES, and 010 mM Na+ (with reciprocal changes in Cs+), pH 7.3, with CsOH. After filling tips, pipettes were back-filled with the same solution supplemented with 520 µM amphotericin B, dispensed from concentrated aliquots (12 mg/100 µl DMSO). Amphotericin Bcontaining internal solutions were kept on ice and used within 2 h. After achieving a high resistance seal, series resistance declined over 510 min to <10 M
. Cells were exposed to an extracellular solution containing (in mM): 130 TEACl, 10 HEPES, 10 glucose, 2 CaCl2, 1 MgCl2, pH = 7.3, with TEAOH. Currents were measured with an Axopatch 200A voltage clamp (Axon Instruments) using series resistance compensation (~90%) and were filtered at 5 kHz. Cells were held at -70 mV and depolarized to voltages between -15 and 0 mV, while current and fluorescence intensity were measured at 0.15 kHz just before and 0.210 s after changes in voltage, and at 45 Hz otherwise, and saved on a laboratory computer. Currents were corrected for a linear leak based on responses to small hyperpolarizing voltage steps. [Ca2+]i elevations evoked under voltage clamp were somewhat larger than those elicited by high K+ at comparable membrane potentials, presumably because of more rapid depolarization and more efficient Ca2+ channel activation under voltage clamp. However, the kinetics of the [Ca2+]i recovery after repolarization were similar for the two techniques, provided that pipette solutions contained mM levels of Na+.
Measurement of Ca2+ Fluxes
To study the Ca2+ transport systems that restore [Ca2+]i to its resting level after depolarization-evoked Ca2+ entry, cells were depolarized either by exposure to high K+ Ringer's (equimolar substitution for Na+) or under voltage clamp, and the [Ca2+]i recovery that followed repolarization was examined. Cells were treated with 200500 nM thapsigargin (Tg) for 1020 min before beginning measurements to inhibit SERCA pump activity and minimize Ca2+ accumulation by the endoplasmic reticulum. Such treatments rendered cells completely insensitive to other SERCA pump inhibitors, including CPA (50 µM) and BHQ (10 µM), and to caffeine (110 mM), each of which consistently elicited [Ca2+]i transients in cells that had not been treated with Tg.
Cells typically responded to high K+ depolarization with [Ca2+]i responses that were quite reproducible, making it possible to compare, in single cells, responses elicited under several different conditions. Under voltage clamp, intracellular ion concentrations could be manipulated and Ca2+ fluxes could be measured over a wider range of [Ca2+]i, but after depolarizations in the presence of FCCP, [Ca2+]i recovered to values that were ~50100 nM higher than those measured in the presence of FCCP before depolarization. The reason for this increase is not clear, but it is consistent with the development of a small Ca2+ leak (~5 nM/s), which would introduce a small error in the measured FCCP-sensitive component of the total flux, leading to a slight overestimation of this flux (~5% at 500 nM [Ca2+]i).
The net cytosolic Ca2+ flux per unit volume during the recovery (J, units: nM/s) was calculated as the time derivative of [Ca2+]i at each intermediate sample time ti according to ([Ca2+]i(ti + t/2) - [Ca2+]i(ti -
t/2))/
t, where
t (400500 ms) is twice the sampling interval. For the first and last sample points, the flux was estimated by computing the slope of a fitted line over the first and last sets of three sample points, respectively, or by fitting an exponential over 4
t and calculating the slope of the fitted exponential at the endpoints. The total Ca2+ flux during the recovery (Jcont) was separated into mitochondrial and nonmitochondrial components based on their differing sensitivities to FCCP. The net mitochondrial flux was determined by taking the difference between the total cytosolic Ca2+ flux in the presence and absence of FCCP at corresponding values of [Ca2+]i. The net mitochondrial Ca2+ flux was then separated into components based on sensitivity to CGP and intracellular Na+. This approach is described and validated in Results. Data were acquired at discrete times so that flux measurements in the presence and absence of an inhibitor were not always made at identical values of [Ca2+]i. Therefore, linear interpolation was used to approximate each measured flux at equally spaced values of [Ca2+]i. Before calculating difference fluxes, the measured fluxes were smoothed 13 times with a binomial filter that replaced each intermediate flux value Ji with a weighted average of Ji and its nearest neighbors (Ji-1 + 2Ji + Ji+1)/4.
Data Analysis
Quantifying the plateau level during the [Ca2+]i recovery.
The plateau level was defined as the value of [Ca2+]i where the first inflection point occurs during the recovery. It was measured by fitting a 912th order polynomial to the [Ca2+]i recovery and determining where the second derivative of the fitted curve changed sign. This provided a suitable way to quantify the plateau level and its sensitivity to CGP. At high concentrations of CGP, an inflection point was sometimes difficult to resolve, so in these cases the plateau level was defined as the value of [Ca2+]i where the second derivative fell below 0.001 nM/s2.
Statistics.
Population results are expressed as mean ± SEM and statistical significance was assessed using Student's t test (
Drugs
CGP 37157 was a kind gift from Anna Suter (Novartis). Purified ruthenium red was generously provided by Dr. M. A. Matlib. Unless indicated otherwise, all other compounds were obtained from Sigma Chemical Co.
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RESULTS |
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Mitochondrial and Nonmitochondrial Components of the Total Ca2+ Flux
Fig 1 compares [Ca2+]i responses elicited by weak and strong depolarization in an intact fura-2loaded sympathetic neuron that was pretreated with Tg to inhibit ER Ca2+ accumulation by SERCA pumps. Exposure to a solution containing 30 mM K+, which depolarizes Vm from a typical resting potential of -69.9 ± 2.5 mV to ~-35 mV (
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Four observations indicate that mitochondria play a role in shaping [Ca2+]i responses elicited by strong depolarization in these cells. First, the responses are greatly modified if cells are stimulated during maintained exposure to the proton ionophore carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP, 1 µM; Fig 1 A, right). In this case, [Ca2+]i rises to a higher level during stimulation, and the ensuing recovery lacks both the initial rapid decline and the slow plateau phase (30 cells). Similar modifications are observed after treatment with antimycin A1 and oligomycin (see below), and after microinjection of ruthenium red (not shown). Second, rapid exposure to FCCP (10 µM) in Ca2+-free Ringer's elicits a large [Ca2+]i transient during the plateau phase of the recovery, but not in the same cells after [Ca2+]i returns to basal levels, arguing that depolarization reversibly increases the Ca2+ content of an FCCP-releasable Ca2+ store. Third, FCCP has little or no effect on resting [Ca2+]i or on responses to weak depolarization which raise [Ca2+]i to ~300 nM or below (
To illustrate mitochondrial and nonmitochondrial contributions to the complex [Ca2+]i recovery, Fig 1 B plots the total cytosolic Ca2+ flux (J = -d[Ca2+]i/dt) vs [Ca2+]i for each of the three recoveries in A, (positive values of J represent outward fluxes from the cytosol). In each case, J is positive, indicating that Ca2+ removal is dominant, but there is a striking difference between the recoveries that follow small and large depolarization-evoked [Ca2+]i elevations. After weak depolarization (Fig 1 B, left), J is nearly proportional to [Ca2+]i below ~300 nM, as expected if Ca2+ is removed from the cytosol by a simple first order process. In contrast, after stronger depolarizations that elevate [Ca2+]i to higher levels (Fig 1 B, middle), J varies with [Ca2+]i in a complex manner that mirrors the temporal complexity of the recovery (Fig 1 A, middle). During phase i, when [Ca2+]i is high, J is much larger than the extrapolated linear flux (Fig 1 B, dashed line), but then declines so that over the range of [Ca2+]i associated with the plateau (phase ii), it is smaller than the linear flux. J then rises during phase (iii), approaching and ultimately coinciding with the linear flux as [Ca2+]i nears its prestimulation level (phase iv). Fig 1 B (right) shows J during the recovery after 50 K+ depolarization in the presence of FCCP. For [Ca2+]i up to ~300 nM, the FCCP-resistant flux (JFCCP-res) closely resembles the linear flux that restores [Ca2+]i to its resting level after weak depolarization (dashed line). However, at higher [Ca2+]i, JFCCP-res is smaller than the extrapolated linear flux, indicating that Ca2+ removal by the underlying transporters becomes limited when [Ca2+]i is high, or that a source of Ca2+ is active just after repolarization (see also
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A simple interpretation of the complex [Ca2+]i recovery after large [Ca2+]i elevations is that the underlying Ca2+ flux consists of two components. One component represents Ca2+ removal by nonmitochondrial transporters at a rate that increases saturably with [Ca2+]i, while the other component represents reversible net Ca2+ transport by mitochondria: net Ca2+ accumulation at high [Ca2+]i followed by net Ca2+ release at low [Ca2+]i. An obvious approach to separating J into mitochondrial and nonmitochondrial components is to take the difference between J in the presence and absence of FCCP at corresponding values of [Ca2+]i to give the FCCP-sensitive flux (JFCCP-sens). This method has been used in previous studies (e.g.,
Properties of the FCCP-resistant component of the total Ca2+ flux. Fig 1 B (right) illustrates the [Ca2+]i dependence of the FCCP-resistant flux (JFCCP-res). Since this flux is seen under conditions where Ca2+ transport by both mitochondria and the endoplasmic reticulum should be largely inhibited, it presumably represents the parallel combination of plasma membrane Ca2+ extrusion and a background leak. The net mitochondrial Ca2+ flux can be determined from the control flux by subtracting JFCCP-res at corresponding values of [Ca2+]i if: (a) FCCP specifically and completely inhibits net mitochondrial Ca2+ transport, and (b) the rate of nonmitochondrial Ca2+ transport at each instant in time depends only on [Ca2+]i at that time. If these conditions are satisfied, JFCCP-res gives the contribution of nonmitochondrial Ca2+ transport to the control flux. Moreover, at each time during the recovery, the control flux is the sum of the net mitochondrial flux and JFCCP-res at the corresponding value of [Ca2+]i, making it possible to calculate Jmito by subtracting JFCCP-res from Jcont.
To test for specificity of FCCP, cells were treated with antimycin A1 and oligomycin as an independent way to inhibit mitochondrial Ca2+ transport, and then depolarized both in the presence and absence of 1 µM FCCP. If FCCP directly influences nonmitochondrial Ca2+ transport, it should modify the recovery in cells treated with antimycin A1 and oligomycin. Fig 2 A compares recoveries in a cell that was treated with antimycin A1 and oligomycin and then depolarized in the presence (left) and absence of FCCP (middle). FCCP has little or no effect on the recovery kinetics, which can be seen more clearly by superimposing the recoveries (right). To assess potential nonmitochondrial effects of FCCP quantitatively, recoveries in the presence and absence of FCCP were fit with two decaying exponentials and the parameters of the fitted curves were compared. FCCP did not influence any of the parameters (five cells, data not shown). Therefore, FCCP (1 µM) does not influence antimycin/oligomycin-resistant Ca2+ transport, arguing that it specifically inhibits mitochondrial Ca2+ transport in these cells.
To determine if JFCCP-res is influenced by long lasting effects of the [Ca2+]i elevation that precedes the recovery, responses to strong and weak depolarization were elicited in the continued presence of 1 µM FCCP and the recoveries were compared (Fig 2 B). Despite following [Ca2+]i elevations of very different magnitude and time course, the recoveries were essentially identical over the common range of [Ca2+]i (see superimposed traces, Fig 2 B). To examine the impact of prior [Ca2+]i elevations on recovery kinetics quantitatively, recoveries after weak and strong depolarization were fit over the common range of [Ca2+]i with the sum of two decaying exponentials and the parameters of the fitted curves were compared, which indicated that the recoveries were indistinguishable (four cells, data not shown). This shows that at each time during the recovery, the rate of Ca2+ removal by FCCP-resistant transporters depends on [Ca2+]i at that time but not on the history of [Ca2+]i (or membrane potential). Thus, given an initial value of [Ca2+]i, the time course of the [Ca2+]i recovery is determined. These results also indicate that inhibition of mitochondrial Ca2+ transport by FCCP is nearly complete at 1 µM: otherwise, the recovery after strong depolarization would be slower than that after weak depolarization.
It is concluded that JFCCP-res represents the activity of nonmitochondrial Ca2+ transport systems that restore resting [Ca2+]i after depolarization. Collectively, these transporters generate an outward net Ca2+ flux whose magnitude at each instant in time is defined by [Ca2+]i at that time. Therefore, the FCCP-sensitive flux, which will be referred to below as the net mitochondrial Ca2+ flux (Jmito), can be calculated from the control flux (Jcont) by subtracting JFCCP-res at corresponding values of [Ca2+]i.
Properties of the mitochondrial Ca2+ flux. Fig 3 compares mitochondrial and nonmitochondrial components of Jcont during the recovery after a 9 s 50 K+ depolarization from a representative cell (left column) along with collected results from 10 cells (right column). The measured fluxes (Jcont, JFCCP-res) are shown in Fig 3 B, while the difference flux (Jmito) is shown in (C). Jmito is large and outward at high [Ca2+]i but small and inward when [Ca2+]i is low (see Fig 3 D). The properties of JFCCP-res and Jmito provide an explanation of the four phases of recovery after strong depolarization (see Fig 3A and Fig D). During phase i, Jmito is large and outward, indicative of strong mitochondrial Ca2+ accumulation over this [Ca2+]i range, and is largely responsible for the rapid decline in [Ca2+]i. As [Ca2+]i declines, Jmito falls, changing sign to become a small but prolonged inward flux over the [Ca2+]i range associated with the plateau (Fig 3 A, phase ii, see arrow). During this phase, Jmito and JFCCP-res have opposite signs but nearly equal magnitudes (D), accounting for the small magnitude of Jcont and the slow rate of recovery. Since Jmito decays with [Ca2+]i more rapidly than JFCCP-res (Fig 3 D), Jcont rises, accounting for the accelerated recovery during phase iii. Finally, as Jmito approaches zero, Jcont is dominated by nonmitochondrial Ca2+ removal systems that define the slow final phase of recovery (phase iv). Note that while Jmito is plotted against [Ca2+]i, it may also depend on other quantities that change during the recovery, such as the intramitochondrial Ca concentration (see below). However, since the flux subtraction used to measure Jmito requires only that the nonmitochondrial flux is defined by [Ca2+]i, it is valid even if Jmito depends on variables other than [Ca2+]i.
Two main conclusions can be drawn from these results. First, mitochondrial Ca2+ accumulation provides the major mechanism for cytosolic Ca2+ clearance when [Ca2+]i is high, in general agreement with the findings of
Separation of the Net Mitochondrial Ca2+ Flux into Uptake and Release Components
Effects of the mitochondrial Na+/Ca2+ exchange inhibitor CGP 37157.
To understand how mitochondrial Ca2+ transport contributes to Ca dynamics, it is necessary to separate Jmito into its components. Neuronal mitochondria accumulate Ca2+ via a uniporter and release Ca2+ via a Na+/Ca2+ exchanger (
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If CGP completely inhibits mitochondrial Ca2+ release, it might be expected to enhance mitochondrial Ca2+ accumulation when [Ca2+]i is high enough to activate the uniporter, slowing the rise in [Ca2+]i during stimulation and speeding the initial phase of recovery after repolarization. This was not observed, arguing that when [Ca2+]i is high, net mitochondrial Ca2+ transport is dominated by the uptake pathway. The slow [Ca2+]i tail observed during the recovery in the presence of CGP may represent residual mitochondrial Ca2+ release due to incomplete block of the Na+/Ca2+ exchanger or release by a CGP-insensitive pathway. The observation that the slow tail is not seen at higher CGP concentrations (see Fig 6 C) support the former explanation.
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The actions of CGP are concentration dependent. When cells are depolarized in the presence of CGP at increasing concentrations, the [Ca2+]i plateau level is progressively lowered and the recovery is prolonged (Fig 4 B). This effect was quantified by measuring the [Ca2+]i level where the rate of recovery reaches a minimum (see Materials and Methods). Plotting the normalized plateau level vs CGP concentration shows that the plateau level falls with concentration in a manner consistent with a single binding site model with IC50 ~ 560 nM (Fig 4 C, smooth curve). This value agrees with the IC50 for CGP-induced inhibition of Na+-dependent Ca2+ efflux from isolated cardiac mitochondria (360800 nM;
The CGP-sensitive component of the recovery requires intracellular sodium.
If the [Ca2+]i plateau reflects Ca2+ release via the mitochondrial Na+/Ca2+ exchanger, both the plateau level and its sensitivity to CGP should depend on intracellular Na+. To examine this point, cells were depolarized under voltage clamp (perforated patch conditions) with or without Na+ added to the pipette solution (Fig 5). When 10 mM Na+ was included in the pipette solution, brief depolarization elicited [Ca2+]i elevations followed by recoveries showing pronounced plateaus (Fig 5 A, 323 ± 19 nM, n = 12) much like those seen in non-voltage-clamped cells after high K+ depolarization; peak [Ca2+]i elevations are larger than those elicited by high K+ presumably because depolarization and Ca2+ channel activation are more rapid under voltage clamp, leading to higher Ca2+ entry rates. This concentration of Na+ would be expected to enable mitochondrial Na+/Ca2+ exchange based on studies of isolated mitochondria (half-maximal activation at ~23 mM [Na+]i, maximal activation at ~10 mM [Na+]i;
As with cells depolarized with high K+, the plateau observed under voltage clamp is depressed by CGP. Fig 5 B illustrates a [Ca2+]i response elicited by a depolarizing step from -70 to -10 mV, first in the presence of CGP (left) and then after washout (right). In the presence of CGP, the plateau is largely suppressed and there is a prolonged tail, possibly reflecting incomplete inhibition of Ca2+ release and/or release via a CGP-insensitive pathway. After washing out CGP, depolarization elicits a rise in [Ca2+]i followed by a pronounced plateau, even though the rise in [Ca2+]i was smaller, reflecting current rundown during the washout period (see Fig 5 B, right, inset). These responses are representative of ten cells in which Na+ was included in the pipette solution: in the presence of CGP, recoveries were marked by plateaus (123 ± 13 nM) that were significantly lower than those measured in the same cells in the absence of CGP (305 ± 13 nM, P < 0.001) (Fig 5 D).
When Na+ was not added to pipette solutions, depolarization elicited [Ca2+]i elevations that were followed by simple recoveries (Fig 5 C, left; n = 4) like those seen in Na+-containing cells in the presence of CGP (Fig 4 A). A second response elicited after treatment with CGP exhibited a recovery that was very similar to that seen in the absence of CGP (Fig 5 C, right), indicating that CGP has little effect in the absence of intracellular Na+ (D), supporting the conclusion that CGP specifically blocks the mitochondrial Na+/Ca2+ exchanger. The failure of [Ca2+]i to recover completely to prestimulation levels (Fig 5B and Fig C) is consistent with the development of a Ca2+ leak over these long experiments.
Relationship between the actions of CGP and FCCP.
If CGP specifically inhibits mitochondrial Ca2+ release via the Na+/Ca2+ exchanger, it should have no additional effect on [Ca2+]i responses elicited in the presence of FCCP. Fig 6 A shows responses induced by high K+ before and during exposure to FCCP, and then in the combined presence of FCCP and CGP. CGP had no additional effect after treatment with FCCP, indicating that it only modifies FCCP-sensitive (mitochondrial) Ca2+ transport and does not affect nonmitochondrial Ca2+ transport systems. Similar results were obtained in each of three cells. A different conclusion was reached by
When FCCP is applied at a higher concentration (10 µM) in the absence of extracellular Ca2+ during the plateau phase of recovery, it elicits a large [Ca2+]i transient ([Ca2+]i = 1,438 ± 396 nM, n = 7) but only a small [Ca2+]i rise when applied after [Ca2+]i recovers to basal levels (35 ± 14 nM, n = 7), providing another way to monitor Ca2+ loss from loaded mitochondria during the recovery. If CGP effectively inhibits mitochondrial Ca2+ release, then in the presence of the inhibitor, depolarization should still increase mitochondrial Ca2+ concentration, but the increase should persist even after resting [Ca2+]i is restored. To test this, cells were depolarized in the continued presence of a nearly saturating concentration of CGP, and then after [Ca2+]i recovered, they were challenged with FCCP (Fig 6 C). In the presence of CGP, FCCP elicited a large [Ca2+]i transient, in striking contrast to the small [Ca2+]i elevation seen under similar conditions in the absence of the blocker (Fig 6 D). Therefore, CGP does not prevent mitochondrial Ca2+ accumulation but does cause these organelles to retain their Ca2+ load. The ability of FCCP to discharge mitochondria under these conditions implicates a Ca2+ release pathway that senses mitochondrial membrane potential and is not blocked by CGP, possibly the Ca2+ uniporter. The observation that FCCP elicits a larger [Ca2+]i transient when applied at 10 µM in the presence of 4 µM CGP (Fig 6 C) than at 1 µM in the presence of 1 µM CGP (B) probably reflects a combination of incomplete block by CGP at the lower concentration (see prolonged tail during the recovery after the second depolarization in B) and slower Ca2+ release induced by FCCP at the lower concentration. Slow FCCP-induced Ca2+ release would also explain why rapid application of the protonophore at the lower concentration during the plateau only prolongs the recovery (four cells) in contrast to the large and rapid rise elicited by 10 µM FCCP (not shown).
Properties of the CGP-sensitive and -resistant components of Jmito. Taken together, the observations presented above indicate that CGP is a specific inhibitor of mitochondrial Ca2+ efflux via the Na+/Ca2+ exchanger. This compound was therefore used to dissect the net mitochondrial Ca2+ flux into its components. Fig 7 A shows [Ca2+]i responses elicited under control conditions (left), in the presence of a nearly saturating concentration of CGP (2 µM, middle), and in the presence of 1 µM FCCP after CGP washout (right). Using a strategy like that employed to separate the total Ca2+ flux into mitochondrial and nonmitochondrial components, CGP was used to separate Jmito into CGP-sensitive and -resistant components that are associated with mitochondrial Ca2+ release and uptake pathways.
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Fig 7 B plots Jmito and its CGP-resistant component (JCGP-res) versus [Ca2+]i during the recovery from the cell shown in panel A and collected results from ten cells (C). JCGP-res was calculated by subtracting JFCCP-res from the total flux measured in the presence of CGP (J+CGP) at corresponding values of [Ca2+]i. JCGP-res is an outward flux that increases steeply and montonically with [Ca2+]i, as expected for the mitochondrial uniporter under conditions where the electrochemical driving force for Ca2+ favors uptake. Importantly, this flux is non-zero even when bulk [Ca2+]i is as low as 200300 nM. Since these measurements were made after voltage-gated Ca2+ channels have closed and radial Ca2+ gradients have largely dissipated (
The CGP-sensitive component of Jmito, determined by subtracting JCGP-res at corresponding values of [Ca2+]i, is easily interpreted if CGP and JCGP-res satisfy conditions like those described above for FCCP and JFCCP-res: (a) CGP specifically and completely inhibits mitochondrial Ca2+ release, and (b) the CGP-resistant flux depends only on the magnitude of [Ca2+]i at each instant in time during the recovery and not on the history of [Ca2+]i. If these conditions are satisfied, JCGP-res gives the rate of mitochondrial Ca2+ uptake, and the net mitochondrial flux is the sum of JCGP-res and the CGP-sensitive component of Jmito (JCGP-sens) at corresponding values of [Ca2+]i, making it possible to calculate JCGP-sens as the difference between Jmito and JCGP-res.
Regarding specificity, the results described above indicate that CGP inhibits Na+-dependent mitochondrial Ca2+ release, and the following observations show that if CGP influences any other Ca2+ transport systems that contribute to the recovery, its effects are small. CGP does not alter resting [Ca2+]i (14 µM CGP, 50 cells) and therefore has little effect on Ca2+ transporters responsible for setting this [Ca2+]i level. Also, CGP does not influence [Ca2+]i recoveries after depolarization in cells already treated with FCCP (1 µM; e.g., Fig 6 A): in the absence of CGP, the fast and slow time constants of recovery were (s) 14.7 ± 3.8 and 249.7 ± 117.4, while in the presence of 1 µM CGP they were 17.7 ± 3.9 s and 271.3 ± 83.1, n = 3, NS). Finally, CGP does not modify [Ca2+]i recovery kinetics under perforated patch conditions when pipette solutions lack Na+ (Fig 5 C).
Two complementary approaches were used to determine if the CGP-resistent flux is defined by [Ca2+]i during the recovery. In these experiments, J+CGP was analyzed since it should be the sum of JCGP-res and JFCCP-res, and the latter flux component has already been shown to have this property. For each approach, mitochondrial Ca2+ release via the Na+/Ca2+ exchanger was inhibited and [Ca2+]i was elevated to different levels by weak and strong depolarization so that the subsequent recoveries could be compared. The approaches differed in the way Ca2+ release was inhibited. When release was inhibited by CGP (2 µM), recoveries were nearly identical over the common range of [Ca2+]i despite being preceded by very different [Ca2+]i elevations (Fig 7 D, see superimposed recoveries at right). Therefore, under the conditions of these experiments, the rate of Ca2+ removal in the presence of CGP depends only on the magnitude of [Ca2+]i at each time. The second approach used Na+-free pipette solutions under voltage clamp to suppress mitochondrial Ca2+ release without relying on CGP. Large and small [Ca2+]i responses were elicited and the ensuing recoveries were compared. Under these conditions, both fast and slow components of the recoveries were indistinguishable (Fig 7 E, recoveries are compared at right), demonstrating that, like the CGP-resistant component of the total flux, the Na+-insensitive component depends on [Ca2+]i but not its history or the state of mitochondrial Ca2+ loading.
Fig 7 B shows the CGP-sensitive flux (JCGP-sens) calculated by subtracting J+CGP from Jcont at corresponding values of [Ca2+]i; averaged results from 10 cells are presented in C. JCGP-sens represents net mitochondrial Ca2+ release and exhibits a U-shaped dependence on [Ca2+]i. As [Ca2+]i declines during the recovery, the magnitude of JCGP-sens rises from a small value near zero to a maximum when [Ca2+]i is near the plateau level (see arrow) and then declines as [Ca2+]i approaches the prestimulation level. The biphasic dependence of JCGP-sens on [Ca2+]i is also evident from the [Ca2+]i responses: CGP has little effect on the recovery rate during the initial rapid phase when [Ca2+]i is high, or on the final approach to prestimulation levels, when [Ca2+]i is low. It is only when [Ca2+]i is at intermediate levels that CGP-sensitive flux is a significant fraction of the total Ca2+ flux, rendering the recovery rate sensitive to CGP.
Components of the mitochondrial flux measured under voltage clamp. The components of the total Ca2+ flux were also measured under voltage clamp, which made it possible to examine the [Ca2+]i dependence of the fluxes over a wider [Ca2+]i range. The first set of experiments was designed to measure the nonmitochondrial Ca2+ flux and the uptake component of Jmito. Fig 8 A shows results from a cell with low internal Na+ to inhibit mitochondrial Ca2+ release via the Na+/Ca2+ exchanger. The total Ca2+ flux was then measured during the recovery after raising [Ca2+]i by a 2.3-s depolarization from -70 to -10 mV before (Jcont) and after exposure to 1 µM FCCP (JFCCP-res). The FCCP-sensitive component of the total flux was outwardly directed and increased steeply with [Ca2+]i, closely resembling JCGP-res measured in cells after high K+ depolarization (compare with Fig 7B and Fig C). Similar results were observed in 3/3 cells. The second set of experiments examined the CGP-sensitive component of Jmito (Fig 8 B). Ca2+ fluxes were measured in Na+-containing cells by depolarizing before (Jcont) and after exposure to CGP (J+CGP). The CGP-sensitive flux, obtained by subtraction, was inwardly directed and displayed a U-shaped [Ca2+]i dependence qualitatively like that seen with JCGP-sens measured in cells stimulated with high K+ (compare with Fig 7B and Fig C). Similar results were obtained in 4/4 cells. Overall, the similarity between these results and those obtained from cells depolarized with high K+ indicate that the properties of the component fluxes are largely independent of the method used to evoke voltage-sensitive Ca2+ entry, and depend primarily on the size of the cytosolic Ca2+ load.
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To summarize, Jmito can be separated into uptake and release components based on their sensitivity to CGP: Mitochondrial Ca2+ uptake is steeply dependent on [Ca2+]i and becomes the dominant mitochondrial Ca2+ transport pathway when [Ca2+]i is high (>~500 nM). Uptake still occurs when [Ca2+]i is as low as 200300 nM, but it is opposed by release at comparable rate, accounting for the small net mitochondrial Ca2+ flux. Under these conditions, the relative rates of uptake and release are critical in defining the net mitochondrial Ca2+ flux. Since the uniporter is the main pathway for mitochondrial Ca2+ uptake and the Na+/Ca2+ exchanger is the principle route for neuronal mitochondrial Ca2+ release (
Properties of the Component Fluxes Explain the Complex Time Course of Recovery
The kinetics of the [Ca2+]i recovery can be understood in terms of Jcont and its components. Fig 9 A shows a [Ca2+]i response elicited by a 70 mM K+ depolarization with the four phases of recovery (iiv) indicated. Fig 9 B shows the time course of Jcont (thick trace) and its mitochondrial and nonmitochondrial components (thin traces); Fig 9 C illustrates Jmito and its components on the same scale. Also shown is the time course of the integrated mitochondrial Ca2+ flux (Fig 9 A, dotted trace) which provides a measure of the change in mitochondrial Ca2+ concentration ([Ca2+]m(i)) from its resting value after recovery is complete (see Appendix). During recovery phase i (see Fig 9A and Fig B), Jcont is large and outward because of the combined effects of strong mitochondrial Ca2+ uptake and weak extrusion across the plasma membrane. As a result,
[Ca2+]m(i) rises and [Ca2+]i falls rapidly. As [Ca2+]i falls, JFCCP-res declines and Jmito changes sign to become an inward flux, which causes
[Ca2+]m(i) to fall and the [Ca2+]i recovery to be slowed (phase ii). Jcont reaches a minimum near zero when the opposing fluxes JFCCP-res and Jmito are nearly balanced, and then rises because the inward flux Jmito decays more rapidly than the outward flux JFCCP-res, causing the recovery to accelerate. Finally, during phase iv, Jmito approaches zero and the [Ca2+]i recovery is controlled by net Ca2+ extrusion. Note that while the initial rapid phase of recovery is dominated by mitochondrial Ca2+ transport, and the final phase is dominated by net Ca2+ extrusion, the intermediate phases (iiiii) are influenced similarly by net mitochondrial Ca2+ release and net Ca2+ extrusion.
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The dynamics of Jmito can be understood in terms of its components (Fig 9 C). During phase i, Jmito is large and outward because Juni is much larger in magnitude than the inward flux JNa/Ca, which is close to zero. Mitochondrial Ca2+ accumulation causes [Ca2+]i to fall, which is accompanied by a decline in Juni and a rise in the inward flux JNa/Ca. Together, these changes cause Jmito to change sign to become an inward flux. Then Juni and JNa/Ca decline at approximately the same rate, so that for a time Jmito is a nearly constant inward flux that maintains [Ca2+]i near the plateau level. As both fluxes approach zero during phase iii, the inward flux Jmito declines, net Ca2+ extrusion becomes unopposed and the recovery accelerates (see Fig 9 B). Note that while Ca2+ uptake via the uniporter is the dominant component of the net mitochondrial flux during phase i of the recovery, uptake and release fluxes have similar magnitudes during phases iiiii.
The results presented above show that even when [Ca2+]i is as low as 200300 nM during the recovery, the mitochondrial Ca2+ uptake pathway is active (Fig 7 B and 9 C). Thus, uptake should occur even during weak depolarizations that elevate [Ca2+]i to levels within this range. However, previous work has shown that FCCP has little effect under these conditions (
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DISCUSSION |
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Summary of Main Results
This study describes the interplay between Ca2+ transport systems that restore the resting distribution of Ca in sympathetic neurons after depolarization-evoked Ca2+ entry. Cells were treated with thapsigargin to inhibit SERCA pump activity and minimize contributions from ER Ca2+ transport, and the remaining Ca2+ flux was separated into mitochondrial and nonmitochondrial components. It was found that mitochondria are powerful Ca2+ sequestration organelles (
The net mitochondrial Ca2+ flux was separated into components representing distinct Ca2+ uptake and release components. It was found that mitochondrial Ca2+ uptake is steeply dependent on [Ca2+]i, as expected for the mitochondrial uniporter, and occurs even when [Ca2+]i is as low as 200300 nM. Mitochondrial Ca2+ release requires intracellular Na+ and is blocked by CGP 37157, a specific inhibitor of the mitochondrial Na+/Ca2+ exchanger, indicating that release depends on activity of this transporter. Because of its high transport rate and steep [Ca2+]i dependence, the uptake pathway dominates the net mitochondrial Ca2+ flux when [Ca2+]i is high (>~400500 nM), while both uptake and release pathways make comparable contributions to the net flux when [Ca2+]i is lower. Mitochondrial Ca2+ transport also occurs during weak depolarization when [Ca2+]i rises to low levels (~300 nM). Under these conditions, the net mitochondrial Ca2+ flux is small, representing the sum of much larger uptake and release fluxes that nearly cancel one another. Recent studies have shown that similar [Ca2+]i elevations stimulate mitochondrial Ca2+ accumulation in heart cells (e.g.,
Properties of the Measured Fluxes
The FCCP-resistant flux was measured after treatment with Tg and FCCP, so it probably represents predominantly net Ca2+ transport across the plasma membrane. Additional support for this conclusion is provided by the finding that JFCCP-res is reduced by ~90% after removal of extracellular Na+ and addition of La3+ (15 mM, unpublished observations). This flux increases with [Ca2+]i and levels off at high [Ca2+]i, possibly indicating saturation of the underlying extrusion systems. Since JFCCP-res was defined by [Ca2+]i at each instant in time during the recovery, the underlying transport systems appear to have little intrinsic time dependence under the conditions of our experiments.
The CGP-resistant component of the net mitochondrial Ca2+ flux represents mitochondrial Ca2+ uptake and shows a steep dependence on [Ca2+]i (Hill coefficient ~2, see accompanying study) as expected for the mitochondrial uniporter (
The CGP-sensitive flux represents mitochondrial Ca2+ release and shows an apparent U-shaped dependence on [Ca2+]i. However, even though JNa/Ca varies with [Ca2+]i, it is not clear that it actually depends on [Ca2+]i (i.e., is a function of [Ca2+]i) and a dependence on other factors is likely, such as the concentration of intramitochondrial free Ca ([Ca2+]m). [Ca2+]m(i) as a basis for estimating changes in mitochondrial Ca concentration, such a model provides a reasonable quantitative description of JNa/Ca, although regulation by other factors (e.g., intramitochondrial Na+) is also possible (see accompanying study).
Interplay between the Components of the Total Ca2+ Flux
This study illustrates how net mitochondrial Ca2+ transport and Ca2+ transport across the plasma membrane contribute to [Ca2+]i dynamics. It also shows how the rate of net mitochondrial Ca2+ transport depends on the relative rates of uptake and release. When [Ca2+]i is high (<~300400 nM) uptake is much more powerful than release, accounting for strong mitochondrial Ca2+ accumulation. When [Ca2+]i is low (~200300 nM), mitochondrial Ca2+ uptake and release occur at comparable rates, accounting for the small net mitochondrial Ca2+ flux. Moreover, since the component fluxes are large compared with the net flux, modulation of either the uptake or release rate would have a large impact on the net mitochondrial Ca2+ flux. Also, since the rate of release depends on the intramitochondrial Ca2+ concentration, which in turn depends on the history of [Ca2+]i (see accompanying study), the net mitochondrial Ca2+ flux at low [Ca2+]i should be sensitive to stimulus history (see accompanying study).
The interplay between net mitochondrial Ca2+ transport and net Ca2+ extrusion across the plasma membrane is also important in determining the [Ca2+]i level reached during depolarization, but in this case the relative rates of mitochondrial Ca2+ accumulation and net Ca2+ entry are critical. Assuming an initial steady-state before depolarization in which [Ca2+]i is at its resting level and the net mitochondrial flux is zero, a small steady rise in [Ca2+]i induced by weak depolarization would be expected to stimulate the mitochondrial Ca2+ uptake pathway, creating an imbalance between uptake and release which leads to net Ca2+ accumulation. The resulting increase in [Ca2+]m would be expected to increase the rate of release, eventually leading to a new steady-state in which release and uptake balance. Indeed, during maintained exposure to 30 mM K+ which raises [Ca2+]i to ~300 nM, mitochondrial Ca accumulation does occur (
In the accompanying study, JFCCP-res, Juni and JNa/Ca are described quantitatively and incorporated into a model of Ca2+ dynamics. The model reproduces the recovery time course with its four distinct phases, the effects of graded inhibition of the Na+/Ca2+ exchanger by CGP (Fig 4), and accounts for the actions of CGP and FCCP on responses to weak depolarization. The model also clarifies the relationship between the [Ca2+]i plateau level and the previously described mitochondrial set-point.
Comparison with Other Studies
We found that mitochondria accumulate Ca at a rate of ~400 nM/s (nmol Ca2+/li effective cytosolic vol/s) when [Ca2+]i ~0.8 µM (Fig 8 A), in general agreement with results from rat chromaffin cells (iT, see Appendix) of total to free cytosolic Ca concentration (~200; Friel, D.D., unpublished observations) provides an estimate of the free cytosolic Ca2+ flux of 92 nM/s, placing a lower limit on the rate of uptake via the uniporter that is consistent with the value obtained in the present study, ~100 nM/s at ~500 nM, (Fig 7 B and 8 A).
Our measurements of Juni and JNa/Ca can only be compared with results obtained from isolated mitochondria, since measurements of these fluxes in situ have not been reported previously. At a membrane potential of 150 mV and external Ca2+ concentration of 500 nM, uptake by isolated rat liver mitochondria occurs at ~4 nmol/mg prot/min (
Integrating the net mitochondrial Ca2+ flux during the entire recovery provides a measure of the depolarization-evoked increase in mitochondrial total Ca concentration referred to the effective cytosolic volume ([Ca2+]m(i), Appendix Eq. 9; e.g., dotted trace in Fig 9 a). This quantity can be compared with measured changes in total mitochondrial Ca concentration induced by high K+ depolarization. For example, during the recovery that follows a ~13-s exposure to 50 mM K+,
[Ca2+]m(i) declines by ~1,000 nM. This may be interpreted as the change in [Ca2+]i that would result if the entire stimulus-evoked mitochondrial Ca load at the instant of repolarization were distributed within a closed compartment having the same effective volume as the cytosol. Estimating the ratio of mitochondrial and cytosolic volume as above (~0.1) and the ratio of mitochondrial and cytosolic Ca2+ buffering strength as ~4,000/200 = 20 (
What Is the Physiological Role of Mitochondrial Calcium Transport at Low [Ca2+]i?
Mitochondrial Ca2+ transport may play a role in modulating cytosolic [Ca2+] signals (
Traditionally, mitochondrial Ca2+ uptake has been viewed as a low affinity process that comes into play only when [Ca2+]i reaches high levels (>~0.51 µM). However, results in the present study indicate that mitochondrial Ca2+ uptake via the uniporter occurs even when [Ca2+]i is much lower (200300 nM). What is the physiological role of Ca2+ uptake at such low [Ca2+]i? One possibility arises in the context of Ca2+-regulated mitochondrial ATP production (
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Acknowledgements |
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The authors thank Drs. S.B. Andrews, S.W. Jones, D. Kunze, R.S. Lewis, and R.W. Tsien for their helpful comments on an earlier version of the manuscript.
This work was supported by grants from the American Heart Association (no. 96011490) and from the National Institutes of Health (NS 33514-03).
Submitted: 23 September 1999
Revised: 30 December 1999
Accepted: 5 January 2000
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Appendix |
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Relationship between Mitochondrial and Cytosolic Ca Fluxes
The net mitochondrial Ca2+ flux (Jmito) measured in this study can be compared with the rate of total mitochondrial Ca transport deduced from electron probe microanalysis (be the total net Ca2+ flux between all mitochondria and the cytosol at an instant in time (e.g., in mmol/s). This flux would cause mitochondrial total Ca concentration to change at a rate
/vm (e.g., mM/s), where vm is the mitochondrial volume. It would also cause the total cytosolic Ca concentration to change at a rate
/vi (vi is the cytosolic volume). If Ca2+ binding to cytosolic buffers reaches equilibrium rapidly and conforms to a single binding site model, this flux would cause the cytosolic free Ca concentration to change at a rate Jmito =
,where
iT is the ratio of the change in total Ca concentration that accompanies an infinitesimal change in [Ca2+]i (Equation 1):
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(1) |
Btotal,j is the total concentration of the jth cytosolic buffer and Kd,j is the corresponding dissociation constant. The superscript specifies that iTis the ratio of total to free Ca concentration to distinguish it from the bound to free ratio (e.g.,
/vm) is then:
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(2) |
If [Ca2+]i < < Kd,j for each buffer (Equation 3),
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(3) |
so that iTis independent of [Ca2+]i. Studies in several cell types have provided evidence that
iTis approximately constant when [Ca2+]i < 1 µM (e.g.,
The time integral of Jmito during the recovery provides information about the change in total mitochondrial Ca concentration [Ca]m from the instant of repolarization (t = 0) to time t. It is convenient to define the difference [Ca]m(t) between [Ca]m at time t and the resting value measured when t is large [Ca]m(
) (Equation 4):
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(4) |
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(5) |
Solving for /vm in Equation 2 above and substituting in Equation 5 gives Equation 6:
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(6) |
Assuming that the ratio of changes in total to free intramitochondrial Ca concentration (mT) is constant, the change in free mitochondrial Ca concentration is (Equation 7 and Equation 8):
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(7) |
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(8) |
where = (vm/vi)(
)is the ratio of effective mitochondrial and cytoplasmic volumes. Multiplying both sides of this equation by
shows that the integral of Jmito gives the change in [Ca2+]i that would result if the total mitochondrial flux from time 0 to t were deposited in a closed compartment having the same effective volume as the cytosol:
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(9) |
where the superscript (i) identifies this as a change in cytosolic Ca concentration that accompanies a change in [Ca]m.
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