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(Circulation Research. 1996;79:194-200.)
© 1996 American Heart Association, Inc.

Articles

Ca²⁺ Influx During the Cardiac Action Potential in Guinea Pig Ventricular Myocytes

C.J. Grantham, M.B. Cannell

the Department of Pharmacology and Clinical Pharmacology, St George's Hospital Medical School, London, UK.

Correspondence to M.B. Cannell, Department of Pharmacology and Clinical Pharmacology, St George's Hospital Medical School, Cranmer Terrace, London SW13 0RE, UK. E-mail mcannell@mbcsg1.sghms.ac.uk.

	Abstract

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The relative contributions of L-type Ca²⁺ current (I_Ca) and Na⁺/Ca²⁺ exchange to Ca²⁺ influx during the cardiac action potential (AP) are unknown. In this study, we have used an AP recorded under physiological conditions as the command voltage applied to voltage-clamped ventricular myocytes. I_Ca (measured as nifedipine-sensitive membrane current) had a complex multiphasic time course during the AP. Peak I_Ca was typically 4 pA/pF, after which it rapidly declined (to about 60% of peak) during the rising phase of the cell-wide Ca²⁺ transient before increasing to a second, more sustained component. The initial decline in I_Ca was sensitive to the amount of Ca²⁺ released by the sarcoplasmic reticulum (SR), and conditions that reduce the amplitude of the Ca²⁺ transient (such as rest or brief application of caffeine) increased net Ca²⁺ influx via I_Ca. Dissection of the Na⁺/Ca²⁺ exchange current at the start of the AP suggested that Ca²⁺ influx via Na⁺/Ca²⁺ exchange is less than 30% of that due to I_Ca. From these data, we suggest that I_Ca is the primary source of Ca²⁺ that triggers SR Ca²⁺ release, even at the highly depolarized membrane potentials associated with the AP. However, Ca²⁺ influx via Na⁺/Ca²⁺ exchange is not negligible and may activate some Ca²⁺ release from the SR, especially when I_Ca is reduced. We propose that SR Ca²⁺ release inhibits I_Ca within the same beat, thereby providing a negative feedback mechanism that may serve to limit Ca²⁺ influx as well as to regulate the amount of Ca²⁺ stored within the SR.

Key Words: action potential • I_Ca • Na⁺/Ca²⁺ exchange • sarcoplasmic reticulum Ca²⁺ release • heart

	Introduction

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In cardiac muscle, it is widely accepted that Ca²⁺ release from the SR and E-C coupling are tightly coupled to Ca²⁺ influx across the sarcolemma via a process called "Ca²⁺-induced Ca²⁺ release" (CICR).¹ Although Ca²⁺ influx via voltage-dependent L-type Ca²⁺ channels has been considered to be the main mechanism by which CICR is triggered during depolarization,^{2 3 4 5} Na⁺/Ca²⁺ exchange can also bring Ca²⁺ into the cell.^{6 7 8} Thermodynamic calculations indicate that, during the upstroke of the cardiac AP, the exchanger may reverse transport direction and bring Ca²⁺ into the cell before [Ca²⁺]_i rises.^{9 10} The ability of "reverse-mode" Na⁺/Ca²⁺ exchange to trigger SR Ca²⁺ release was first reported by Berlin et al¹¹ in a Ca²⁺-overloaded cardiac preparation, and Bers et al¹² demonstrated that twitch contractions could be evoked when I_Ca was blocked by nifedipine if [Na⁺]_i was elevated to between 15 and 20 mmol/L (a condition which enhances Ca²⁺ influx via the exchanger). More recently it has been proposed that Na⁺/Ca²⁺ exchange may have a role in triggering SR Ca²⁺ release under physiological conditions as a result of the elevation of subsarcolemmal [Na⁺]_i by the Na⁺ current,^{13 14 15} and Levi et al^{16 17} have suggested that the exchanger could be a major trigger for CICR during the AP. Additional support for a role of the exchanger in triggering SR Ca²⁺ release has come from experiments using exchanger-inhibitory peptide¹⁸ and temperature changes.¹⁹ However, other studies have suggested that I_Ca is more important than I_Na/Ca in triggering SR Ca²⁺ release.^{20 21 22 23} Therefore, the relative contribution of I_Na/Ca and I_Ca to cardiac Ca²⁺ influx during the AP remains unclear.

A voltage-clamp technique which uses the physiological cardiac AP as the voltage command has been used to examine some of the membrane currents underlying the cardiac AP.^{24 25} We have used this technique to examine Ca²⁺ influx in guinea pig cardiac ventricular myocytes during the AP at physiological temperatures and [Na⁺]_i. Our data suggest that I_Ca is the major source of Ca²⁺ influx during the cardiac AP, although the contribution of I_Na/Ca is not insignificant. Furthermore, we propose that there is tight local control of Ca²⁺ influx into the cardiac ventricular myocyte on a beat-to-beat basis, mediated by a feedback mechanism resulting from the interaction between I_Ca and the SR.

	Materials and Methods

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Cell Dissociation
Ca²⁺-tolerant guinea pig ventricular myocytes were isolated according to methods described in detail elsewhere.²⁶ Briefly, male guinea pigs (Dunkin-Hartley, Harlan Interfauna, Huntingdon, Cambridgeshire, UK; 250 to 300 g) were killed by cervical dislocation followed by exsanguination. Hearts were removed and perfused for 5 minutes at 35°-37°C as a Langendorff preparation, with a nominally Ca²⁺-free, oxygenated isolation solution containing (in mmol/L): NaCl 120, KCl 5.4, MgCl₂ 5, HEPES 10, glucose 20, sodium pyruvate 5, taurine 20; pH 7.1 (adjusted with NaOH). Protease (type XXIV, 0.2 mg/mL) was then introduced, together with 80 µmol/L CaCl₂, into the isolation solution for 2 minutes. The solution was switched to one containing collagenase (type II, 0.3 mg/mL) in isolation solution containing 80 µmol/L CaCl₂ for 5 minutes. The ventricles were cut free, minced in fresh isolation solution (100 µmol/L CaCl₂), and stored at room temperature for up to 6 hours after isolation.

Electrophysiology
AP Recording
Myocytes were placed in a perfusion chamber on the stage of a Nikon Diaphot inverted microscope (Nikon Instruments Inc) and superfused with a bathing solution containing (in mmol/L): NaCl 135, KCl 5.4, CaCl₂ 2.5, MgCl₂ 1, HEPES 10, glucose 10, NaH₂PO₄ 0.33, sodium pyruvate 1; pH 7.4, at 35°-37°C. APs were recorded with conventional microelectrodes with resistances >5 M, made from thin-walled borosilicate glass with a Flaming-Brown P-87 micropipette puller (Sutter Instrument Co) and filled with a pipette solution containing (in mmol/L): KCl 30, aspartic acid 80 to 110, HEPES 10, NaCl 10, MgATP 5; pH 7.2 (titrated with KOH). Cardiac APs were evoked by 2-ms depolarizing current steps from an Axoclamp-2 amplifier (Axon Instruments Inc) in bridge mode. Myocytes were stimulated at 3 Hz, and steady state APs (after 20 to 30 stimuli) were recorded digitally via a 12-bit digital-to-analog converter (DT 2801A, Data Translation) in a 386 PC computer running custom software. A representative AP was selected on the basis of the average properties of the APs obtained from at least six cells. Before using this AP, the measured junction potential (+10 mV) and the stimulus artifact at the leading edge of the upstroke were removed by subtraction. Although some AP clamp experiments have used the AP recorded from a cell as the voltage command for the same cell,²⁴ in these experiments we used the representative AP as the command voltage waveform. The rationale for this approach is that in intact tissue the cells experience the electrical behavior of a large number of cells (within the electrical space constant) and so do not necessarily experience the AP which would be recorded from an individual cell in isolation. Since we are interested in the amplitude of Ca²⁺ influx under "normal" conditions, we therefore used the representative AP waveform. (A similar approach was used by Arreolla et al²⁵ and Bouchard et al²³ ).

Whole-Cell Voltage Clamp
Cells were superfused at 35°-37°C with the same solution used for AP recording and voltage clamped in the whole-cell configuration²⁷ with 1 to 2 M electrodes containing a K⁺-based intracellular pipette solution (as described above except where noted in the text). K⁺ conductances were suppressed by addition of either 20 mmol/L CsCl or 0.1 mmol/L BaCl₂ to the bathing solution. Cells were clamped with either the representative AP waveform or voltage steps generated by pCLAMP software (Axon Instruments Inc). All electrical records were digitized and recorded on videotape for off-line analysis. Membrane capacitance currents were subtracted and current densities calculated from the cell capacitance.

Intracellular Ca²⁺ Measurement With Fluo 3
In some experiments fluo 3 (30 to 100 µmol/L) was included in the intracellular solution so that changes in intracellular free Ca²⁺ could be monitored. The inclusion of fluo 3 had no noticeable effect on the time course of membrane currents reported here. The fluo 3 was illuminated at 470 to 480 nm with light derived from a xenon arc lamp (Amco) and delivered to the epifluorescence port of the Nikon Diaphot microscope via a fiber-optic light guide. Emitted light was measured at 525 nm with a photomultiplier tube (Thorn EMI) and recorded as a photocurrent. For analysis, fluorescence records were corrected by subtracting background fluorescence (measured after gigaseal formation) and normalized to the average resting fluorescence during the prestimulation period (F_stimulus/F_rest).

Chemicals
Chemicals were of analar grade and were supplied by Sigma-Aldrich Company Ltd, BDH, and Mallinckrodt, with the exception of collagenase, which was from Worthington Biochemical Corp and fluo 3, which was from Molecular Probes Inc.

	Results

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To provide a representative AP for this study, APs which met the criteria of a resting potential <-80 mV accompanied by vigorous contractions (without aftercontractions) were selected for analysis (see the Table). From these data, an AP which fell in the middle of the range of values observed was selected for use (Table and Fig 1A) in all experiments described below.

View this table: [in this window] [in a new window]   Table 1. Data From Six Cardiac Action Potentials at 3 Hz and 37°C

View larger version (19K): [in this window] [in a new window]   Figure 1. Whole-cell currents in guinea pig ventricular myocytes voltage clamped with either an AP or voltage steps spanning the range of AP plateau potentials in the presence of 10 mmol/L BAPTA with no Na+ in the pipette. A, Voltage command protocols and cadmium-sensitive membrane currents evoked by the AP or voltage steps. The first 2 ms of each stimulus were ignored (due to saturation of the digital-to-analog converter) and have been replaced by a dotted line. B, Whole-cell current-voltage relationship of the same cell. C, Integrated Ca2+ influx in response to an AP or voltage steps (as indicated) from the experiment shown in A. Cell capacitance, 150 pF. Similar results were obtained in at least four other cells. Vm indicates membrane potential; Im, membrane current.

I_Ca During the AP in the Presence of BAPTA
We first examined the I_Ca time course during an AP under conditions that are frequently used to isolate I_Ca. Thus the pipette filling solution contained BAPTA (see Fig 1 legend) to suppress Ca²⁺-dependent currents, and no Na⁺, to prevent outward I_Na/Ca. Fig 1A shows the cadmium-sensitive current (I_Ca) elicited by either an AP or voltage steps. In response to the AP, the I_Ca activated rapidly to a peak of -6.8 pA/pF and then decayed to 80% of the peak within 20 ms. Thereafter, I_Ca increased gradually to reach a second peak of -6.4 pA/pF before decaying to zero at a membrane potential of -39 mV. For comparison, a voltage step to +5 mV generated a rapidly activating I_Ca whose peak value was -21 pA/pF and which decayed monotonically throughout the pulse. Voltage steps to more depolarized potentials produced progressively smaller peak inward currents with similar time courses until, at +50 mV, the peak I_Ca was -5.6 pA/pF. The whole-cell current-voltage relationship under these conditions is summarized in Fig 1B. The threshold for I_Ca activation was about -40 mV; it reached a peak value of -21 pA/pF at +5 mV and reversed at +70 mV.

The time course of Ca²⁺ influx during these voltage-clamp protocols was examined by integrating the current records (Fig 1C). These data show that nearly all of the Ca²⁺ entry (4.5 amol/pF) occurred within 150 ms of the start of the AP. Similar quantities of Ca²⁺ entered during voltage-clamp steps to between +5 and +35 mV (4.7 and 4.0 amol/pF, respectively, at 150 ms). However, the entry of Ca²⁺ was larger at earlier times during voltage steps than during the AP except during steps to +50 mV, when the total influx was much smaller than that associated with the AP. APs at either extreme of the range recorded (see the Table) produced qualitatively similar results (data not shown). The AP ultimately produces a Ca²⁺ flux comparable with that resulting from steps to the peak of the I_Ca activation curve, an observation that can be explained by the more sustained nature of Ca²⁺ influx during the AP.

Fig 2A shows the steady state membrane current recorded when the Na⁺/Ca²⁺ exchange and SR release were active (ie, with 10 mmol/L Na⁺ and no BAPTA in the pipette). Under these conditions, the AP stimulated a [Ca²⁺]_i transient which increased biphasically throughout most of the AP (Fig 2A) before starting to decline about 140 ms after the AP start (when the membrane potential was about 0 mV). The membrane current recorded under these conditions would include components due to I_Na/Ca as well as I_Ca. To dissect these components, nifedipine (10 µmol/L) was rapidly applied between stimuli, which revealed a residual outward current during the plateau of the AP. This current reversed at about -50 mV, to become an inward tail current decaying toward zero along a time course with fast and slow components (Fig 2A, right side). The [Ca²⁺]_i transient was not abolished by the application of nifedipine, although its time course and amplitude were quite different (see below).

View larger version (15K): [in this window] [in a new window]   Figure 2. Membrane currents and Ca2+ influx in a myocyte with intact Na+/Ca2+ exchange and SR release in response to an AP. The myocyte was at steady state, having been stimulated continuously at 0.2 Hz with a train of at least 20 APs. A (top), AP command; (middle), membrane current; and (bottom), [Ca2+]i-transient time course in the absence of nifedipine or, on the right, immediately after blockade of ICa by 10 µmol/L nifedipine. B, Time course of AP-evoked nifedipine-sensitive current (ICa) and calculated INa/Ca. C, Cumulative Ca2+ influx via ICa () and the calculated INa/Ca () at the beginning of the AP. Cell capacitance, 150 pF. Similar results were obtained in at least four other cells.

The nifedipine-sensitive current (labeled as I_Ca in Fig 2B) was multiphasic, the initial component being an early transient peak of -3.9 pA/pF, which decayed within 20 ms to a long-lasting plateau component with a peak amplitude of -2.4 pA/pF. In the presence of nifedipine, Na⁺/Ca²⁺ exchange should be the primary mechanism moving Ca²⁺ across the sarcolemma²⁸ so that the quantity of Ca²⁺ entering via the exchanger should be equal to its extrusion (assuming the SR content is constant). Thus, the time integral of the residual outward exchange current should equal the time integral of the inward exchange current. However, the outward current recorded in the presence of nifedipine was larger than the inward current (Fig 2A), an observation that can be explained by the presence of a residual leak current.

To account for this leak current, we solved the equation by adjusting G_leak (the leak conductance) and E_leak (the reversal potential of the leak current) so that the time integral of I_Na/Ca was zero (with the assumption that the leak was linear). For the experiment shown in Fig 2, G_leak was calculated to be 13.5 pS/pF, with a reversal potential of -81 mV. This value for G_leak is equivalent to an input resistance of 0.5 G. From this analysis, the calculated I_Na/Ca (Fig 2B) increased to reach a peak value of 1.39 pA/pF 25 ms after the peak of the I_Ca, before declining and reversing 170 ms after the start of the AP. The calculated I_Na/Ca had a value of 0.55 pA/pF when peak I_Ca occurred, which would correspond to a Ca²⁺ influx rate equivalent to an I_Ca of 1.1 pA/pF. In addition, I_Na/Ca reversed direction at about the time that I_Ca had decreased to zero, a relationship that would limit futile cycling of Ca²⁺ across the sarcolemma.

The rate of activation of the Ca²⁺ transient immediately after the application of nifedipine was markedly decreased, being five times slower than the control Ca²⁺ transient (Fig 2A). A similar reduction in the rate of rise of the transient was observed in six other cells. From these results, we conclude that I_Ca provides a larger trigger for SR Ca²⁺ release than I_Na/Ca. This view is consistent with the current densities calculated above, with the exchanger producing a Ca²⁺ influx of about 28% of that due to I_Ca 3.5 ms after the start of the AP. Fig 2C shows that the influx due to I_Ca was always greater than that due to the exchanger during the rapid phase of SR Ca²⁺ release. In five cells, the mean Ca²⁺ influx (±SEM) 6 ms after the beginning of the AP was 0.057±0.005 amol/pF for I_Ca and 0.012±0.005 amol/pF for I_Na/Ca, suggesting that Na⁺/Ca²⁺ exchange contributes about 20% of the total Ca²⁺ influx during the upstroke of the [Ca²⁺]_i transient. Ca²⁺ influx via the exchanger should be less than that estimated above, since in the presence of nifedipine, reverse-mode exchange should have increased in response to the reduced SR release (see "Discussion").

It has been suggested that I_Ca inactivation may be partly due to Ca²⁺ release from the SR.^{21 29} If this suggestion is correct, the rapid early phase of I_Ca inactivation observed during the AP (see Fig 2B) may be related to the time course of Ca²⁺ release from the SR. In mammalian cardiac preparations, SR Ca²⁺ content changes with periods of rest,^{30 31} and resumption of stimulation in guinea pig myocytes gradually loads the SR with Ca²⁺. Fig 3A shows a [Ca²⁺]_i transient and nifedipine-sensitive current from a rested myocyte in response to an AP. With the first stimulus, the [Ca²⁺]_i transient rose with a slow time course. After 30 APs, the [Ca²⁺]_i transient was in steady state (solid line, Fig 3B) and increased more rapidly after depolarization to much higher levels. The nifedipine-sensitive current associated with the first AP (Fig 3A) was larger (peak -4.5 pA/pF) and decayed almost monotonically throughout the AP. The steady state nifedipine-sensitive current (solid line, Fig 3B) was similar to that shown in Fig 2B and displayed rapid inactivation from a peak of -3.5 pA/pF to a more sustained plateau. The differences in time course and amplitudes of the Ca²⁺ transient and I_Ca are apparent from comparison of the two superimposed traces (see Fig 3B); the larger [Ca²⁺]_i transient is associated with a current that shows rapid early inactivation followed by a "hump" that persisted for most of the duration of the AP. Thus, after rest, I_Ca inactivated to only 80% of its peak value, whereas at steady state, the rapid inactivation resulted in I_Ca decreasing to 58% within 20 ms of the peak of the I_Ca.

View larger version (12K): [in this window] [in a new window]   Figure 3. Comparison of ICa and SR release in a myocyte in the rested state or at steady state. A, AP-evoked [Ca2+]i transient and nifedipine-sensitive current in a myocyte which had not been previously stimulated. B, [Ca2+]i transient and nifedipine-sensitive current in the same cell at steady state (SS) after 30 APs at 0.2 Hz. The rested-state responses from A are superimposed as dashed lines. Cell capacitance, 140 pF. Similar results were observed in five other cells.

The simplest interpretation of these data would be that the rapid phase of Ca²⁺ release by the SR produces a rapid inactivation of I_Ca. The difference between the current records cannot be explained by changes in I_Na/Ca magnitude (arising from changes in the amplitude of the Ca²⁺ transient) because outward I_Na/Ca should have been reduced by the increase in the amplitude of the [Ca²⁺]_i transient. In addition, when Na⁺ was omitted from the pipette (thereby disabling reverse-mode Na⁺/Ca²⁺ exchange) the nifedipine-sensitive current still decreased with stimulation from rest (not shown). Nevertheless, it is possible that changes in I_Ca gating may play some role in observed change in I_Ca time course from rest.³² To examine whether such an effect could explain the changes in I_Ca time course, we used caffeine to change the amount of Ca²⁺ released by the SR in response to depolarization.

A myocyte was stimulated until a steady state [Ca²⁺]_i transient and membrane current were attained (Fig 4A). The nifedipine-sensitive current exhibited a peak value of -6.3 pA/pF, which decayed within 16 ms to a plateau value of -2.8 pA/pF. At this point, a low-Ca²⁺ bathing solution containing caffeine (see legend to Fig 4) was briefly applied to the myocyte to deplete the SR Ca²⁺ content. The action of caffeine was confirmed by the concomitant increase in fluorescence and a transient inward current (not shown) due to the Na⁺/Ca²⁺ exchange extruding the released Ca²⁺.³³ Immediately after the caffeine exposure, there was a decrease in both the rate of activation and the absolute magnitude of the [Ca²⁺]_i transient (Fig 4A, right side), as would be expected from the depletion of the SR Ca²⁺ content. There was also a small increase in the peak I_Ca (to -6.6 pA/pF), but a much larger increase in the plateau I_Ca (-3.5 pA/pF). With subsequent APs, the plateau phase of I_Ca declined in amplitude, whereas there was an increase in the rate of activation and absolute magnitude of the [Ca²⁺]_i transient toward control (precaffeine exposure) levels. The marked difference in effect of SR depletion on the peak and plateau phases of I_Ca can be seen more clearly in Fig 4B, in which I_Ca under control conditions, after SR depletion, and after the SR had reloaded are superimposed, showing that the main effect of SR depletion on I_Ca was an increase in the amplitude of the plateau phase of I_Ca. From these data, it is clear that the general pattern in the changes in I_Ca time course was very similar to that shown earlier, in which the SR Ca²⁺ load varied during the positive staircase; in both cases, increasing SR Ca²⁺ release leads to a significant decrease in the magnitude of the plateau of I_Ca.

View larger version (25K): [in this window] [in a new window]   Figure 4. Comparison of ICa and SR release in a myocyte in which the SR was depleted by application of caffeine (10 mmol/L) and low-Ca2+ (0.1 mmol/L) bathing solution. A, Steady state nifedipine-sensitive current and [Ca2+]i transient after 10 APs at 1 Hz. The right side of the panel shows the nifedipine-sensitive current associated with the first AP after the caffeine/low-Ca2+ solution was removed. B, Comparison of the time course of control ICa with ICa immediately after caffeine had unloaded the SR Ca2+ store and after a further 10 APs at 1 Hz. Cell capacitance, 112 pF. Similar results were obtained in three cells. C, Time course of conductance change due to SR depletion during the AP. The upper panel shows that the conductance changed rapidly, within 15 ms of the start of the AP. The lower panel shows that the change in fractional conductance precedes the increase in cytosolic Ca2+ measured with fluo 3. D, Comparison of ICa time course with and without Ca2+-dependent inactivation. The time course of ICa without Ca2+-dependent inactivation (d.f only), calculated by computing the Hodgkin-Huxley gating variables d and f from the equations given by Luo and Rudy34 and our AP time course, decayed almost monotonically. When a Ca2+-dependent gating variable, calculated from C, was included in the model, early inactivation of ICa (d.f.fCa) became pronounced, and the current decayed with a multiphasic time course. ICa is expressed in arbitrary units. G indicates change in Ca2+ conductance; G/G, fractional change in conductance.

The change in Ca²⁺ channel conductance observed after SR depletion was used to examine the time course of the development of Ca²⁺-dependent inactivation. The Ca²⁺ conductance G_t and Ca²⁺-dependent inactivation variable (f_Ca) are related by the following equation:

(E1)

where d_t,v and f_t,v are the Hodgkin-Huxley activation and inactivation gating variables, respectively, and G_max is the maximum Ca²⁺ conductance. Since the cell was stimulated by a constant AP waveform, d_t, f_t, and G_max can be combined into a single function of time [k(t)]:

(E2)

Hence,

(E3)

where G_t and f_Ca are the changes in G_t and f_Ca due to SR depletion. Fig 4C (upper panel) shows that the effect of altering SR load on G_t occurs very rapidly after the start of the AP and reaches a maximum within 15 ms. As shown by Equation 3, it is possible to examine the fractional change in the Ca²⁺-dependent gating variable (f_Ca/f_Ca) by examining the fractional change in conductance (G_t/G_t). Fig 4C (lower panel) shows that G_t/G_t starts to change within 1.6 ms of the start of the AP and rises more rapidly than the cytosolic Ca²⁺ level (as reported by the fluo 3 signal, which did not start to change until 2.4 ms after the start of the AP).

To examine the extent to which such changes in G_t, and hence f_Ca, can explain the time course of I_Ca recorded in these experiments, we computed d and f from the equations given by Luo and Rudy³⁴ and our AP time course. Fig 4D shows that the I_Ca calculated without any Ca²⁺-dependent inactivation rapidly declines to 25% of its peak value before decaying monotonically throughout the plateau of the AP. Adding a Ca²⁺-dependent gating variable (f_Ca) calculated from Fig 4C to this model gives an I_Ca which is remarkably similar to that shown in Fig 2B. We therefore conclude that rapid Ca²⁺-dependent inactivation of I_Ca due to SR Ca²⁺ release is an important determinant of I_Ca time course.

	Discussion

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Relative Contributions of I_Ca and I_Na/Ca to Ca²⁺ Influx
It has recently been suggested that Ca²⁺ influx via the Na⁺/Ca²⁺ exchanger, rather than voltage-dependent Ca²⁺ channels, could be a major mechanism for triggering SR release of Ca²⁺ and cardiac contraction.^{13 14 15 16 17} However, to our knowledge there have been no previous measurements of the current carried by the exchanger during the cardiac AP nor has there been a comparison between Ca²⁺ current density and Na⁺/Ca²⁺ exchange current density. Our data suggest that reverse I_Na/Ca provides <30% of the Ca²⁺ influx provided by I_Ca (at the time of peak I_Ca, I_Na/Ca was 0.55 pA/pF and I_Ca was 3.9 pA/pF). Therefore, although the I_Ca decreases and reverse I_Na/Ca increases with increasing depolarization,¹⁶ at plateau potentials, Ca²⁺ influx through Ca²⁺ channels is greater than via Na⁺/Ca²⁺ exchange. Nevertheless, the contribution to Ca²⁺ influx from the exchanger is not negligible and may become more important if I_Ca is reduced.

Since our estimate of exchanger current density was obtained at a time when I_Ca and SR Ca²⁺ fluxes were inhibited, it is likely that we have overestimated the exchanger current density. SR Ca²⁺ release follows the activation of the Ca²⁺ current with a delay <<2 ms,³⁵ therefore the local Ca²⁺ in the region where E-C coupling occurs must rise rapidly to high levels. It has been estimated in a theoretical study³⁶ that [Ca²⁺]_i within 20 nm of an open Ca²⁺ channel reaches 10 µmol/L. For comparison, Cannell et al⁵ calculated that the local [Ca²⁺]_i required to trigger CICR would have to be about 15 µmol/L at the SR release site. Another study³⁷ calculated that the local [Ca²⁺]_i 20 nm from an open SR release channel would be about 56 µmol/L. Although the exact proximity of the Na⁺/Ca²⁺ exchange molecule to either the Ca²⁺ channel or the SR release channel is unknown, such increases in [Ca²⁺]_i in the vicinity of the exchanger would reduce its ability to import Ca²⁺ and so should reduce the exchanger current density to less than that estimated above. More direct measurement of I_Na/Ca during the Ca²⁺ transient is problematic because of the concomitant activation of Ca²⁺-dependent nonselective cation current.^{38 39} Since we measured the exchanger current density when SR release was reduced, the contribution of Ca²⁺-activated nonselective current should have been minimized. However, any activation of nonselective current in our experiments would also cause the exchanger current density to be overestimated. These considerations further reinforce our view that the actual exchanger current density must be less than that estimated above.

I_Na/Ca and Its Role in E-C Coupling
The Ca²⁺ transient recorded here consisted of two phases: an initial rapid upstroke followed by a slower rise. The initial rapid component appeared to reflect the amount of SR Ca²⁺ release,⁴⁰ since it was severely depressed after exposure to caffeine (Fig 4A), ryanodine, and cylcopiazonic acid (not shown). This component was strongly inhibited by nifedipine, as the rate of rise of the Ca²⁺ transient was reduced by a factor of about 5 when I_Ca was inhibited (see Fig 2A). This observation (together with relative current densities of I_Ca and I_Na/Ca at the beginning of the AP) supports the view that I_Ca is the primary trigger for SR Ca²⁺ release, although a possible decrease in SR Ca²⁺ content during nifedipine exposure may have also contributed this effect.

It is possible that the Ca²⁺ influx via I_Ca (as well as Ca²⁺ released from the SR) may stimulate the exchanger by a catalytic effect,⁴¹ which would explain why the exchanger current reached a peak after [Ca²⁺]_i started to rise. If this suggestion is correct, then it raises the possibility that the exchanger may be effectively controlled by I_Ca and SR Ca²⁺ fluxes to increase Ca²⁺ efflux when Ca²⁺ influx or SR Ca²⁺ content is increased (and would not simply follow the electrochemical gradient for the Na⁺/Ca²⁺ exchange reaction). Such a catalytic mechanism would provide an additional negative feedback pathway for the local control of Ca²⁺ transient amplitude^{5 20} and help ensure the cell-wide uniformity of the Ca²⁺ transient.

Time Course of I_Ca During the AP
We were able to predict the observed steady state I_Ca time course (Fig 3B) with d and f Hodgkin-Huxley gating variables computed from the model of Luo and Rudy,³⁴ together with a Ca²⁺-dependent component derived from our experiments (Fig 4C). When the Ca²⁺-dependent variable was omitted from the model, the I_Ca time course became similar to that seen in cells in which SR load was depleted (Fig 3A), suggesting that SR Ca²⁺ release exerts a rapid and strong inactivation of I_Ca. This, together with the observation that the fractional change in Ca²⁺-dependent inactivation precedes a measurable rise in cytosolic Ca²⁺, supports the idea that SR Ca²⁺ release regulates I_Ca in a subsarcolemmal space,²¹ where local Ca²⁺ gradients are more important than bulk intracellular Ca²⁺.

SR-Mediated I_Ca Inactivation and Its Role in E-C Coupling
Comparison of the Ca²⁺ transients obtained from rest and immediately after caffeine exposure (Figs 3A and 4A) suggests that when the rate of Ca²⁺ release from the SR is reduced, the rapid inactivation of I_Ca is also reduced. It is possible that the inactivation of I_Ca during the rested-state response represents Ca²⁺-dependent inactivation of I_Ca via Ca²⁺ entry through the Ca²⁺ channel itself.^{42 43 44} However, the SR release–mediated component of I_Ca inactivation appears to be larger than that due to I_Ca, a fact that may be explained by the relatively higher Ca²⁺ flux associated with SR release than by I_Ca^{40 45}

From previous discussion, the decrease in I_Ca inactivation seen after caffeine exposure would be expected from the reduction in SR Ca²⁺ release. However, caffeine application also resulted in a small increase in peak I_Ca (4%). While we cannot exclude the possibility that this small effect on peak I_Ca may be related to inhibition of phosphodiesterase,⁴⁶ the disproportionate increase in the plateau phase of I_Ca suggests that such a mechanism is not responsible for the reduction in I_Ca inactivation. This view is supported by the lack of effect of prior caffeine exposure on the holding current (which would be altered if cAMP levels increased⁴⁷ ), suggesting that possible cAMP-mediated effects were not significant when the records were obtained.

The rapid inactivation of I_Ca results in a significant decrease in the net Ca²⁺ entry during the AP. For example, in Fig 3 SR-mediated inactivation of I_Ca resulted in a 30% reduction in the integrated Ca²⁺ influx. We suggest that the SR-mediated inactivation of I_Ca serves three important regulatory functions: First, over a number of beats, the SR-mediated changes in I_Ca will help ensure a constant SR Ca²⁺ load due to the negative feedback between Ca²⁺ influx across the surface membrane (I_Ca) and SR Ca²⁺ release. Thus, if the SR Ca²⁺ content is reduced, the increase in I_Ca will help reload the SR Ca²⁺ store. Second, this mechanism helps ensure that E-C coupling will take place, while minimizing Ca²⁺ influx via I_Ca. If SR release is delayed, the trigger Ca²⁺ influx would be maintained at a higher level (than if SR release occurs) because there would be less inactivation of I_Ca. As soon as SR release takes place, the probability that local sarcolemmal Ca²⁺ channels will (re)open is reduced, which then makes (re)activation of the SR release less likely. Finally, the changes in I_Ca will provide a modest offset to possible changes in SR Ca²⁺ content within the same beat. Although I_Ca is not a major contributor to the Ca²⁺ transient, an increase in its amplitude when SR release is reduced will help raise cytosolic Ca²⁺ directly.

	Selected Abbreviations and Acronyms

 

AP = action potential CICR = Ca2+-induced Ca2+ release E-C = excitation-contraction ICa = L-type Ca2+ current INa/Ca = Na+/Ca2+ exchange current SR = sarcoplasmic reticulum

	Acknowledgments

 
This study was supported by a project grant from the British Heart Foundation.

	Footnotes

 
Correspondence to M.B. Cannell, Department of Pharmacology and Clinical Pharmacology, St George's Hospital Medical School, Cranmer Terrace, London SW13 0RE, UK. E-mail mcannell@mbcsg1.sghms.ac.uk.

Received December 11, 1995; accepted April 19, 1996.

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