Low-voltage triggering of Ca2+ release from the sarcoplasmic reticulum in cardiac muscle cells

Fabien Brette, Jean-Yves Le Guennec, and Ian Findlay

Centre National de la Recherche Scientifique, Unité Mixte de Recherche 6542, Faculté des Sciences, Université de Tours, 37041 Tours Cedex, France

Submitted 11 April 2003 ; accepted in final form 18 August 2003


    ABSTRACT
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
This study investigated the interaction between L-type Ca2+ current (ICaL) and Ca2+ release from the sarcoplasmic reticulum (SRCR) in whole cell voltage-clamped guinea pig ventricular myocytes. Quasiphysiological cation solutions (Nao+:KI+) were used for most experiments. In control conditions, there was no obvious interaction between ICaL and SRCR. In isoproterenol, activation of ICaL from voltages between -70 and -50 mV reduced the amplitude and accelerated the decay of the current. Short (50 ms), small-amplitude voltage steps applied 60 or 510 ms before stimulating ICaL inhibited and facilitated the current, respectively. These changes were blocked by ryanodine. Low-voltage activated currents such as T-type Ca2+ current, TTX-sensitive ICa (ICaTTX), or slip mode Ca2+ conductance via INa+ were not responsible for low-voltage SRCR. However, L-type Ca2+ currents could be distinguished at voltages as negative as -45 mV. It is concluded that in the presence of isoproterenol, Ca2+ release from the SR at negative potentials is due to activation of L-type Ca2+ channels.

heart; calcium current; low-voltage activation


DEPOLARIZATION of the cell membrane activates sarcolemmal L-type Ca2+ channels in cardiac ventricular myocytes. The resulting Ca2+ influx causes Ca2+-induced Ca2+ release (CICR) from the adjacent junctional sarcoplasmic reticulum (SR). The gain of this system provides the Ca2+ required for effective contraction. In addition, the closely opposed sarcolemmal and junctional membranes at the t-tubules (15) allow feedback regulation of the L-type Ca2+ current (ICaL): Ca2+ released from the SR causes inactivation of ICaL (1, 28, 36, 37, 39) so that Ca2+ influx is limited and the contribution of ICaL to the ventricular action potential is reduced.

Ca2+-induced inactivation of L-type Ca2+ channels arises from activation of calmodulin bound to the intracellular COOH terminus of the {alpha}1-subunit of the channel (3, 19). It has been suggested that inactivation of the L-type Ca2+ channel, whether by voltage or Ca2+, is an absorptive state; that is, once the channel has opened in response to depolarization and subsequently assumed an inactivated state, this state is irreversible during continued depolarization and is reversed only on hyperpolarization. Although reasonable for the molecular changes associated with voltage-dependent inactivation, this is not consistent with the known interaction between Ca2+ and calmodulin, which is readily reversible and dependent only upon Ca2+ concentration. Thus Ca2+-dependent inactivation should reflect the increase of intracellular Ca2+ concentration after Ca2+ influx and CICR and the subsequent reduction due to Ca2+ diffusion and buffering, reuptake, and efflux by intracellular buffers, sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), and the Na+/Ca2+ exchanger, respectively.

However, the idea that Ca2+ is the only trigger for SR Ca2+ release (SRCR) in cardiac muscle has been challenged by the suggestion that voltage-gated SR Ca2+ release (VSRM) (see Ref. 14 for recent review) may also occur in these cells. It has been proposed that such release is activated at voltages negative to the threshold for activation of ICaL, so that Ca2+ release from the SR may coincide with, or even precede, activation of ICaL, which may have important implications for Ca2+-dependent inactivation of ICaL. It has, however, also been suggested that VSRM is an artifact of particular in vitro conditions (44), and alternative means of evoking low-voltage CICR have been proposed (11, 35, 40): it is possible that ICaL is activated at low voltages in the presence of increased cAMP concentrations (32), which are required to demonstrate VSRM.

The initial objective of this study was to characterize the relationship between the activation of ICaL and CICR and the consequent Ca2+-induced inactivation of ICaL. In particular, we were interested in the early, rapid, and reversible inactivation of ICaL (9, 39); this multiphasic decay can be blocked by ryanodine and thus appears to be due to SRCR, which suggests that Ca2+-induced inactivation as distinct from voltage-dependent inactivation might not be an absorbing state. We also found that in the presence of isoproterenol, the degree of Ca2+-induced inactivation caused by SR depended not only upon the activation voltage of ICaL but also upon the voltage from which ICaL was stimulated. This appeared to be due to CICR evoked by ICaL at negative potentials.


    METHODS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell preparation. All animal experiments were conducted according to the ethical standards of the Ministère de l'Agriculture (license no. B37-261-4). Male guinea pigs (250-400 g) were killed by cervical dislocation and the hearts were removed. Single ventricular myocytes were isolated using collagenase and protease digestion as described elsewhere (26). Myocytes isolated from the left ventricle were aliquoted into 35-mm diameter plastic petri dishes, which served as experimental chambers. The cells were stored in standard extracellular solution (below) at room temperature and used within 8 h of isolation.

A plastic petri dish containing isolated myocytes was placed on the stage of an Olympus CK2 inverted microscope. The myocytes were superfused with experimental solutions via a system of parallel tubes positioned adjacent to the cells. Fluid flow was maintained by gravity from syringe barrel reservoirs, and the exchange of solutions was achieved by manual displacement of the tubes. Solution exchange around the myocyte was estimated to be complete in 4-5 s. All experiments were conducted at room temperature (~23°C).

Experimental procedures. Whole cell current voltage-clamp experiments were conducted using an Axon Instruments 202A patch-clamp amplifier in resistive feedback mode (Axon Instruments). Pipettes were fabricated from thin-walled borosilicate glass capillary tubes (Clark Electro-medical Instruments, Pangbourne, England) with a Narishige PP7 double-stage puller (Narishige Instruments, Tokyo, Japan). Pipettes were coated with Sylgard (Dow Corning) and then heat polished. Finished pipettes had a resistance of <2 M{Omega} when filled with standard intracellular solution. Experimental voltage-clamp protocols and data acquisition were controlled with Acquis1 software (Dipsi Industrie, Chatillon, France) installed on a 386-20 PC computer. Data were filtered at either 1 or 2 kHz and acquired at 2 or 5 kHz, respectively. Data analysis was performed with either Acquis1 or Origin 4.1 (Microcal Software). Results are shown as means of data obtained from n myocytes with bars representing SE when these are larger than their respective symbol. Cell capacitance and series resistance were compensated (~80%) with the Axon Instruments amplifier so that the maximum voltage error was <3 mV. Only cell current recordings with a series resistance of <5 M{Omega} were accepted for study. Cell currents are expressed in cell current density, pA/pF. Once the whole cell configuration of the patch-clamp cell current recording technique (18) had been achieved, isolated myocytes were voltage clamped at -80 mV. Voltage-clamp protocols were delivered to the cells from this holding potential.

Experimental solutions. The standard extracellular solution used to fill the petri dishes and store myocytes contained (in mM) 140 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 glucose, and 10 HEPES, pH 7.4, with NaOH. TTX citrate salt, 10 µM (from either Alomone Labs, Jerusalem, Israel, or Latoxan, Valence, France) was added to this standard solution when required. In some experiments, extracellular NaCl was replaced by an equimolar amount of N-methyl-D-glucamine (NMDG), and the pH was adjusted with HCl. The standard intracellular solution used to fill the patch pipettes contained (in mM) 140 KCl, 10 NaCl, 5 EGTA-KOH, 1.4 MgCl2, 0.1 CaCl2, 2 ATP-Mg2+, 10 glucose, and 10 HEPES, pH 7.3, with KOH. The estimated free concentrations of Mg2+ and Ca2+ in this solution were 1 mM and 1 nM, respectively. One series of experiments (Fig. 2A) was conducted with Na+- and K+-free solutions where extracellular Na+ was replaced by 140 mM TEA-Cl, and K+ was replaced by 5 mM CsCl2, and intracellular K+ and Na+ were replaced by 100 mM CsCl2 and 40 mM TEA-Cl. In experiments in which {beta}-adrenergic stimulation used, the pipette solutions also contained 100 µM GTP. Cadmium, barium, and nickel were added to extracellular solutions as their chloride salts. Isoproterenol was prepared daily as a 100-µM stock solution in distilled water and added to extracellular solutions to give a final concentration of 100 nM. Ryanodine was dissolved as a 2-mM stock solution in distilled water and added to extracellular solution to give a final concentration of 10 µM. Forskolin was dissolved as a 10-mM stock solution in DMSO and added to extracellular solution to give a final concentration of 10 µM. When the effects of forskolin were tested, an equivalent amount of vehicle was added to all experimental solutions.



View larger version (22K):
[in this window]
[in a new window]
 
Fig. 2. Multiphasic decay of ICaL. A: these experiments were conducted in Na+- and K+-free experimental solutions (see METHODS). Cell currents recorded from a representative myocyte during voltage steps to -20 and +20 mV from a holding potential of -80 mV. Cell currents were recorded first in control conditions (left) and then after the application of 10 µM forskolin (right). The time and current scales apply to each record. The horizontal dotted lines indicate the cell current level recorded at the end of the 500-ms duration voltage step. B: the background current evoked by the activation of adenylate cyclase by 10 µM forskolin. The symbols and bars represent means ± SE (n = 11) of the difference between cell currents recorded under control conditions and after the application of forskolin. The currents activated by forskolin reversed between 0 and +10 mV, which was close to the equilibrium potential for an anionic current under these experimental conditions. It is concluded that these currents represent IClcAMP. C and D: the effects of extracellular Ni2+ and Ba2+. Cell currents were recorded from representative myocytes in the presence of 100 nM isoproterenol (Iso) during voltage steps to -20 mV. Currents recorded evoked from prior voltages of -55 and -45 mV (arrow) have been superimposed. Cell currents were recorded first in the presence of isoproterenol alone (left) and then after either the addition of 50 µM Ni2+ (C, right) or the replacement of extracellular Ca2+ with Ba2+ (D, right).

 


    RESULTS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Effects of conditioning voltage upon inactivation of ICaL. Figure 1 illustrates the conditions in which multiphasic decay of ICaL was recorded: voltage steps to negative potentials (-20 mV) from negative conditioning voltages (prepulse -65 mV) in the presence of {beta}-adrenergic stimulation. It was abolished by the application of ryanodine and by evoking ICaL from more positive conditioning voltages (prepulse -45 mV). Multiphasic decay was not observed when ICaL was evoked by voltage steps to positive potentials (+20 mV), although more positive conditioning voltages and ryanodine still slowed the decay of ICaL. These results suggest that conditioning voltages that are below the threshold for activation of ICaL nevertheless affect ICaL via SRCR. However, it is also possible that SRCR evoked other currents. The cell current records shown in Fig. 1 were recorded with quasiphysiological cation gradients, and the appearance of a kink in the time course of decay of ICaL could have resulted from the superposition of outward current, such as Ca2+-activated K+ current, Ca2+-activated anion current (48), and Na+/Ca2+ exchange current. To address these possibilities, the experiments shown in Fig. 2A were conducted in Na+- and K+-free extracellular and intracellular solutions; multiphasic decay of ICaL was still recorded during negative voltage steps (-20 mV) after activation of adenylate cyclase by forskolin. The absence of Na+ excluded the contribution of the Na+/Ca2+ exchange current. The absence of K+ excluded the contributions of either a Ca2+-activated, K+-selective current or inwardly rectified, transient outward K+ current (27). Activation of IClcAMP by forskolin is shown in Fig. 2B. This current was essentially time independent and slightly outwardly rectified: an inwardly directed current was recorded at negative voltages and an outwardly directed current was recorded at positive voltages. Thus the outward inflection in the decay of ICaL at negative voltages could not represent a Cl- current. The experiment shown in Fig. 2C was to investigate the influence of T-type Ca2+ current (ICaT) on the decay of ICaL. The application of 50 µM Ni2+, which blocks ICaT in guinea pig ventricular myocytes (30), had no effect upon the multiphasic decay of ICaL or on the effect of a depolarized conditioning voltage abolishing multiphasic decay of ICaL. We conclude that ICaL at -20 mV is not contaminated by ICaT and, thus, that under these experimental conditions (100 nM isoproterenol), ICaT was not responsible for the effects of conditioning voltage upon ICaL (see also Fig. 7) (40). Figure 2D tests whether multiphasic decay of ICaL after {beta}-adrenergic stimulation is an artifact resulting from loss of control of the voltage clamp or to transient depletion of extracellular Ca2+ or transient accumulation of intracellular Ca2+, either of which could result in a reduction of the electrochemical gradient for Ca2+ influx and, thus, a reduction in the amplitude of ICaL recorded at a given voltage. These propositions were tested by recording ICaL carried by Ca2+ (Fig. 2D, left) or Ba2+ (Fig. 2D, right). At -20 mV, ICaL carried by Ba2+ was -42.2 ± 2.8 pA/pF compared with -10.9 ± 1.1 pA/pF (n = 3) when carried by Ca2+. ICaL-Ba2+ did not show multiphasic decay, and the current recorded during a voltage step to -20 mV was not influenced by the prior conditioning voltage. If loss of control of voltage clamp had been responsible for the multiphasic decay of ICaL-Ca2+ (Fig. 2D, left), this should have been visible or even enhanced with the much larger current recorded as ICaL-Ba2+ (Fig. 2D, right). Thus it is unlikely that the multiphasic decay of ICaL-Ca2+ is due to loss of voltage-clamp control. The larger ICaL-Ba2+ should also have enhanced extracellular depletion and/or intracellular accumulation of the permeant cation. However, no kink was observed in the decay of ICaL-Ba2+, suggesting that transient displacement of the electrochemical gradient for ion entry is not responsible for the multiphasic decay of ICaLCa2+. We conclude that the multiphasic decay (Figs. 1 and 2) reflects a reversible Ca2+-dependent inactivation of ICaL and does not result from overlapping membrane currents.



View larger version (20K):
[in this window]
[in a new window]
 
Fig. 1. The effects of conditioning voltage, ryanodine, and isoproterenol upon L-type Ca2+ current (ICaL). A and B: cell currents obtained from 2 representative myocytes during voltage steps to -20 and to +20 mV. Cell current records were obtained from each myocyte after 500-ms duration conditioning voltage steps to either -65 mV (solid lines) or -45 mV (dashed lines). These traces have been superimposed. A: currents were recorded first under control conditions (left) and then in the presence of 100 nM isoproterenol (right). B: cell currents were recorded first in the presence of 100 nM isoproterenol alone (left) and then after the addition of 10 µM ryanodine (right). The time and current scales in A and B apply to all 4 groups of traces in A and B, respectively. The horizontal dotted lines in each figure indicate the zero level, which was measured as the current level that had been recorded after a 500-ms prepulse to +10 mV. This had been assessed to have evoked >98% inactivation of ICaL (not shown).

 


View larger version (11K):
[in this window]
[in a new window]
 
Fig. 7. Neither the T-type Ca2+ current nor slip-mode conductance of INa+ are involved in the low-voltage effect upon ICaL. Experiments were conducted in Na+-free, NMDG-rich extracellular solution that also contained 100 nM isoproterenol. A: cell current records were obtained from 1 representative myocyte during voltage steps to -55 mV (a), -45 mV (b), and -35 mV (c). Superimposed in each group are 3 traces that were recorded after 500-ms duration conditioning voltage steps to -110, -90, and -50 mV. Schematic representations of the voltage-clamp protocols are shown below each set of traces. The dotted lines indicate the 0 pA current level. The time and current scales apply to all 3 groups of records. B: amplitude of the peak current (n = 3) recorded during voltage-clamp steps to between -90 and 0 mV after conditioning voltages of -50 mV ({blacksquare}), -90 mV ({bullet}), and -110 mV ({blacktriangleup}).

 

The effect of conditioning voltage upon ICaL was analyzed by recording the amplitude and the rate of decay of ICaL at -20 mV (Fig. 3A) and +20 mV (Fig. 3B) after voltage steps to between -70 and -40 mV. Maximal ICaL amplitude at either voltage was recorded from a conditioning voltage of -50 mV, and all data were therefore normalized to this value. The amplitude of ICaL was reduced when conditioning voltages were positive to -50 mV. This reflects the onset of voltage-dependent inactivation of ICaL (31) and was independent of the experimental conditions. On the other hand, the effect of conditioning voltages negative to -50 mV depended upon the experimental condition. In basal conditions, irrespective of the presence (circles) or absence (squares) of ryanodine, the amplitude and the rate of decay of ICaL were essentially unchanged by conditioning voltages negative to -50 mV. But, in isoproterenol (triangles) the amplitude of ICaL was progressively reduced and the rate of decay of the current progressively increased with conditioning voltages negative to -50 mV. These effects were abolished by ryanodine (inverted triangles).



View larger version (40K):
[in this window]
[in a new window]
 
Fig. 3. Characterization of the effects of isoproterenol upon low-voltage and ryanodine-sensitive properties of ICaL. Normalized peak inward ICaL (left) and the maximum rate of decay of ICaL (right) were extracted from experiments where double-pulse voltage-clamp protocols were applied to myocytes with test-pulse voltage steps to -20 mV (A) and +20 mV (B). Conditioning voltage steps of 500-ms duration were applied to between -70 and -40 mV in 5-mV increments (inset schematic diagrams). {blacksquare}, control conditions (n = 8). {bullet}, control conditions with 10 µM ryanodine (Ryan) (n = 5). {triangleup}, 100 nM isoproterenol (n = 8). {triangledown}, 100 nM isoproterenol and 10 µM ryanodine (Ryan) (n = 5).

 

These results could arise from an effect of conditioning voltage or from the repetitive application of voltage-clamp stimuli (12). If repetitive stimulation were responsible for the increase in the amplitude of ICaL and the reduction of its rate of decay, this would occur irrespective of the order of presentation of the conditioning voltage. Figure 4A illustrates results obtained when conditioning voltage steps were applied sequentially from -65 to -40 mV, and Fig. 4B illustrates results obtained when conditioning voltage steps were applied sequentially from -40 to -65 mV. In both cases, ICaL evoked from -60 mV showed multiphasic decay, whereas ICaL evoked from -40 mV showed sequential decay. There was no difference in the relationship between conditioning voltage and either peak current amplitude (Fig. 4C) or rate of inactivation (Fig. 4D) when conditioning voltage steps were applied sequentially from -40 to -65 mV (open symbols) or from -65 to -40 mV (closed symbols). A second test of the effect of repetitive stimulation of ICaL applied a stimulus from -65 mV to -20 mV 14 times every 6 s. Neither the amplitude nor the rate of inactivation of ICaL was influenced by repetitive stimuli at this frequency (n = 8). It is concluded that the effects of conditioning voltage upon the amplitude and rate of inactivation of ICaL did not result from repetitive application of voltage-clamp stimuli or repetitive activation of ICaL. Therefore ICaL amplitude and rate of decay were influenced by conditioning membrane voltages that were apparently subthreshold for its activation and inactivation.



View larger version (33K):
[in this window]
[in a new window]
 
Fig. 4. The effect of repetitive stimulation upon ICaL. A double-pulse voltage-clamp protocol was applied to myocytes either with prepulse voltage steps applied sequentially from -65 to -40 mV (A and filled symbols) or sequentially from -40 to -65 mV (B and open symbols). The test-pulse voltage step was to -20 mV. A schematic representation of the voltage-clamp protocol is inset in B. The cells were bathed in 100 nM isoproterenol. A and B: cell currents evoked by a voltage step to -20 mV after prepulse (pp) voltage steps to -60 and -40 mV have been superimposed. In A, the protocol progressed from -65 to -40 mV and these traces represent respectively the second and sixth stimuli. In B, the protocol progressed from -40 to -65 mV and the traces represent respectively the first and fifth stimuli. The records shown in A and B were obtained from the same myocyte. The time and current scale bars apply to both groups of records. The dotted lines indicate the zero level assessed as in Fig. 1, C and D. The peak current amplitude (C) and maximum rate of inactivation (D) of currents evoked by test-pulse voltage steps to -20 mV were normalized to the values recorded after a prepulse voltage step to -50 mV (n = 18).

 

Calcium release from the SR and inactivation of ICaL. The experiments shown in Fig. 5 addressed two issues. The first concerned the multiphasic decay of ICaL observed during voltage steps to negative voltages during {beta}-adrenergic stimulation (Figs. 1, 2, and 4). Recovery of ICaL from Ca2+-induced inactivation during sustained depolarization has not been a frequent observation (9, 39) but implies that Ca2+-induced inactivation is not an absorptive state. This led us to question whether SR might be capable of inactivating ICaL before the current had been activated. The second issue concerned the loss of the multiphasic decay and increase in amplitude observed with more positive conditioning voltages or ryanodine (Fig. 1B), which suggests that triggering SRCR might relieve a subsequent ICaL from inhibition by its own CICR.



View larger version (36K):
[in this window]
[in a new window]
 
Fig. 5. The importance of the timing of the trigger for sarcoplasmic reticulum Ca2+ release (SRCR) and ICaL. These experiments were conducted in the presence of 100 nM isoproterenol. The voltage-clamp protocols are indicated schematically above each column of data (A-C). The voltage-clamp protocols were double-pulse stimuli that consisted of prepulse voltage steps to between -65 and 0 mV in 5-mV increments followed by a test pulse to 0 mV. A: prepulse voltage steps had a duration of 500 ms, and a 10-ms interval at -70 mV was intercalated between the pre- and test-pulse voltage steps. B: prepulse voltage steps had a duration of 50 ms with a 10-ms interval at -70 mV between pre- and test-pulse voltage steps. C: 50-ms duration prepulses were situated 510 ms before the test-pulse voltage step. There was an interval of 460 ms between the end of the prepulse and the onset of the test-pulse voltage step. Illustrated cell currents were recorded after prepulse (pp) voltage steps to -60 (solid lines) and -40 mV (dotted lines) and superimposed. The time and current scales are applicable to all traces. The horizontal dotted lines indicate the zero level that was assessed as described in Fig. 1. Upper row: test-pulse cell currents obtained from a representative myocyte bathed in 100 nM isoproterenol alone. Middle row: different representative myocyte that was bathed in 10 µM ryanodine, as well as 100 nM isoproterenol. Bottom row: The amplitude of the cell current evoked by the test pulse to 0 mV was normalized to that recorded following a prepulse voltage step to -65 mV. {blacksquare}, 100 nM isoproterenol (n = 9). {square}, 100 nM isoproterenol and 10 µM ryanodine (n = 6).

 

Figure 5A represents the standard conditions for this series of experiments and illustrates results obtained from the application of a double pulse voltage-clamp protocol consisting of 500-ms prepulse voltage steps, a 10-ms interval, and a test pulse, so that there were 510 ms between the onset of the prepulse voltage step and the onset of the test pulse. Although this particular series of cells showed little or no facilitation of the amplitude of ICaL, a prepulse to -40 mV, without inducing inactivation, resulted in reduced decay of ICaL. This effect was abolished by ryanodine.

Figure 5B illustrates results obtained using 50-ms prepulse voltage steps, so that the onset of the prepulse voltage step was separated from the onset of the test pulse by 60 ms. These short prepulse voltage steps applied to between -55 and -40 mV resulted in a clear reduction in amplitude of the following ICaL. This effect was abolished by ryanodine. This result strongly suggests that voltage steps to between -55 and -40 mV were associated with SRCR, which inhibited sarcolemmal Ca2+ channels before their activation. That neither Ca2+ influx during, nor membrane voltage of, the prepulse voltage step was responsible for the reduction of the following ICaL was shown by their lack of effect in the presence of ryanodine.

Figure 5C illustrates results obtained when short prepulse voltage steps were situated with their leading edge 510 ms before the onset of the test voltage pulse. In the presence of isoproterenol alone, short voltage steps to -50 mV and more positive voltages provoked an increase in the amplitude of the following ICaL and reduced its decay. These effects were abolished by ryanodine. It is concluded from these results that triggering SRCR during one voltage step decreased inhibition and inactivation of the following ICaL due to its own CICR (Figs. 5, A and C). Whether this resulted from inactivation of the SRCR mechanism (22), of the ryanodine receptors (38), or transient exhaustion of the junctional SR (1, 12, 13) remains to be determined.

These data also show that the triggering of SRCR has different effects upon ICaL depending upon the temporal relation between the increase in intracellular Ca2+ and the activation of ICaL (Figs. 5, B and C).

The low-voltage trigger for Ca2+ release from the SR? The data shown in Figs. 1 and 5 suggest SRCR at membrane voltage steps that are normally considered too negative to activate ICaL and consequent CICR (22) but which could be explained by VSRM (14). We therefore investigated events during low-voltage steps to determine what might be responsible for triggering SRCR.

Figure 6A shows currents triggered from -70 mV in the presence of isoproterenol. A voltage step to -40 mV evoked a small transient inward current. The I-V relationship of currents evoked from -70 mV shows a clear inflection of inward currents between -50 and -20 mV in addition to ICaL.



View larger version (31K):
[in this window]
[in a new window]
 
Fig. 6. Characterization of a transient, inward low-voltage gated current. A, left: cell currents were evoked with 50-ms voltage steps to -60 and -40 mV in 1 representative myocyte that was bathed in 100 nM isoproterenol. The horizontal line indicates the zero level. A, right: peak amplitude of cell currents evoked by voltage steps between -70 and 0 mV in the presence of 100 nM isoproterenol (n = 11). The voltage-clamp protocol is indicated schematically. B: effect of extracellular Na+ upon the low-voltage-activated transient inward current was examined with a double-pulse voltage-clamp protocol. The 500-ms duration prepulses were to between -90 and -20 mV in 5-mV increments, the 200-ms duration test pulse was to -40 mV as indicated schematically at right. These recordings represent superimposed cell currents recorded from one representative myocyte during test-pulse voltage steps to -40 mV after prepulse voltage steps to between -90 and -50 mV. The cell was bathed in extracellular solution that contained 100 nM isoproterenol and 10 µM ryanodine. Upper traces: extracellular solution contained 140 mM NaCl. Lower traces: extracellular NaCl was replaced by 140 mM NMDG.HCl. The time and current scales apply to both groups of traces. The horizontal lines indicate the 0 pA current level. Right: amplitude of the test-pulse (-40 mV) evoked current (n = 5). {blacksquare}, Na+ bath solution. {square}, NMDG bath solution.

 

The nature of this low-voltage-activated transient inward current was examined (Fig. 6B). The availability of this current was tested using a double pulse voltage-clamp protocol with a test pulse to -40 mV. When prepulse voltage steps were applied to membrane potentials more negative than -70 mV, there was a considerable increase in the amplitude of the transient low-voltage-activated current recorded at -40 mV (Fig. 6B and filled symbols). The amplitude of this current did not saturate even with prepulse voltage steps as negative as -90 mV. Although all bathing solutions contained 10 µM TTX, it was decided to test whether this large transient inward current might depend upon extracellular Na+. When extracellular Na+ was replaced by NMDG, the transient inward current recorded at -40 mV was abolished (Fig. 6B and open squares) and could not be recovered by prior voltage steps to values as negative as -90 mV. It is concluded that this low-voltage-activated transient inward current corresponded to INa+ that had not been entirely blocked by the inclusion of 10 µM TTX in the solutions bathing the cells.

The next series of experiments was to investigate whether this residual TTX-insensitive portion of INa+ might be responsible for triggering low-voltage-associated SRCR. The three experimental protocols used to investigate the effect of prior voltage duration and temporal position upon ICaL (Fig. 5) were applied to myocytes bathed first in normal Na+-containing solution containing TTX and then in extracellular solution in which Na+ had been replaced by NMDG. Neither the 500-ms availability curve (n = 9), the prior low-voltage reduction of ICaL (n = 7), nor the 500-ms leading edge prior voltage facilitation of ICaL (n = 8) were affected by the removal of extracellular Na+ (data not shown). Thus it appears that INa+ is not responsible for low-voltage-associated SRCR.

In the following experiments (Figs. 7 and 8), cells were bathed in Na+-free, NMDG-rich extracellular solution. Cells were exposed to this solution for only as long as required to apply the different voltage-clamp protocols; otherwise, they were superfused with the standard Na+-rich extracellular solution. Insufficient time was allowed for the absence of extracellular Na+ to influence the {beta}-adrenergic response (6, 20).



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 8. Activation of ICaL occurs at low voltages. These experiments were conducted in NMDG extracellular solution that also contained 100 nM isoproterenol and 10 µM ryanodine. A: contribution of ICaL to the ensemble whole cell current was assessed by replacing extracellular Ca2+ with the impermeant divalent cation Mg2+. Illustrated are superimposed cell currents evoked by voltage steps to 0 mV from a conditioning voltage of -80 mV in the presence (+Ca2+, a) and absence (-Ca2+, b) of extracellular Ca2+. The time and current scales apply to both traces. The dotted lines indicate the 0 pA current level. B: I-V relations of peak currents evoked by voltage steps to between -90 and 0 mV in the presence ({blacksquare}) and absence ({bullet}) of extracellular Ca2+ (n = 16). C: superimposed cell currents evoked by voltage-clamp steps to -50 (a), -45 (b), -40 (c), and -35 mV (d) in 1 myocyte in the presence (+Ca2+) and absence (-Ca2+) of extracellular Ca2+. The time and current scales apply to all 4 groups of traces. The arrows to the left of each trace indicate the 0 pA current level.

 

Guinea pig ventricular myocytes express the low-voltage-activated transient T-type Ca2+ current (5), and during {beta}-adrenergic stimulation, INa+ might allow Ca2+ influx via slip-mode conductance (35). Either of these currents could cause Ca2+ entry at negative potentials and, hence, trigger SRCR, thus influencing ICaL. Figure 7 shows experiments designed to test for the presence of these currents. The voltage-clamp protocol promotes the detection of the T-type Ca2+ current (5, 40), whereas the experimental conditions promote slip-mode conductance of INa+ (35). Whether voltage steps were applied from conditioning voltages of -50, -90, or -110 mV, no transient inward currents could be detected (Fig. 7A) and no inflection of the total I-V relation could be seen in the low-voltage range (Fig. 7B). Because neither ICaT nor slip-mode Ca2+ conductance via INa+ could be detected under these conditions, it is concluded that these currents would not have provoked CICR to account for the effects of low-voltage-associated SRCR upon ICaL.

Another possible source of low-voltage gated Ca2+ entry that could trigger SRCR is ICaTTX (2, 11, 21). The presence of ICaTTX was investigated in experiments similar to those shown in Fig. 7 in the presence and absence of 10 µM TTX. No TTX-sensitive current could be distinguished in the recordings (n = 3), so it is unlikely that ICaTTX could be responsible for low-voltage-associated SRCR.

It remained to test for the presence of ICaL at low voltages during {beta}-adrenergic stimulation. ICaL was detected by conducting experiments in extracellular solutions that contained either 2 mM CaCl2 and 1 mM MgCl2 (Fig. 8Aa, +Ca2+, and squares in Fig. 8B) or 3 mM MgCl2 and 250 µM EGTA.NaOH (Fig. 8Ab, -Ca2+, and circles in Fig. 8B). ICaL was abolished by replacement of extracellular Ca2+ with Mg2+ (Fig. 8A) without effect upon membrane surface charge screening. In the absence of extracellular Ca2+, the I-V relation of isolated ventricular myocytes showed the N-shape characteristic of the background K+ current IK1 (Fig. 8B). Close comparison of current records obtained in the presence and absence of extracellular Ca2+ (Fig. 8C) revealed that steps to membrane potentials positive to -50 mV resulted in essentially time-independent Ca2+ currents superimposed upon IK1. Although these currents did not attain a level that corresponded to the gross inward current carried by Ca2+ when voltage was stepped to -35 mV, the difference between the currents recorded at -45 and -40 mV in the presence and absence of Ca2+ represents a net influx of Ca2+.


    DISCUSSION
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The results of the present study show that Ca2+-dependent inactivation of ICaL is reversible (not an absorbing state) on a physiological time scale (tens of milliseconds). In addition, during {beta}-adrenergic stimulation, ICaL could be activated at voltages negative to -40 mV, which could evoke CICR.

Experimental approach used. It is well known that Ca2+-dependent inactivation of ICaL in cardiac cells is due to Ca2+ flowing via the channel and Ca2+ release by the SR (1, 9, 36, 39). The presence of a slow Ca2+ buffer (EGTA) appears not to interfere with the local Ca2+ signaling between L-type Ca2+ channels and ryanodine receptors, which takes place in a restricted diffusion space (24, 3, 12, 36). In a previous study, we showed that by using a faster Ca2+ chelator (BAPTA), such local Ca2+ signaling is stopped (9). Furthermore, using confocal microscopy, Song et al. (41) showed that in the presence of 10 mM EGTA, ICaL-induced release of Ca2+ in the dyadic space, (intracellular Ca2+ recorded using Oregon Green 5N), providing direct evidence that Ca2+ is released in the dyadic space in the presence of EGTA.

In recent years, it has been shown that Ca2+-dependent inactivation of the L-type calcium channel is due to Ca2+ binding to calmodulin tethered to the COOH terminus of the channel (see Ref. 33 for review). In the present study, we used Ca2+-dependent inactivation of ICaL as an assay of Ca2+ binding to this calmodulin, as other investigators use contraction as an assay of Ca2+ binding to troponin C.

Fast and reversible calcium-dependent inactivation of ICaL. Previous work has shown that the decay of ICaL in cardiac myocytes can be multiphasic (9, 39). The evidence strongly suggests that multiphasic decay of ICaL results from SRCR because it is blocked by ryanodine. Multiphasic decay did not seem to result from the accumulation or the depletion of Ca2+ because the decay of ICaL was sequential in the presence of ryanodine and when ICaL was carried by Ba2+; in both conditions, the current was larger and accumulation or depletion would have been expected to be greater. It did not seem to be associated with activation of IClCa (47, 48, 49, 50), first, because an equivalent and opposite current was not observed at membrane potentials, which were on the other side of the equilibrium potential for chloride, and second, because activation of ICl under the experimental conditions used here evoked an inward rather that an outward current. Multiphasic decay of ICaL could be observed in the absence of extracellular and intracellular Na+, which inhibits Na+-Ca2+ exchange (9), arguing against the superposition of Na+-Ca2+ exchange current upon ICaL. We propose that multiphasic decay of ICaL in cardiac myocytes reflects the voltage dependence of the gain between Ca2+ influx and SRCR predicted by local control theory (42) and supported by direct observation (34). Analysis of the rate of decay of ICaL (Fig. 3) showed that multiphasic decay represents one extreme of the general process of the acceleration of inactivation of ICaL by SRCR (1, 9, 29, 36, 37, 39).

Although abolition of the overshoot of inactivation of ICaL was the most evident demonstration of the effects of conditioning voltage steps and ryanodine, the equivalent of increased current amplitude and reduced rate of decay were found in ICaL with simpler forms of decay (Fig. 3). Thus the effect of conditioning voltage on the current was not a characteristic confined to a particular set of conditions. The particular effect of SRCR depended upon its temporal relation with the activation of ICaL. Thus release of Ca2+ just before the activation of ICaL inhibited the current (Fig. 5B) in a manner reminiscent of the inhibitory effects of Ca2+ release evoked either by caffeine (1, 28) or illumination of caged compounds (8, 17). The release of Ca2+ ~500 ms before activation of the current liberated the following ICaL from CICR and enhanced ICaL amplitude with reduced decay (Fig. 5C) in a manner similar to frequency dependent facilitation of ICaL (12, 25). This could reflect the refractory period for SRCR that may result either from transient reduction of junctional SR Ca2+ content (1, 12, 13) and/or refractoriness in the behavior of ryanodine receptors (38). Alternatively, conditioning voltage steps may have inactivated the VSRM mechanism (22).

Possible mechanism of low-voltage-triggering Ca2+ release. The importance of coordinating Ca2+ influx via sarcolemmal Ca2+ channels and release of Ca2+ from the SR is indicated by studies that show SRCR can limit not only Ca2+ entry into the cell but also reconfigure the contribution of ICaL to the ventricular action potential (12, 29). In this context, the potential importance of VSRM (14) is evident: the simultaneous rather that the sequential occurrence of Ca2+ influx and Ca2+ release could severely impair the function of ICaL. The mechanism of low-voltage-activated release of Ca2+ from the SR has been controversial. The evidence for a purely voltage-dependent release mechanism has been recently challenged by experiments that could not record SRCR in the absence of ICaL (16, 45). In the present study, a number of candidates for low-voltage-activated Ca2+ entry to provoke CICR were tested, but neither ICaT (5, 40), ICaTTX (11, 21), nor slip mode Ca2+ current through INa+ (35) could be recorded under experimental conditions where prior voltage clearly influenced ICaL in a ryanodine-dependent manner. On the other hand, ICaL was activated at voltages negative to -40 mV in the presence of the {beta}-adrenergic agonist isoproterenol (Fig. 8). These currents were small and essentially time independent, in contrast to the transient current reported in cat myocytes (32) but similar to those recorded in the rat and rabbit (16, 43). However, opening of single L-type Ca2+ channels, particularly with the large driving force at such negative voltages, can provoke CICR (7, 10, 23, 34, 41, 46).

In conclusion, the data in this study do not resolve the controversy between CICR and VSRM as mechanisms causing SR Ca2+ release (4). The data do, however, contribute to the developing consensus of experimental evidence (16, 45) that suggests that VSRM may be accounted for by CICR evoked by activation of ICaL at negative potentials.


    ACKNOWLEDGMENTS
 
We thank Dr S. C. Calaghan and Prof. C. H. Orchard for critical reading of the manuscript.

Present addresses: F. Brette, School of Biomedical Sciences, University of Leeds, UK; J.-Y. Le Guennec, INSERM EMI 0211, Faculté de Médicine, Université de Tours, France.


    FOOTNOTES
 

Address for reprint requests and other correspondence: F. Brette, School of Biomedical Sciences, Univ. of Leeds, Leeds LS2 9NQ, UK (E-mail: bmsfpb{at}bms.leeds.ac.uk).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


    REFERENCES
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
1. Adachi-Akahane S, Cleeman L, and Morad M. Cross-signalling between L-type Ca2+ channels and ryanodine receptors in rat ventricular myocytes. J Gen Physiol 108: 435-454, 1996.[Abstract]

2. Aggarwal R, Shorofsky SR, Goldman L, and Balke CW. Tetrodotoxin blockable calcium currents in rat ventricular myocytes: a third type of cardiac cell sodium current. J Physiol 505: 353-369, 1997.[Abstract]

3. Anderson ME. Ca2+-dependent regulation of cardiac L-type Ca2+ channels: is a unifying mechanism at hand? J Mol Cell Cardiol 33: 639-650, 2001.[ISI][Medline]

4. Balke CW and Goldman L. Excitation-contraction coupling in cardiac myocytes: is there a purely voltage-dependent component? J Gen Physiol 121: 349-352, 2003.[Free Full Text]

5. Balke CW, Rose WC, Marban E, and Wier WG. Macroscopic and unitary properties of physiological ion flux through T-type Ca2+ channels in guinea pig heart cells. J Physiol 456: 247-265, 1992.[Abstract]

6. Balke CW and Wier WG. Modulation of L-type calcium channels by sodium ions. Proc Natl Acad Sci USA 89: 4417-4421, 1992.[Abstract]

7. Bassani JWM, Yuan W, and Bers DM. Fractional SR Ca release is regulated by trigger Ca and SR Ca content in cardiac myocytes. Am J Physiol Cell Physiol 268: C1313-C1329, 1995.[Abstract/Free Full Text]

8. Bates SE and Gurney AM. Ca2+-dependent block and potentiation of L-type calcium current in guinea pig ventricular myocytes. J Physiol 466: 345-365, 1993.[Abstract]

9. Brette F, Lacampagne A, Salle L, Findlay I, and Le Guennec J-Y. Intracellular Cs+ activates the PKA pathway revealing a fast reversible calcium dependent inactivation of L-type Ca2+ current. Am J Physiol Cell Physiol 285: C310-C318, 2003.[Abstract/Free Full Text]

10. Cleemann L, Wang W, and Morad M. Two-dimensional confocal images of organization, density, and gating of focal Ca2+ release sites in rat cardiac myocytes. Proc Natl Acad Sci USA 95: 10984-10989, 1998.[Abstract/Free Full Text]

11. Cole WC, Chartier D, Martin F, and Leblanc N. Ca2+ permeation through Na+ channels in guinea pig ventricular myocytes. Am J Physiol Heart Circ Physiol 273: H128-H137, 1997.[Abstract/Free Full Text]

12. Delgado C, Artiles A, Gomez AM, and Vassort G. Frequency-dependent increase in cardiac Ca2+ current is due to reduced Ca2+ release by the sarcoplasmic reticulum. J Mol Cell Cardiol 31: 1783-1793, 1999.[ISI][Medline]

13. Delprincipe F, Egger M, and Niggli E. Calcium signalling in cardiac muscle: refractoriness revealed by coherent activation. Nat Cell Biol 1: 323-329, 1999.[ISI][Medline]

14. Ferrier GR and Howlett SE. Cardiac excitation-contraction coupling: role of membrane potential in regulation of contraction. Am J Physiol Heart Circ Physiol 280: H1928-H1944, 2001.[Abstract/Free Full Text]

15. Franzini-Armstrong C and Prostasi F. Ryanodine receptors of striated muscles: a complex channel capable of multiple interactions. Physiol Rev 77: 699-729, 1997.[Abstract/Free Full Text]

16. Griffiths H and MacLeod KT. The voltage-sensitive release mechanism of excitation-contraction coupling in rabbit cardiac muscle is explained by calcium-induced calcium release. J Gen Physiol 121: 353-373, 2003.[Abstract/Free Full Text]

17. Hadley RW and Lederer WJ. Ca2+ and voltage inactivate Ca2+ channels in guinea pig ventricular myocytes through independent mechanisms. J Physiol 444: 257-268, 1991.[Abstract]

18. Hamill OP, Marty A, Neher E, Sakmann B, and Sigworth FL. Improved patch-clamp techniques for high resolution current recording from cells and cell-free membrane patches. Pflügers Arch 395: 85-100, 1981.

19. Hamilton SL, Serysheva I, and Strasburg GM. Calmodulin and excitation-contraction coupling. News Physiol Sci 15: 281-284, 2000.[Abstract/Free Full Text]

20. Harvey RD, Jurevicius JA, and Hume JR. Intracellular Na+ modulates the cAMP-dependent regulation of ion channels in the heart. Proc Natl Acad Sci USA 88: 6946-6950, 1991.[Abstract]

21. Heubach JF, Kohler A, Wettwer E, and Ravens U. T-type and tetrodotoxin-sensitive Ca2+ currents coexist in guinea pig ventricular myocytes and are both blocked by mebefradil. Circ Res 86: 628-635, 2000.[Abstract/Free Full Text]

22. Howlett SE, Zhu JQ, and Ferrier GR. Contribution of a voltage-sensitive calcium release mechanism to contraction in cardiac ventricular myocytes. Am J Physiol Heart Circ Physiol 274: H155-H170, 1998.[Abstract/Free Full Text]

23. Hussain M and Orchard CH. Sarcoplasmic reticulum Ca2+ content, L-type Ca2+ current and the Ca2+ transient in rat myocytes during {beta}-adrenergic stimulation. J Physiol 505: 385-402, 1997.[Abstract]

24. Lederer WJ, Niggli E, and HadleyRW. Sodium-calcium exchange in excitable cells: fuzzy space. Science 248: 283, 1990.[ISI][Medline]

25. Lee KS. Potentiation of the calcium channel currents of internally perfused mammalian heart cells by repetitive depolarization. Proc Natl Acad Sci USA 84: 3941-3945, 1987.[Abstract]

26. Le Guennec JY, Peineau N, Esnard F, Lacampagne A, Gannier F, Argibay J, Gauthier F, and Garnier D. A simple method for calibrating collagenase/pronase E ratio to optimize heart cell isolation. Biol Cell 79: 161-165, 1993.[ISI][Medline]

27. Li GR, Yang B, Sun H, and Baumgarten CM. Existence of a transient outward K+ current in guinea pig cardiac myocytes. Am J Physiol Heart Circ Physiol 279: H130-H138, 2000.[Abstract/Free Full Text]

28. Lipp P, Mechmann S, and Pott L. Effects of calcium release from sarcoplasmic reticulum on membrane currents in guinea pig atrial cardioballs. Pflügers Arch 410: 121-131, 1987.[ISI][Medline]

29. Mitchell MR, Powell T, Terrar DA, and Twist VW. Ryanodine prolongs Ca currents while suppressing contraction in rat ventricular cells. Br J Pharmacol 81: 13-15, 1984.[Abstract]

30. Pascarel C, Brette F, and Le Guennec J-Y. Enhancement of the T-type calcium channel by hyposmotic shock in isolated guinea pig ventricular myocytes. J Mol Cell Cardiol 33: 1363-1369, 2001.[ISI][Medline]

31. Pelzer D, Pelzer S, and McDonald TF. Properties and regulation of calcium channels in muscle cells. Rev Physiol Biochem Pharmacol 114: 107-207, 1990.[ISI][Medline]

32. Piacentino V, Dipla K, Gaughan JP, and Houser SR. Voltage-dependent Ca2+ release from the SR of feline ventricular myocytes is explained by Ca2+-induced Ca2+ release. J Physiol 523: 533-548, 2000.[Abstract/Free Full Text]

33. Saimi Y and Kung C. Calmodulin as an ion channel subunit. Annu Rev Physiol 64: 289-311, 2002.[ISI][Medline]

34. Santana LF, Cheng H, Gomez AM, Cannell MB, and Lederer WJ. Relation between the sarcolemmal Ca2+ current and Ca2+ sparks and local control theories for cardiac excitation-contraction coupling. Circ Res 78: 166-171, 1996.[Abstract/Free Full Text]

35. Santana LF, Gomez AM, and Lederer WJ. Ca2+ flux through promiscuous cardiac Na+ channels: slip mode conductance. Science 279: 1027-1033, 1998.[Abstract/Free Full Text]

36. Sham JSK. Ca2+-release-induced inactivation of Ca2+ current in rat ventricular myocytes: evidence for local Ca2+ signalling. J Physiol 500: 285-295, 1997.[Abstract]

37. Sham JSK, Cleemann L, and Morad M. Functional coupling of Ca2+ channels and ryanodine receptors in cardiac myocytes. Proc Natl Acad Sci USA 92: 121-125, 1995.[Abstract]

38. Sham JSK, Song LS, Chen Y, Deng LH, Stern MD, Lakatta EG, and Cheng H. Termination of Ca2+ release by a local inactivation of ryanodine receptors in cardiac myocytes. Proc Natl Acad Sci USA 95: 15096-15101, 1998.[Abstract/Free Full Text]

39. Sipido KR, Callewaert G, and Carmeliet E. Inhibition and rapid recovery of Ca2+ current during Ca2+ release from sarcoplasmic reticulum in guinea pig ventricular myocytes. Circ Res 76: 102-109, 1995.[Abstract/Free Full Text]

40. Sipido KR, Carmeliet E, and Van de Werf F. T-type Ca2+ current as a trigger for Ca2+ release from the sarcoplasmic reticulum in guinea pig ventricular cells. J Physiol 508: 439-451, 1998.[Abstract/Free Full Text]

41. Song LS, Sham JSK, Stern MD, Lakatta EG, and Cheng H. Direct measurement of SR release flux by tracking Ca2+ spikes in rat cardiac myocytes. J Physiol 512: 677-691, 1998.[Abstract/Free Full Text]

42. Stern MD. Theory of excitation-contraction coupling in cardiac muscle. Biophys J 63: 497-517, 1992.[Abstract]

43. Talo A, Stern MD, Spurgeon HA, Isenberg G, and Lakatta EG. Sustained subthrehold-for-twitch depolarization in rat ventricular myocytes causes sustained calcium channel activation and sarcoplasmic reticulum calcium release. J Gen Physiol 96: 1085-1103, 1990.[Abstract]

44. Trafford AW and Eisner DA. Another trigger for heart beat. J Physiol 513: 1, 1998.[Abstract/Free Full Text]

45. Trafford AW and Eisner DA. No role for a voltage sensitive release mechanism in cardiac muscle. J Mol Cell Cardiol 35: 145-151, 2003.[ISI][Medline]

46. Wang SQ, Song LS, Lakatta EG, and Cheng H. Ca2+ signalling between single L-type Ca2+ channel and ryanodine receptors in heart cells. Nature 410: 592-596, 2001.[ISI][Medline]

47. Xu Y, Dong PH, Zhang Z, Ahmmed GU, and Chiamvimonat N. Presence of a calcium-activated chloride current in mouse ventricular myocytes. Am J Physiol Heart Circ Physiol 283: H302-H314, 2002.[Abstract/Free Full Text]

48. Zygmunt AC. Intracellular calcium activates a chloride current in canine ventricular myocytes. Am J Physiol Heart Circ Physiol 267: H1984-H1995, 1994.[Abstract/Free Full Text]

49. Zygmunt AC and Gibbons WR. Calcium-activated chloride current in rabbit ventricular myocytes. Circ Res 68: 424-437, 1991.[Abstract]

50. Zygmunt AC and Gibbons WR. Properties of the calcium-activated chloride current in heart. J Gen Physiol 99: 391-414, 1992.[Abstract]





This Article
Abstract
Full Text (PDF)
All Versions of this Article:
285/6/C1544    most recent
00145.2003v1
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Search for citing articles in:
ISI Web of Science (1)
Google Scholar
Articles by Brette, F.
Articles by Findlay, I.
Articles citing this Article
PubMed
PubMed Citation
Articles by Brette, F.
Articles by Findlay, I.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2003 by the American Physiological Society.