INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Although inactivation and facilitation of whole-cell L-type Ca²⁺ currents have been thoroughly studied (see McDonald et al., 1994, for a review), the single channel mechanisms underlying these important regulatory processes remain less clear. Moreover, previous studies concerning single L-type Ca²⁺ channel gating properties have used Ba²⁺ ions (Pietrobon and Hess, 1990; Hirano et al., 1999), a high concentration of Ca²⁺ ions with Ba²⁺ ions (Imredy and Yue, 1994), and/or the addition of an L-type Ca²⁺ channel agonist (Yue et al., 1990; Imredy and Yue, 1994) to increase the amplitude and/or duration of the channel openings. Such nonphysiological interventions may produce marked differences in Ca²⁺ channel behavior, including alterations in the single Ca²⁺ channel conductance (see Guia et al., 2001) and the voltage-dependent kinetics of channel gating.

The L-type Ca²⁺ channel is activated by membrane depolarization, and subsequent Ca²⁺ ion influx through the channel is self-limited during a sustained depolarization. In myocardial cells, as in other types of excitable cells, regulation of Ca²⁺ influx through L-type Ca²⁺ channels is achieved by controlling channel opening and closing in response to membrane potential and prior Ca²⁺ ion entry (Brehm and Eckert, 1978; Brown et al., 1981; Josephson et al., 1984; Lee et al., 1985; Hadley and Hume, 1987; Yue et al., 1990; Imredy and Yue, 1994). Thus, the inactivation of the L-type Ca²⁺ channel during depolarization results from both voltage-dependent and ion-dependent mechanisms (Brehm and Eckert, 1978; Brown et al., 1981; Josephson et al., 1984; Lee et al., 1985; Hadley and Hume, 1987). On the single channel level, ion-dependent inactivation has been characterized as a shift of gating from relatively frequent, brief openings (mode 1) to a lower open probability, termed "mode Ca" (Imredy and Yue, 1994).

Conversely, several types of facilitation are known to produce an enhancement of the L-type Ca²⁺ current (see Dolphin, 1996, for a review). Of these, strong conditioning depolarization has been shown to result in a (Ca²⁺-independent) increase, or facilitation of the macroscopic cardiac L-type Ca²⁺ current that is related to an increase in the number of long-duration (mode 2) openings of the Ca²⁺ channel (Pietrobon and Hess, 1990).

Therefore, in the present paper and in the accompanying paper (Josephson et al., 2002) we have compared the effects of a nearly physiological concentration of Ca²⁺ ions, with a range of concentrations of Ba²⁺ ions (in the absence of any Ca²⁺ channel agonists), to investigate the single channel mechanisms involved in prepulse-dependent and ion-dependent inactivation and facilitation of the L-type Ca²⁺ channel. In the accompanying paper (Josephson et al., 2002) we report that in addition to alterations in gating, strong prepulses modulate the conductance of the Ca²⁺ channel, suggesting a more intimate association between these channel functions than was previously thought. Together with the results of the accompanying paper, these results suggest that the molecular mechanisms involved in prepulse-mediated alterations in both gating and conductance of single L-type Ca²⁺ channels are strongly influenced by the nature and concentration of the divalent ionic species permeating the channel. A preliminary report of some of the results has been presented in abstract form (Josephson et al., 2001).

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Myocyte preparation

Cells were isolated in accordance with National Institutes of Health guidelines for the care and use of animals. Male Sprague-Dawley rats (250-300 g, 2-3 months old) were anesthetized with pentobarbital (80-100 mg/kg, i.p.) and their hearts were removed via a transverse incision over the diaphragm. The hearts were washed in a nominally calcium-free modified Krebs solution (in mM: 120 NaCl; 5.4 KCl; 1.6 MgSO₄; 1 NaH₂PO₄; 20 NaHCO₃; 5.6 glucose; 5 taurine; gassed with 95% O₂, 5% CO₂) and then suspended and perfused via the aorta (constant pressure, 100 cm H₂O) on a heated (37°C) Langendorff apparatus. The hearts were cleared of extracellular calcium by nonrecirculating retrograde perfusion of the same solution for 5 min, then switched to a recirculating solution of similar content with the addition of protease (0.02 mg/ml, type XIV, Sigma Chemical Co., St. Louis, MO) and collagenase (1 mg/ml; type B, 220-230 U/mg, Boehringer-Mannheim, Indianapolis, IN, or type 2, Worthington, Lakewood, NJ), and, after 3 to 4 min in the enzymes, 50 µM CaCl₂ was added to the perfusate. At the end of the first digestion, the ventricles were chopped into several chunks and then placed into fresh Krebs solution containing 100 µM CaCl₂ and collagenase (1 mg/ml). This second digestion was allowed to proceed in a shaker (60-70 rpm) at 37°C until a satisfactory yield was obtained (10-15 min). The second digestion was quenched by filtering the supernatant for centrifugation at 500 × g and three subsequent washes with a modified Tyrode's solution (in mM: 137 NaCl; 4.9 KCl; 15 glucose; 1.2 MgSO₄; 1.2 NaH₂PO₄; 20 HEPES; NaOH, pH 7.4) with successively increasing calcium concentrations (250, 500, 1000 µM). The cells were allowed to settle from the supernatant for 10 min between washes. Cells were stored at room temperature in a similar Tyrode's solution containing 1 mM CaCl₂. The myocytes isolated in this manner were relaxed and rod-shaped, with clear sarcomeric striations and smooth, clean membranes.

Chemicals and solutions

All chemicals used in the cell isolation procedure were purchased from Mallinckrodt Chemicals Co. (Paris, KY), except for HEPES (ICN Biochemicals Inc., Aurora, OH) and MgSO₄ (Mallinckrodt Baker Inc., Phillipsburg, NJ). Chemicals used for physiological recordings were purchased from Sigma except for sucrose (ICN) and NaOH (Mallinckrodt). Pentobarbital (Sigma) was dissolved 30 mg/ml in a 10% ethanolic aqueous solution.

Single channel recording and analysis

We have previously demonstrated that unitary L-type Ca²⁺ channel currents can be reliably recorded with a low concentration of Ca²⁺ ions permeating the channel, and in the absence of channel agonists (Guia et al., 2001). Recording of unitary L-type Ca²⁺ channels was performed as previously described (Guia et al., 2001). Aliquots of cells were placed in a 0.1 ml bath mounted on the stage of a conventional inverted microscope. At least 10 min was allowed for the cells to attach to the coverslip on the bottom of the bath. The cells were then perfused with a high potassium depolarizing solution (HiK) at an approximate rate of 2-3 ml/min. The HiK solution (in mM: 120 potassium aspartate; 25 KCl; 10 HEPES; 10 glucose; 2 MgCl₂; 1 CaCl₂; 2 EGTA; 6 KOH, pH 7.2, 290 mOsm) was used to depolarize the cells to near 0 mV so that V_m was equal to Vpatch. The free calcium concentration in the HiK solution was calculated to be ~80 nM. To allow stabilization in their new milieu, the cells were perfused with HiK for at least 20 min before unitary current measurements were conducted. All experiments were performed at room temperature (22.5-23.5°C).

Borosilicate pipettes made from Corning 7052 glass (1.5 OD, 0.86 ID, Model 5968, A-M Systems, Inc., Carlsborg, WA) were pulled in 3 or 4 heating cycles using a horizontal Flaming-Brown pipette puller (model P-97, Sutter Instrument Co., Novato, CA) or a CO₂ laser-based puller (model P-2000, Sutter Instrument Co.) to yield tips ~1 µm in diameter. The pipette tips were firepolished (model MF-83, Narishige Instrument Lab., Tokyo, Japan) to produce 8 to 15 M tip resistances when filled with the pipette solutions, and were painted with a thick layer of silicone elastomer (Sylgard, Dow-Corning 184, Essex Brownell, Fort Wayne, IN, polymerized under a heat gun) to within 100 µm of the tip. Pipettes were filled with a solution containing BaCl₂ or CaCl₂ of the desired concentration, 10 mM CsCl and 5 mM 4-aminopyridine to block K⁺ currents, 10 mM HEPES, and TEA-OH to pH 7.4, with sucrose added to maintain normal osmolarity. Pipettes were stored in a covered container and were back-filled with pipette solution and used immediately. Seal resistances of 50 to >300 G were obtained by applying slight pressure with the pipette tip on the membrane, then applying gentle suction inside the pipette using a gas-tight glass syringe. For each seal, the pipette potential was offset to 0 mV with the pipette positioned near the membrane before initiating a seal. Formation of a stable seal was usually accomplished within a 20 to 30 s after the pipette potential was nulled. No other corrections were made for junction potentials. Membrane and pipette capacitances were corrected electronically. The noise at a bandwidth of 5 KHz was measured and only seals quieter than 250 fA RMS were used.

Current amplification was accomplished with an Axopatch 200B patch clamp (Axon Instruments Co., Burlingame, CA) and recorded on a computer hard disk using PClamp software (v. 6 and v. 8, Axon Instruments Co.) via a Digidata 1200A signal acquisition system. Data were filtered at 2 kHz (3 dB, 4-pole Bessel) and digitized at 10 kHz sampling rate. A 100 ms prepulse that varied from 50 to +130 mV (in 20 mV increments) was immediately followed by a 300 or 400 ms test voltage step to 10 or 0 mV. These double voltage-step protocols were applied at a rate of 0.5 Hz (allowing for complete recovery between runs), from a holding potential (HP) of 50 mV. The entire 10-step double-pulse protocol was repeated 100-200 times, or until channel rundown was observed.

Each file from a series of repeated protocols was parsed and transposed into 10 files, using software developed in the laboratory. Each of these 10 files contained the episodes recorded at a given prepulse potential. The current traces were corrected for leakage and capacity currents by subtraction of an average of episodes devoid of single channel activity during the test voltage step (null sweeps). The identification of single channel opening and closing transitions using a 50% amplitude threshold (set constant for each experiment) was accomplished using Fetchan 6.0/PClamp (Axon Instruments). The rise time of our recording system (0.166 ms at 2 kHz) limited resolution of kinetic events to those lasting >0.2 ms. Thus, events shorter than 0.2 ms were not included in the kinetic analysis. The number of active Ca²⁺ channels in a given patch (N) was estimated by the maximum number of overlapping currents recorded upon repolarization following a prestep to +130 mV (maximal activation, and synchronization of mode 2 openings) and the probability of opening was calculated by dividing by N. Data were pooled from myocytes from multiple rat hearts; the total number of events analyzed was 111,500 for 105 mM Ba²⁺, 54,826 for 10 mM Ba²⁺, 30,345 for 5 mM Ba²⁺, 12,714 for 2 mM Ba²⁺, and 5,464 for 5 mM Ca²⁺. The analysis of the probability of opening, open-time distributions and their exponential fits, amplitude distributions and their Gaussian fits, and scatterplots of amplitude versus duration were done using a modified version of pSTAT (PClamp, Axon Instruments). Data are reported as means ± SEM. Testing for statistical significance was accomplished using an analysis of variance (ANOVA), Dunnett's method, or Student's paired t-test, as was appropriate.

In this and the accompanying paper (Josephson et al., 2002) we use the "modal" nomenclature developed by Hess et al. (1984) and Yue et al. (1990) in describing the single L-type Ca²⁺ channel currents. Thus, relatively frequent, brief-duration openings are referred to as "mode 1," and relatively infrequent, longer-duration openings are referred to as "mode 2."

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Voltage prepulses modulate the gating of single Ca²⁺ channels: Ca²⁺ versus Ba²⁺ ions

Fig. 1 A displays representative current traces of single L-type Ca²⁺ channel activity (corrected for leakage and capacity currents) using 105 mM Ba²⁺ ions in the patch pipette solution, and recorded during test voltage steps to 10 mV from a holding potential of 50 mV. The test voltage steps were preceded by either no prepulse (50 mV) in column a, a prepulse to +10 mV in column b, or a prepulse to +110 mV in column c. It is evident from an examination of the current traces that the pattern of Ca²⁺ channel activity was influenced by the relatively brief prepulse (100 ms in duration). In the absence of a prepulse (a) the Ca²⁺ channel openings were quite frequent and were nearly time-invariant during the test pulse. Following a prepulse to +10 mV (b) the channel openings during the test step were less frequent than in (a), consistent with a partial inactivation of the current at this moderate level of prepulse depolarization. Most striking, however, was the increased activity of the Ca²⁺ channel following strong predepolarization to +110 mV (c). The signature of this prepulse-mediated facilitation of the Ca²⁺ channel activity is an enhancement in the number of long-duration (mode 2-type) channel openings upon the return to the test voltage.

View larger version (22K): [in this window] [in a new window]   FIGURE 1   Effects of voltage prepulses on single L-type Ca2+ channel currents during test steps using 105 mM Ba2+, 5 mM Ba2+, and 5 mM Ca2+ ions. (A) Representative single L-type Ca2+ channel currents recorded during 400-ms test pulses to 10 mV in the absence of a 100-ms prepulse (a, left column), following a 100-ms prepulse to +10 mV (b, middle column) or a 100-ms prepulse to +110 mV (c, right column). The pipette contained 105 mM Ba2+ ions. The holding potential was 50 mV, the cell resting potential was zeroed as described in the Methods. The patch contained one active channel. The current traces were corrected for capacity and leakage currents. (B) Examples of the effects of prepulses on single L-type Ca2+ channel currents recorded using 5 mM Ba2+ ions in the patch pipette. The voltage-step protocol was identical to that shown in Fig. 1, except the test step was 300 ms in duration. The currents recorded in response to a test pulse to 10 mV are shown in the absence of a prepulse (a, left column), following a prepulse to +10 mV (b, middle column) or a prepulse to + 110 mV (c, right column). The patch contained one active channel. Note the expanded current calibration as compared with part A. (C) The effects of prepulses on single L-type Ca2+ channel currents recorded with 5 mM Ca2+ ions in the patch pipette. Voltage protocols as described in part A. The currents recorded in response to a test pulse are shown in the absence of a prepulse (a, left column), following a prepulse to +10 mV (b, middle column) or a prepulse to + 110 mV (c, right column). The patch contained one active channel. Note the expanded current calibration as compared with parts A and B.

A similar, but slightly different, pattern of behavior is observed when a lower concentration of Ba²⁺ ions permeates the Ca²⁺ channel. Fig. 1 B displays representative single Ca²⁺ channel activity recorded with 5 mM Ba²⁺ ions in the pipette solution, using identical protocols as shown in Fig. 1 A. Under these conditions, the channel openings during the test step, which were frequent in the absence of a prepulse (a), were strongly reduced by a prepulse to +10 mV (b), and were strongly enhanced by a prepulse to +110 mV (c). The facilitation with strong predepolarization was more pronounced with this lower Ba²⁺ concentration (5 mM), with some reopenings lasting the entire duration of the sweep.

In contrast, the substitution of Ca²⁺ ions for Ba²⁺ ions in the pipette solution resulted in a substantial overall decrease in Ca²⁺ channel opening and reopening frequency, as well as dramatic changes in the prepulse-mediated behavior of the channel. Fig. 1 C shows examples of the L-type Ca²⁺ channel currents (recorded during a test step to 10 mV) using 5 mM Ca²⁺ ions, in the absence of a prepulse (a), following a prepulse to +10 mV (b), and after strong depolarization to +110 mV (c). With 5 mM Ca²⁺, prepulses to +10 mV resulted in a marked decrease in the number of Ca²⁺ channel openings. Upon strong predepolarization, long-duration single Ca²⁺ currents were observed upon return to the test potential (c); however, the subsequent frequency of reopening was reduced as compared with Ba²⁺ ions (compare with Fig. 1, A and B).

The probability of opening during the test pulse: Ca²⁺ versus Ba²⁺ ions

To examine the voltage-dependent effects of conditioning prepulses on the overall activity of the Ca²⁺ channel currents we analyzed the averaged probability of opening during an ensemble of test steps (P_avg), as a function of the permeant divalent ion. Fig. 2 A presents the voltage-dependent effects of 100-ms prepulses on the probability of opening of the single L-type Ca²⁺ channel currents, averaged over multiple test steps. For comparison, data are presented from experiments using 105 mM Ba²⁺ (open squares; n = 1000 episodes, 6 cells), 10 mM Ba²⁺ (open triangles; n = 650 episodes, 6 cells), 5 mM Ba²⁺ (open circles; 300 episodes, 5 cells), 2 mM Ba²⁺ (open diamonds; n = 660 episodes, 7 cells) or 5 mM Ca²⁺ ions (closed circles; n = 400 episodes, 6 cells) in the patch pipette solution. As can be seen in Fig. 2 A, the P_avg during the test pulse decreased with increasing prepulse potential, and reached a minimum at +30 to +50 mV for 105 mM Ba²⁺, +30 mV for 10 mM Ba²⁺, +10 mV for 5 mM Ba²⁺, 10 mV for 2 mM Ba²⁺, and +30 mV for 5 Ca²⁺ mM ions. The shifting of the U-shaped inactivation/facilitation curve on the voltage axis is caused by the effects of screening of membrane surface charges by the type and concentration of divalent ion in the recording solution (e.g., Wilson et al., 1983). Similar amounts of surface charge-related shifts in the threshold potential for Ca²⁺ channel activation were also noted during the prepulse; the threshold for Ca²⁺ channel activation was 40 to 30 mV for 2 mM Ba²⁺, 30 to 20 mV for 5 mM Ba²⁺, 10 to 0 mV for 105 mM Ba²⁺, and 30 to 20 mV for 5 mM Ca²⁺.

View larger version (27K): [in this window] [in a new window]   FIGURE 2   The voltage-dependent effect of prepulses on the probability of Ca2+ channel opening during test steps. (A) The different symbols represent pooled data from experiments conducted using 105 mM Ba2+ (open squares; n = 1000 episodes, 6 cells), 10 mM Ba2+ (open triangles; n = 650 episodes, 6 cells), 5 mM Ba2+ (open circles; n = 300 episodes, 5 cells), 2 mM Ba2+ (open diamonds; n = 660 episodes, 7 cells), and 5 mM Ca2+ (closed circles; n = 400 episodes, 6 cells). Plotted (logarithmically) is the probability of opening (the sum of all open times detected divided by total episode time) averaged over test voltage steps that were preceded by a prepulse to the indicated potential (means ± SEM). Note the greater averaged probability of opening (at all potentials) for Ba2+ ions (open symbols) compared with Ca2+ ions (closed circles). (B) Averaged probability of opening data shown in A were normalized to their values at 50 mV (without a prepulse) and replotted. Values <1.0 represent inactivation, and values >1.0 represent facilitation of the single Ca2+ channel currents.

Prepulses to more positive potentials produced a return of the test pulse P_avg to its initial value, and higher depolarizing prepulses resulted in a further increase or facilitation of the test pulse P_avg to values above those recorded without a prepulse. It is interesting to note that this U-shaped curve appears to be similar to that usually recorded using whole-cell Ca²⁺ channel currents during double-pulse protocols (e.g., Josephson et al., 1984); however, in the macroscopic analysis of inactivation only the magnitude of the peak current is plotted, whereas Fig. 2 presents the entire test pulse P_o, averaged over multiple episodes of single Ca²⁺ channel recordings.

To compare the extent of prepulse-mediated inactivation or facilitation of P_avg recorded with Ba²⁺ or Ca²⁺ ions, the data from Fig. 2 A were normalized to their P_avg obtained at 50 mV, and are presented in Fig. 2 B. Normalization of the data was advantageous for several reasons. First, as noted above, changes in external divalent ion concentrations are well-known to produce a voltage-shift in Ca²⁺ channel gating parameters due to alterations in surface potential (Wilson et al., 1983) that results in changes in P_o at the test potential. Therefore, the transmembrane potential "sensed" by the Ca²⁺ channel gating voltage sensors during the test potential was actually shifted to more negative potentials as a function of increasing divalent ion concentration. Second, the intrinsic variability of activity among Ca²⁺ channels made the comparison of absolute values of P_o between groups problematical. Third, our method for determining the number of active channels (N) present might underestimate N, and therefore overestimate P_o.

The normalized plot (Fig. 2 B) shows that the maximal extent of inactivation of P_avg (over moderate prepulse potentials) was greatest with 5 mM Ca²⁺ (94%); and inactivation with Ba²⁺ ions followed in order of increasing concentration: 2 mM Ba²⁺ (74%), 5 mM Ba²⁺ (63%), 10 mM Ba²⁺ (52%), 105 mM Ba²⁺ (18%). The maximal degree of facilitation (following a +110 mV or +130 mV prepulse) was (in decreasing order): 2 mM Ba²⁺ (341%), 5 mM Ba²⁺ (46%), 10 mM Ba²⁺ (41%), 5 mM Ca²⁺ (29%), and 105 mM Ba²⁺ (20%). Each of the preceding measurements was found to be statistically significant at the p < 0.01 level, when compared with their control values (in the absence of a prepulse). Thus, the degree of prepulse-induced inactivation decreased with increasing Ba²⁺ ion concentration, and the degree of facilitation also decreased with increasing Ba²⁺ ion concentration. Stated differently, the Ba²⁺ currents that inactivated most during the test pulse also displayed the greatest amount of facilitation at high prepulse potentials. This interesting finding will be addressed further in the Discussion.

A more detailed analysis was then conducted to determine by what single channel mechanism(s) the averaged probability of opening (P_avg) was being altered by the application of depolarizing prepulses. First, the probability of channel opening (P_o) of each test pulse was calculated to determine the cumulative fraction of Ca²⁺ channel activity during the episode. Episodic diaries of the P_o during the test pulse were then plotted to decipher the pattern of activity that contributed to the prepulse-mediated inactivation or facilitation of the Ca²⁺ channel. Fig. 3 A displays P_o diaries from episodes recorded using 105 mM Ba²⁺ ions, without a prepulse (a), following a prepulse to +20 mV (b), and following a prepulse to +110 mV (c). Inspection of the diaries reveals that a prepulse to +20 mV (b) results in only a slight overall reduction of P_o, and an increase in the number of null episodes (those test pulses without Ca²⁺ channel opening). However, the prepulse to +110 mV produced an increase in P_o of the active sweeps.

View larger version (27K): [in this window] [in a new window]   FIGURE 3   Prepulse-dependent effects on episodic diaries of the probability of opening of Ca2+ channels during a test pulse. (A) Recorded using 105 mM Ba2+ ions, during a test pulse (0 mV) in the absence of a prepulse (a, top row), with a prepulse to +20 mV (b, middle row), or with a prepulse to +110 mV (c, bottom row). (B) Recorded using 5 mM Ba2+ ions, during a test pulse (0 mV) in the absence of a prepulse (a, top row), with a prepulse to +10 mV (b, middle row), or with a prepulse to +130 mV (c, bottom row). (C) Recorded using 5 mM Ca2+ ions, during a test pulse (0 mV) in the absence of a prepulse (a, top row), with a prepulse to +10 mV (b, middle row), or with a prepulse to +130 mV (c, bottom row).

For comparison with the experiments using 105 mM Ba²⁺, an analogous series of double-voltage step experiments were conducted using 5 mM Ba²⁺ ions. The single Ca²⁺ channel currents recorded during the second, test pulse with 5 mM Ba²⁺ are shown in Fig. 3 B. As shown in the diary, using 5 mM Ba²⁺ ions (a 21-fold reduction in ion concentration, as compared with 105 mM Ba²⁺) prepulses to +10 mV (b) resulted in a more substantial reduction of P_o, and an increased number of null episodes. In addition, prepulses to +130 mV (c) resulted in an even greater enhancement of P_o in individual episodes.

The effects of prepulses on the test pulse diaries of P_o obtained using 5 mM Ca²⁺ (Fig. 3 C) were even more dramatic. In this nearly physiological condition, the P_o of the Ca²⁺ channel (which in the absence of a prepulse (a) was ~10-fold lower than that recorded using Ba²⁺ ions) was further dramatically reduced following a prepulse to +10 mV (b), with most episodes displaying no channel openings. Nevertheless, prepulses to +130 mV (c) produced episodes with P_o occasionally greater than in the absence of a prepulse. However, the number of null traces was also greater at +130 mV than in the absence of a prepulse.

Thus, it is apparent from inspection of the P_o diaries that the number of null test pulse traces (i.e., without channel openings) increased with increasing prepulse depolarization, using Ba²⁺ ions as well as Ca²⁺ ions, as shown in Fig. 4. The ratio of the number of active traces (i.e., containing one or more openings) divided by the total number of traces provides a measure of Ca²⁺ channel availability. By this method, the availability of the Ca²⁺ channels during the test pulse can be seen to decrease with increasing prepulse potential, but then increase again at higher prepulse potentials. The increase at the higher prepulse potentials reflects the contribution of traces containing one or more long-duration openings.

View larger version (18K): [in this window] [in a new window]   FIGURE 4   The availability of single Ca2+ channel currents during the test pulse, plotted as a function of prepulse potential. Mean availability data from experiments using Ba2+ ions are shown by the open symbols (105 mM, squares (1000 episodes, 6 patches); 5 mM, circles (300 episodes, 5 patches); 2 mM, triangles (660 episodes, 7 patches)); and those using 5 mM Ca2+ are shown by the closed circles (400 episodes, 6 patches). Availability is defined as the fraction of episodes displaying Ca2+ channel openings.

The prepulse-induced reduction in availability was most marked using 5 mM Ca²⁺ ions, and was less prevalent with Ba²⁺ ions. However, the prepulse-mediated reduction in availability was inversely related to Ba²⁺ ion concentration, with 2 mM Ba²⁺ having a greater effect on availability than 5 mM Ba²⁺, which was greater than 105 mM Ba²⁺. Also, note that the maximum reduction in availability occurred at a prepulse potential of +10 mV for 2 mM Ba²⁺, +30 mV for 5 mM Ba²⁺, +30 to +50 for 5 mM Ca²⁺, and +70 mV in 105 mM. The relative shift in potential most probably was caused by changes in surface potential produced by the permeant ions (Wilson et al., 1983).

It is important to bear in mind that the relatively short-duration prepulse (100 ms) used throughout this study was chosen because it does not produce complete (i.e., absorbing) voltage-dependent inactivation, thus allowing a determination of the relative ion concentration-dependent effects on inactivation and facilitation. Thus, the rebound in availability at high prepulse potentials reflects the decrease in null traces due to an increase in those traces displaying long-openings.

Given the relative decrease in Ca²⁺ channel availability at high prepulse potentials (as compared with the absence of a prepulse) how does strong conditioning depolarization produce a facilitation of the test pulse, P_avg, to values larger than those obtained in the absence of a prepulse? Further examination of the P_o diaries revealed that following a strong prepulse some of the episodes with test pulse activity display a P_o that was greater than those without a prepulse. Thus, the increase in P_o during these highly active sweeps is greater than the decrease in the proportion of active sweeps, resulting in an increase or facilitation of the average P_o following strong prepulses.

The extent of the prepulse-dependent facilitation on the P_avg of the Ca²⁺ channel during the test pulse is even more evident after correcting for the effects of the prepulse on Ca²⁺ channel availability. The resulting corrected P_avg (after removal of null sweeps caused by either voltage-dependent, or ion-dependent inactivation that lasted for the entire test pulse, data not shown) emphasizes the voltage-dependent activation of the prepulse-dependent facilitation of P_avg. The increase, or facilitation of the corrected test pulse P_avg with a prepulse to +130 mV (as compared with no prepulse) was 36% for 105 mM Ba²⁺, 46% for 5 mM Ba²⁺, 258% for 5 mM Ca²⁺, and 392% for 2 mM Ba²⁺ ions (p < 0.01).

Distributions of Ca²⁺ channel open times are prepulse- and ion-dependent

We next investigated the single channel gating mechanisms underlying the prepulse-induced inactivation and facilitation of the test pulse P_o. To address this question, analyses of the distributions of Ca²⁺ channel open-times as a function of prepulse potential were performed with data recorded using 105 mM Ba²⁺ (Fig. 5 A), 5 mM Ba²⁺ (Fig. 5 B), or 5 mM Ca²⁺ ions (Fig. 5 C). Representative distributions of the open-times are displayed in log-log plots to better visualize the long-duration events, and were fit with a sum of three exponentials. A sum of three exponentials provided a better fit than the sum of two exponentials, as judged by comparing the goodness-of-fit criteria (the "F"-value calculated from the sum of squared errors for the two models in pSTAT).

View larger version (33K): [in this window] [in a new window]   FIGURE 5   Analysis of the distribution of Ca2+ channel open-times during a test pulse, as a function of prepulse potential. The distributions of the open-times (during test steps to 10 mV) are displayed in logarithmic plots to visualize the long-duration events, and then fit with a sum of three exponentials in the absence of a prepulse (a, top row), with a prepulse to +10 mV (b, middle row), or with a prepulse to +130 mV (c, bottom row). Examples shown were recorded using 105 mM Ba2+ ions (A); 5 mM Ba2+ ions (B); or 5 mM Ca2+ ions (C).

The extracted fit parameters (fast time constant, ₁; medium time constant, ₂; and slow time constant, ₃) of the exponential functions and their respective proportions are presented in Table 1. Averaged test pulse time constants are displayed for the pooled data using 5 mM Ca²⁺ (6 cells), 5 mM Ba²⁺ (5 cells), and 105 mM Ba²⁺ (6 cells) at three prepulse potentials: 50 mV (no prepulse), +10 mV or +30 mV (the prepulse potential yielding maximal inhibition), and +110 mV or +130 mV (yielding maximal facilitation). A similar pattern of time constants as a function of prepulse potential was found for 5 mM Ca²⁺, 5 mM Ba²⁺, and 105 mM Ba²⁺; however, the values for ₁ and ₂ with 5 mM Ca²⁺ ions were consistently much smaller than those found for Ba²⁺ ions. Under all three ionic conditions moderate depolarization produced a decrease, and high depolarization produced an increase in the values for the three time constants.

Also shown in Table 1 is the prepulse-dependence of the proportion of each time constant; a comparison of these values gives the relative contributions of the short-, medium-, and long-duration openings under each condition. As can be seen, there was a greater contribution of short-duration (₁) events in 105 mM Ba²⁺ as compared with 5 mM Ba²⁺. However, both 105 mM Ba²⁺ and 5 mM Ba²⁺ displayed a prepulse-dependent decrease (with moderate depolarization), and increase (with strong depolarization) in the relative number of long-duration (₃) events. A more striking dependence on prepulse potential is seen with 5 mM Ca²⁺ ions permeating the channel; in this case the relative numbers of short-, medium-, and long-duration events are all decreased at moderate prepulse potentials, whereas the relative numbers of medium- and long-duration events at high prepulse potentials are markedly increased.

The relative contribution of each kinetic component to the overall distribution of Ca²⁺ channel open times, as a function of prepulse potential and permeating ion, is presented in Fig. 6. The data are plotted as the product of the average time constant of each component (in ms) and its average proportion (yielding a dimensionless fraction between 0 and 1). Part A shows the results using 105 mM Ba²⁺ ions (6 cells); part B using 5 mM Ba²⁺ ions (5 cells), and part C using 5 mM Ca²⁺ ions (6 cells). These plots present the fraction of the total Ca²⁺ current conducted by short-, medium-, and long-duration events as a function of prepulse potential.

View larger version (22K): [in this window] [in a new window]   FIGURE 6   The relative contribution of each kinetic component to the overall distribution of Ca2+ channel open times, as a function of prepulse potential and permeating ions. The data are plotted as the product of the average time constant of each component (in ms), and its average proportion (a dimensionless fraction between 0 and 1). (A) Results using 105 mM Ba2+ ions; (B) using 5 mM Ba2+ ions; and (C) using 5 mM Ca2+ ions.

For 105 mM Ba²⁺ ions (Fig. 6 A) a large fraction of the current (73%) is carried by short (₁) openings (open columns) with prepulses to 50 mV, +30 mV, and +130 mV. The fraction carried by medium-duration (₂) openings (hatched columns) is an increasing function of prepulse potential. The fraction carried by long-duration (₃) openings (solid columns) decreases with moderate prepulses (+30 mV), but then increases with strong prepulses (+130 mV). With a prepulse to +130 mV a substantial fraction of the total current (27%) is carried by long (₃) openings.

For 5 mM Ba²⁺ ions (Fig. 6 B) a similar pattern for the kinetic proportions of the currents was obtained. However, as compared with 105 mM Ba²⁺, with 5 mM Ba²⁺ ions a larger fraction of the current is carried by medium-duration openings (₂) at all prepulse potentials (hatched columns). In addition, moderate prepulses (+10 mV) resulted in a decrease in both short- (₁) and medium-duration (₂) openings, compared with no prepulse (50 mV). Strong prepulses (+130) produced an increased contribution of both short and medium durations. Similar to the case of 105 mM Ba²⁺, with 5 mM Ba²⁺ the long-duration (₃) component (solid columns) was decreased with moderate prepulses (+10 mV), and then increased with strong prepulse (+130 mV).

For 5 mM Ca²⁺ ions (Fig. 6 C), in the absence of a prepulse (at 50 mV) a large proportion of the total Ca²⁺ current (52%) was composed of long-duration (₃) events (solid columns). With moderate prepulses to +10 mV all three components were strongly inhibited, consistent with the conspicuous decrease in the probability of opening due to Ca²⁺-dependent inactivation. However, with high prepulses (to +130 mV) there is a large increase in the relative contributions of medium- (₂) and long-duration (₃) events (74% of total current). Thus, even though the relative frequency of ₃ events is lower than ₁ or ₂ events, they conduct a surprisingly large fraction of the total Ca²⁺ current, due to their markedly increased open-duration.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The present study is the first to describe the inhibitory and facilitory effects of a wide range of conditioning voltages on single cardiac L-type Ca²⁺ channel currents with a near-physiological level of Ca²⁺ ions permeating the channel, and in the absence of any Ca²⁺ channel agonists. We have found marked ion-dependent alterations in Ca²⁺ channel gating kinetics with both prepulse-induced inhibition (following moderate depolarization) and facilitation (following strong depolarization). An additional dimension of the study is that we have characterized the concentration-dependent effects of Ba²⁺ ions in modulating single Ca²⁺ channel gating in response to voltage prepulses. These findings support the hypothesis that both the conditioning voltage and the permeating divalent cations alter the gating properties of the L-type Ca²⁺ channel.

The present results reveal that the amount and timing of Ca²⁺ influx into the myocardial cell are exquisitely fine-tuned by the utilization of these opposing facilitory and inhibitory mechanisms, as each of these predominates over a different range of potentials and manifests different kinetics. A "U-shaped" curve for the voltage-dependence of inactivation (where the curve reaches a minimum at moderately depolarizing prepulses, and then increases at higher prepulse depolarization) has traditionally been one of the hallmarks of Ca²⁺-dependent inactivation of the macroscopic L-type Ca²⁺ channel current. It has been widely thought that the U-shaped curve reaches a minimum over prepulse potentials that elicit the maximum Ca²⁺ current, and that inactivation is relieved at higher potentials because I_Ca was diminished as E_Ca was approached as the prepulse voltage was increased. However, the present results point to a more complex single channel mechanism to explain the U-shape of inactivation. Thus, at moderately depolarizing prepulse potentials Ca²⁺ channel availability is at its lowest value, due to a combination of voltage-dependent and Ca²⁺-dependent inactivation. Following higher prepulse potentials, test pulse Ca²⁺ channel availability may still be low, but this inhibition is overcome by an increase in mode 2 openings of active sweeps. Therefore, the U-shape is derived as a product of the decreasing Ca²⁺ channel availability and the increasing activation of mode 2 with increasing prepulse potential.

The interaction between these two opposing regulatory mechanisms of L-type Ca²⁺ channels, inactivation and facilitation, can be observed most clearly when Ca²⁺ ions are permeating the channel. Under these conditions, a moderate predepolarization produces a marked decrease in the test pulse P_o due to a reduction in the frequency of openings of all durations. Conversely, strong predepolarization elicits longer-duration openings upon return to the test potential. The mode 2 openings are more noticeably prolonged with Ba²⁺ ions permeating the channel. The simplest explanation to account for this effect is that Ba²⁺ ion binds, but with a lesser affinity, to a site within the Ca²⁺ channel protein that normally leads to Ca²⁺-dependent closure of the channel. This idea was originally proposed to explain the relative slowing of macroscopic Ba²⁺ current inactivation in neurons (Brown et al., 1981) and in native myocytes (Josephson et al., 1984), and has more recently been applied to results obtained using cloned cardiac Ca²⁺ channels (Ferreira et al., 1997).

An additional novel finding of the present study is that the maximal amount of prepulse-induced inactivation (reduction of P_o) was found to decrease with increasing Ba²⁺ ion concentration, and that the degree of prepulse-induced facilitation (increase in P_o) also decreased with increasing Ba²⁺ ion concentration. In other words, divalent ion conditions that favored the more rapid development of inactivation also promoted the greatest amount of facilitation of the single Ca²⁺ currents.

It is well known from previous whole-cell experiments that the initial component of the biphasic inactivation of the Ba²⁺ current becomes slower as the Ba²⁺ ion concentration is increased (see McDonald et al., 1994, for a review). This peculiar behavior of the macroscopic L-type Ca²⁺ current when using Ba²⁺ ions as the charge carrier is opposite to that observed when using Ca²⁺ ions as the charge carrier. With Ca²⁺ ions permeating the Ca²⁺ channel the fast, or initial, phase of the inactivation of the whole-cell current becomes even more rapid as the Ca²⁺ ion concentration is increased. Indeed, this relationship is held as one of the signatures of Ca²⁺-dependent inactivation of the Ca²⁺ current. Taken together, this very different behavior of the inactivation of the macroscopic Ca²⁺ and Ba²⁺ currents suggests that at least two divalent cation binding sites may be involved to produce these characteristics. One site (site 1), which may be responsible for initiating the ion-dependent inactivation, may have a much lower affinity for Ba²⁺ ions than for Ca²⁺ ions. That would, of course, explain the relatively slower initial inactivation of the macroscopic Ba²⁺ current, as compared to the Ca²⁺ current. A second site (site 2), perhaps located on the extracellular side of the Ca²⁺ channel or in the outer region of its pore, may be involved in determining the reopening frequency of the Ca²⁺ channel. This site may be sensitive to the external divalent ion concentration in a manner such that a higher ion concentration produces a greater number of reopenings of the Ca²⁺ channel during a depolarization. The reopening frequency is a major determinant of burst length, and therefore of the time course for the decay of the ensemble, or macroscopic Ca²⁺ current. The product of the relative contributions of these two mechanisms (and the relative affinities of these two hypothetical sites to Ba²⁺ and Ca²⁺) can then explain the very different inactivation behavior of the currents. For Ba²⁺ ions, the affinity for site 2 would be greater than for site 1, and therefore the current inactivation would be slower with increasing Ba²⁺ ion concentration. Conversely, for Ca²⁺ ions the affinity for site 1 would be greater than for site 2, thus the current inactivation would be faster with increasing Ca²⁺ ion concentration.

As the principal point of Ca²⁺ influx into myocardial cells, the L-type Ca²⁺ channel has evolved to possess numerous control mechanisms to insure efficient regulation over the influx of this crucial ion. At the single L-type Ca²⁺ channel level, a moderate-depolarizing prepulse, with its attendant Ca²⁺ influx, leads to a diminution of the subsequent Ca²⁺ channel activity. This alteration in behavior has been characterized as a shift from a gating mode displaying mostly relatively brief channel openings ("mode 1") into a gating mode of very low opening probability, termed "mode Ca" (Yue et al., 1990; Imredy and Yue, 1994). The molecular locus of this mechanism, the action of prior Ca²⁺ influx to inhibit subsequent Ca²⁺ influx, involves a calmodulin-binding site that has been implicated in Ca²⁺-dependent inactivation of the Ca²⁺ channel (Zuhlke et al., 1999). Previous single channel studies assumed that Ca²⁺-dependent inactivation did not significantly affect the gating charge movement and, therefore, the Ca²⁺ channel voltage sensor(s) (Imredy and Yue, 1994); however, more recent work suggests that gating currents (and therefore transitions among the closed states) are indeed affected by Ca²⁺ influx (Shirokov, 2000). In light of this, revised models of Ca²⁺ channel gating and modal interconversion are needed that take into account these newer findings.

The molecular locus for the high-voltage facilitation of the cardiac L-type Ca²⁺ channel remains somewhat obscure. It was originally suggested that an additional conformational change of one or more of the channel's voltage-sensing S4 regions, or other (as yet unknown) voltage-sensing moieties of the channel, may be responsible for transducing the conformational change produced by strong depolarizing prepulses (Pietrobon and Hess, 1990). In support of this view, several gating current studies have identified a component of Ca²⁺ channel charge movement occurring at very positive voltages in native myocytes (Bean and Rios, 1989; Josephson and Sperelakis, 1992) and for cloned human cardiac L-type Ca²⁺ channels (Josephson and Varadi, 1996; Josephson, 1997). Additionally, the coexpression of a  subunit with the 1C of the Ca²⁺ channel is required for voltage-dependent facilitation (Kamp et al., 2000) and also enhances the occurrence of long-duration single Ca²⁺ channel currents (Constantin et al., 1998). However, it is interesting to note that, in contrast to neuronal N- and P/Q-type Ca²⁺ channels, the facilitation of the cardiac (1C) Ca²⁺ channel by strong depolarization appears to be independent of a G protein pathway (Kamp et al., 2000).

It has previously been observed in whole-cell recordings of L-type Ca²⁺ channel currents (especially when using Ba²⁺ ions as the charge carrier) that the decay of the tail currents elicited upon repolarization of the voltage step could be described by a fast and a slow component (McDonald et al., 1994). Although it has sometimes been explained as the deactivation of a second open state of the Ca²⁺ channel, or even related to the Na⁺-Ca²⁺ exchange current, the present results would indicate that this slow component of deactivation reflects the closure of mode 2 openings at the return voltage. Interestingly, a similar time-dependent recruitment of mode 2-like channel openings, and a slow deactivation time course of the macroscopic current, have recently been reported for Shaker K⁺ channels (Olcese et al., 2000). This finding for K⁺ channels suggests that time-dependent modal conversion may be a general feature of many types of voltage-dependent ion channels.

In the present study we found that a (lower) Ba²⁺ ion concentration that produced a greater degree of inactivation also produced a greater degree of facilitation. This relationship may imply the possibility that these two processes may be linked. In this regard, it may be noted that a feature of high prepulse-dependent facilitation of the L-type Ca²⁺ channel is the relatively long time-dependence (on the order of tens to hundreds of milliseconds) for its activation and deactivation (Pietrobon and Hess, 1990; Hirano et al., 1999). This suggests that following the rapid movements (on the order of milliseconds) of the voltage sensors, additional (and much slower) conformational rearrangements of the Ca²⁺ channel occur during prolonged depolarization, which may permit the long duration openings (mode 2) upon return to the test potential. By analogy, other examples of slow conformational gating changes are the long time course for the development of voltage-dependent inactivation of the Ca²⁺ current and for the associated negative shift in the availability of Ca²⁺ channel gating charge movement (Shirokov, 2000; Josephson, 1996). It is tempting to speculate that the slow deactivation of mode 2 during repolarization of the action potential is a mechanism designed to increase Ca²⁺ ion influx at a time and voltage when mode 1 openings have ceased.

In summary, we have demonstrated, using a low concentration of Ca²⁺ ions and a range of Ba²⁺ ion concentrations, that the application of a conditioning prepulse produces voltage- and ion-dependent alterations in the probability of opening, availability, and gating kinetics of the subsequent single L-type Ca²⁺ channel currents. A greater understanding of these microscopic mechanisms for modulation of inactivation and facilitation of the Ca²⁺ currents will provide a more comprehensive and accurate portrayal of the complex behavior of the L-type Ca²⁺ channel and its role in the local control of cardiac E-C coupling (Stern, 1992).