Abstract have been described for canine atrial cells, 2 rabbit sinoatrial nodal cells, 5 guinea pig ventricular cells, 6 -7 and frog atrial cells. 8 In some neurons, a third type of Ca 2+ channel has also been described: the N current. 9 -10 This latter current differs from the other two by its voltage dependence of inactivation and activation, time course of inactivation, responses to pharmacological agents, and single channel conductance 911 (however, see Reference 12). Abstract Although T and L currents in ventricular cells from guinea pig have been described, 6 ' 7 there is no complete report on these two Ca 2+ channel currents in canine ventricular cells and Purkinje cells (however, see Reference 13). Furthermore, it has recently been shown that in the sinoatrial nodal cells there is a high density of T current and that this current is important for late phase 4 depolarization in these pacemaker cells. 5 Is it possible then that the pacemaker cells of the ventricle, the Purkinje cells, also possess a high density of T current? Since the major functions of ventricular cells and Purkinje cells are different (i.e., contraction for the former and impulse Multiple Types of Ca 2+ Currents in Single Canine Purkinje Cells Gea-Ny Tseng and Penelope A. Boyden Multiple Types of Ca 2+ Currents in Single Canine Purkinje Cells Whole-cell Ca 2+ channel currents were recorded from isolated single canine Purkinje and ventricular cells to determine whether there were multiple types of Ca 2+ channels in these two cell types, as in many other excitable tissues. The experimental conditions were such that currents other than Ca 2 + channel currents were largely suppressed. The charge carrier was either Ca 2+ or Ba 2+ (5 mM). In every canine Purkinje cell studied (n=36), we saw T and L Ca 2+ channel currents that are similar to their counterparts in other tissues. Neither current was affected by tetrodotoxin (30 fiM), but both were reduced by Mn 2+ (5 mM). Ni 1+ (SO fiM) blocked T more than L current. Nisoldipine (1 fiM) apparently abolished the L current but also decreased the T current by 50%. Substitution of Ba 2+ for Ca 2 * augmented and prolonged L current but did not affect T current significantly. At 36° C and with 5 mM (Ca 2+ ] 0 , T current inactivated over a voltage range from -70 to -30 mV whereas L current inactivated between -30 and +20 mY. T current was detectable in only some of the ventricular cells studied (8 out of 12). In these cells the ratio of maximal T current to maximal L current (0.2±0.1, n=S) was lower than the T/L ratio in Purkinje cells (0.6±0.2, n-6). The density of peak L current in ventricular cells (7.5± 1.7 pA/pF, n=8) was higher than that in Purkinje cells (4.4±3.4 pA/pF, n=6). Therefore, in ventricular cells the L current is the main Ca 2 + current whereas in Purkinje cells, the T current also contributes significantly to membrane electrical activity. In Purkinje cells, 0-adrenoceptor stimulation by isoproterenol (1 /JM) increased L current but did not affect T current. On the other hand, in 70% (7 out of 10) of the Purkinje cells, <v-adrenoceptor stimulation by 10 fiM norepinephrine (in the presence of 2 fiM propranolol) increased the T current. Our observations show that the distribution of the two types of Ca 2+ channels in canine ventricle is heterogeneous and that the two types of Ca 2+ channels are modulated by catecholamines by different receptors. (Circulation Research 1989;65:1735-1750) I n various tissues there exist multiple types of Ca 2+ channels. 1 In most of these tissues two types of Ca 2+ channels have been described: T current (also called fast current, 2 SD [for slow deactivation], 3 or type I 4 ) and L current (also called slow current, 2 FD [for fast deactivation], 3 or type II 4 ). These two currents differ in terms of voltage range of inactivation and activation, kinetics of inactivation and deactivation, single-channel conductance, and sensitivity to pharmacological agents. For cardiac tissues, two types of Ca* + channels conduction and pacemaking for the latter), are there differences in the proportions of these two Ca 2+ channels between these two cell types? Multiple Types of Ca 2+ Currents in Single Canine Purkinje Cells In the present study, we recorded whole-cell Ca 2+ channel currents from single canine Purkinje and ventricular cells under conditions that largely suppressed other interfering currents. The following questions were addressed: 1) Are there multiple Ca 2+ channel currents in Purkinje and ventricular cells, as observed in other tissues? 2) If there are, what are their properties? 3) How are these Ca 2+ channel currents modulated by catecholamines? Portions of these results have been reported in abstract form. 14 -15 Materials and Methods Cell Preparations Canine Purkinje cells were prepared by a modification of the method of Sheets et al 16 and described previously. 17 Briefly, free-running Purkinje fiber bundles were removed from both ventricles of a mongrel dog, minced, and placed in low Ca 2+ minimal essential medium (MEM, GIBCO, Grand Island, New York), pH 6.7 with HEPES-NaOH and containing collagenase (5 mg/ml, Worthington type II, 231 units/mg). The tissue fragments were agitated in a 37° C rotary shaker bath under a 100% O 2 canopy at a rate of 1-2 cycles/sec for 45-90 minutes. Afterwards, the tissue fragments were transferred to a "dispersion solution" containing 160 mM K-glutamate and 5.7 mM MgSO 4 , pH 6.7 with HEPES-KOH, and the cells were released by gentle hand pipetting with a Pasteur pipette. Dispersed cells were centrifuged and resuspended in MEM, pH 7.3 with HEPES-NaOH. Cell Preparations [Ca] 0 in this solution was gradually elevated from nominally 0 to 1 mM in a period of 1 hour. The cells were then stored in this solution under a 100% O 2 canopy at room temperature before use. Single ventricular cells were isolated from the left ventricle of a mongrel dog by a method adapted from that of Powell and Twist 19 and described previously. Briefly, a wedge of left ventricular free wall was perfused through the first branch of left circumflex artery with nominally Ca-free Tyrode's solution (for the composition of Tyrode's solution, see below) containing collagenase (0.5 mg/ml, Worthington type II, 190 units/ mg). The deep myocardium was then minced and triturated in the same medium. Isolated cells were washed twice with Tyrode's solution containing 0.1% albumin and stored in Tyrode's solution containing 0.1% albumin, 1 unit/ml insulin, 5 mM mannitol, and 5 mM pymvate at 4° C before use. Electrophysiological Experiments The cells were allowed to adhere to a poly-L-h/sine-coated glass coverslip placed on the bottom of a 0.7-ml Lucite tissue chamber mounted on the stage of a Nikon inverted microscope and superfused continuously with Tyrode's solution. Electrophysiological Experiments The Tyrode's solution had the following composition (mM): NaCl 137, NaHCO 3 12, dextrose 5.5, NaH ? PO 4 1.8, MgCl 2 0.5, KC1 4, and CaCl 2 2. The solution was equilibrated with 5% CO 2 -95% O 2 and maintained at pH 7.1. In some experiments the Tyrode's solution was buffered with 5 mM HEPES-NaOH in which the NaHCO 3 and NaH 2 PO 4 were omitted. No significant differences were noted in recorded currents between cells superfused with these two solutions. The whole-cell Ca 2+ channel currents were recorded by the whole-cell variant of the patch-clamp method. The pipette was made of Pyrex glass (Mercer Glass, New York, New York) or Boralex glass (Rochester Scientific, Rochester, New York) and pulled by a two-stage puller (PE-2 puller, Narishige, Tokyo, Japan). The pipette was heat-polished before use and filled with Tyrode's solution. Tip resistance was 2-3 Mfi. With the pipette in the bath solution, pipette potential was adjusted to give zero current; this was the reference potential for later measurements. After a "gigascal" was formed, the pipette solution was switched to the internal solution by use of a pipette perfusion device. The internal solution had the following composition (mM): Cs-aspartate 125, tctraethylammonium chloride 20, HEPES 10, ATP (Mg salt) 5, EGTA 10, and creatine phosphate 3.6. The patch membrane was then ruptured by an increase in suction pressure. An interval of at least 10 minutes was allowed for the intracellular dialysis to reach equilibrium. By then, the holding current at -30 mV was stable and close to zero. The bath solution was then changed to a Na-and K-free solution that had the following composition (mM): choline Cl 145, dextrose 5.5, MgCl 2 0.5, HEPES 5, and CaCl 2 or BaCl 2 5. The pH was adjusted to 7.3 with CsOH. This solution also contained 4-aminopyridine (2 mM) to block a 4-aminopyridine-sensitive transient outward current. A period of 10 minutes of continuous superfusion with this solution before the start of recording was allowed to make sure that the extracellular Na and K were totally washed out (the time for total exchange of bath solution, judged by monitoring the change in resting membrane potential when the bath [K] was changed from 4 to 16 mM, was 0.5-1 minute). The experiment was carried out during continuous superfusion with the Na + -and K + -free solution at 35-37° C or room temperature (22-25° C). The temperatures of the experiments will be specified in "Results." Electrophysiological Experiments The following drugs were used: isoproterenol (Isuprel, Winthrop-Breon, New York, New York), norepinephrine (bitartrate salt, Sigma, St. Louis, Missouri), propranolol (1 mg/ml propranolol hydrochloride in sterile water, Ayerst, New York, t>few York), prazosin (hydrochloride, Sigma), and nickel chloride (NiCl z , Sigma). For experiments studying the effects of norepinephrine, 10 fiM EDTA and 2 yiM propranolol were added to the control as well as the norepinephrine-containing solutions. Nisoldipine, a generous gift from Dr. R. Kass, was dissolved in polyethylene glycol 400 (Fisher, Springfield, New Jersey) at a concentration of 10 mM and added to the superfusate to reach a final concentration of 0.1-1 fiM. The solvent at a concentration 40 times that used in these experiments did not affect Ca 2+ current. 22 Data Acquisition and Analysis A List EPC-7 amplifier (List Medical, FRG) was used for voltage clamping. The pipette capacitance was compensated by the fast capacitance compensation circuit of the amplifier after the formation of gigaseal and before breaking into the cell. After rupture of cell membrane, the cell capacitance was determined by integrating the area under the capacitative transient and then dividing this area by the small voltage step used to induce this capacitative transient (5 mV hyperpolarizing step from -80 mV). The series resistance was determined by dividing the time constant of the capacitative transient by the cell capacitance. The capacitance from the cell membrane was partially compensated by using the slow capacitance compensation circuit of the amplifier. The series resistance was partially compensated, and the residual series resistance was 3 MO or less. The resultant voltage error was 3 mV or less for a current of 1 nA. Data Acquisition and Analysis We evaluated the spatial homogeneity during voltage clamp by estimating the space constant (A) at the time of peak Ca 2+ channel conductance, using the following equation 23 : Data Acquisition and Analysis where L is cell length, A is cross-sectional area of the cell, R fa is average input resistance, and Rj is specific resistivity of the cell interior. Ru, at peak Ca 2+ channel conductance, calculated from the positive slope of the peak Ca 2+ current-voltage (I-V) relation, was 100-200 Mft. For Purkinje cells, we used the estimated parameters that were likely to shorten the space constant (L=100 jura," A=707 assuming a cell diameter of 30 jim, 16 R in =100 , and R,=600 O-cm 24 ). The calculated A equaled 1,535 jim, 30 times the distance between the pipette (in the center of the cell) and the cell end. Thus, due to the combination of a big cell diameter and low peak Ca 2+ channel conductance, the spatial homogeneity was achieved during the peak Ca 2+ channel current. Data Acquisition and Analysis One problem we encountered when voltage clamping a Purkinje cell was a large cell capacitance due to cell size and, thus, long capacitative transients. The average cell capacitance of canine Purkinje cells was 243±50 pF (n=6). This value is somewhat lower than the value reported by Sheets et al 16 for the same cell type (360±80 pF). Such a cell capacitance coupled with a residual series resistance of 3 Mfl gave an average time constant for the decay of capacitative transients of 0.4 msec. In this study, we chose not to perform digital capacitance subtraction to avoid possible errors in the measurement of peak current amplitudes during such a procedure. Furthermore, we did not perform linear leak current subtraction because in single Purkinje cells the background leak I-V relation was not always linear. This could be seen when the Ca 2+ channels were blocked by a high concentration of Ni 2 ' 1 ". Under such a condition, the leak I-V relation was linear and very flat between -90 and 0 mV; however, at voltages positive to 0 mV, the leak current deviated from the linear I-V relation in an outward direction (data not shown). Subtraction of a so-called linear leak current would cause an underestimation of the Ca 2+ channel current in the voltage range where the leak current deviated from the linear I-V relation. Therefore, we determined the amplitude of peak inward Ca 2+ current as the difference between the inward peak and the current level 300 msec after depolarization. For the T current that decayed with a time constant 40 msec or less (see "Results"), the current reached a steady state at 300 msec. For the L current that decayed with a double-exponential time course (the slow time constant could be longer than 100 msec at certain voltages), the current did not always reach a steady state by 300 msec. However, at the voltage where the L current was maximal, the current level 300 msec after depolarization was only 3-5% of the peak Ca current amplitude. At more positive voltages where leak I-V was nonlinear, our method should give reasonable measurements of Ca currents. At more negative voltages, the degree of underestimation of current amplitudes measured by our method would be more serious due to the slow rate of decay of inward current or the "window" current in this voltage range. The amplitude of peak inward~Ba current was determined as the difference between the inward peak and the holding current level. At positive voltages these current amplitudes would be underestimated due to the nonlinearity of leak I-V relation. We assessed the accuracy of our measurements by comparing current amplitudes measured by our method with those measured as the Nisensitive currents (data not shown). Our measurement of maximal L current was underestimated by about 2%. The maximal T current was underestimated by about 10%. Therefore, we can qualitatively describe our results obtained over a wide voltage range; however, quantitative estimation will be restricted to the voltages of maximal L and maximal T currents in which the uncertainties in the measurement of current amplitude are expected to be minimal. All averaged values are mean±SD. Data Acquisition and Analysis The voltage-clamp protocol was generated by a 12-bit D/A converter (Axon Instruments, Foster City, California) controlled by pClamp software (Axon Instruments) on an IBM-AT. The voltageclamp protocols used for studying the properties of the CV + channel currents (e.g., activation, inactivation, and restitution) are described in the corresponding parts of "Results." The current was filtered at 3 kHz and recorded, along with the voltage, on an FM tape recorder (model B, A.R. Vetter, Rebersburg, Pennsylvania) at a speed of 7.5 inch/ sec (3 dB bandwidth: 0-2.2 kHz). Data analysis was performed on an IBM-AT with pClamp software; current was sampled with a 12-bit A/D converter (Axon Instruments) at a sampling interval of 0.2 msec. The time course of current decay during depolarization was studied by best-fitting the current change with a single-or double-exponential function using the least-squares technique. For studies on the time course of restitution of Ca 2+ channel currents or the sigmoidal voltage dependence of availability and activation of Ca channels, curve fitting was done with a simplex algorithm. 25 Results Ca 2+ Channel Currents in Canine Purkinje Cells For recording Ca 2+ channel currents we superfused the cells with a Na + -and K + -free solution containing 4-aminopyridine. The pipette solution was also Na + -and i C -free and contained Cs + and tetnaethylammonium ions (see "Materials and Methods"). Under such conditions, currents through Na + and K + channels were largely suppressed. These conditions also suppressed currents generated by the operation of Na + -K + pump ((K + ] o =0) 26 and Na + -Ca 2+ exchanger (INa + ] o =0)., 27 The main divalent cation in the bath solution was either 5 mM Ca 2+ or 5 mM Ba 2+ . Current through tetrodotoxinsensitive Na* channels carried by Ca 2+ or Ba 2+ was negligible because tetrodotoxin (30 ^M, a concentration that has been shown to almost completely block cardiac Na + channel 28 ) did not affect the inward currents (data not shown). Figure 1 , left panel, shows selected original current tracings recorded from a canine Purkinje cell at two holding voltages (V h s) of -70 and -30 mV. The complete I-V relations of peak current from this experiment are illustrated in Figure 1 , right panel. There are two maxima in the peak inward current at V h of -70 mV: one at -25 mV and the other at +20 mV. At V h of -30 mV, only one maximum in peak inward current was observed at +20 mV. As mentioned above, tetrodotoxin (30 FIM) did not affect either of the inward currents observed under our experimental conditions. On the other hand, a commonly used Ca 2+ channel blocker, Mn 2+ (5 mM), greatly reduced both currents (data not shown). These results confirm that the inward currents recorded under our experimental conditions were currents through Ca 2+ channels. Thes&observations are similar to those observed in other tissues that display two types of Ca 2 * channels. 1 This similarity, along with other observations on the 0a 2+ channel currents (see below), indicates that in canine Purkinje cells there are at least two types of Ca 2+ channels. In this study we adopted the general terminology for these two Ca 2+ channel currents: T current for the current component seen only at more negative holding voltages (e.g., V h of -70 mV) and L current for the one remaining at less negative holding voltages (e.g., V h of -30 mV). PA In the experiment shown in Figure 1 , the apparent reversal potential for L current was about +75 mV, as determined by extrapolating the ascending limb of the peak I-V relation recorded at a V h of -30 mV to the point where it intercepted the voltage axis. The apparent reversal potential for the T current, as determined by the intersection of the difference current with the voltage axis, is +37 mV. The apparent reversal potential for L current was 68.3±7.6 mV in 5 mM Ca 2+ (/i=3) and 50.0±10.0 mV in 5 mM Ba 2+ (n=3). The apparent reversal potential for T current was 32.3±10.8 mV in 5 mM Ca 2+ (n=3) and 30.0±10.0 in 5 mM Ba 2+ (n=3). The less positive reversal potential for T than L current suggests that the selectivity for Ca 2+ over Cs + is lower for the T channel than for the L channel.-Peak I-V relations similar to those shown in Figure 1 were observed in 23 of 36 Purkinje cells studied (66%). The current densities of the two Ca z+ chan-nel currents were estimated from six Purkinje cells under the same experimental conditions: 5 mM [Ca 2 "^ and 35-37° C. The density of T current was determined by dividing the amplitude of maximal T current recorded from V h of -70 mV by the cell capacitance. The density of T current was 2.9±2.1 pA/pF in Purkinje cells. The density of L current was determined by using the maximal peak L current amplitude recorded from V h of -30 mV. The data used for maxima^ L current amplitude were limited to those collected 20-30 minutes after starting the intraceEular dialysis to minimize underestimation of L current amplitude due to "run-down." PA The density of L current in Purkinje cells was 4.4±3.4 pA/pF. The ratio of maximal T current to maximal L current was 0.60±0. 16 . PA In the remaining 13 Purkinje cells (34%), the peak I-V relations displayed characteristics somewhat different from those shown in Figure 1 . One typical example is illustrated in Figure 2 . The amplitude of inward current at V b of -30 mV was significantly smaller than that at V h of -70 mV. The smaller inward current seen at V h of -30 mV was not due to run-down of L current because the inward currents recorded at V h of -70 mV after the recording at V h of -30 mV were still the larger. The difference current in Figure 2 , right panel, decreased as the voltage step became more positive from -15 to +35 mV. However, unlike the difference current shown in Figure 1 , right panel, the difference current was still significantly inward even at +35 mV. Moreover, the decay of the difference current was rapid at -15 and -5 mV but slowed again at +15 and +35 mV (Figure 2 , left panel). For example, the difference current at -15 mV decayed with a time constant of 4.8 msec. At +35 mV, the difference current decayed with a double-exponential time course: T,=7.2 msec and r=34.0 msec with the fast component accounting for 36% of total current. Furthermore, the decay of the difference current at +35 mV was faster than that of the L current at the same voltage (recorded at V=-30 mV, T,=22.5 msec, and T 2 =50.0 msec with the fast component accounting for 56% of total current) ( Figure 2 , left panel). Thus, the difference current decayed at a rate intermediate between those of the T and L currents. Figure 2 , right panel, shows the peak I-V relations of this experiment. Currents recorded at V of -70 and at V of -30 mV and the difference current were all extrapolated to cross the voltage axis at +75 mV. This is unlike the results shown in Figure 1 , right panel, in which the apparent reversal potentials for L and T currents differed markedly. There are at least two possible explanations for the discrepancies between the two groups of cells exemplified in Figures 1 and 2 : there is a slow inactivation of L current at -30 mV in some, but not all, Purkinje cells, 22 -30 or there is a third type of Ca 2+ channel current in some Purkinje cells that resembles the N-type Ca9 2+ current described for neurons-10 (see "Discussion"). Ca 2+ Channel Currents in Canine Ventricular Cells The Ca 2+ channel currents were recorded from ventricular cells under the same conditions as those for Purkinje cells (5 mM [Ca 2^ and 35-37° C). Unlike Purkinje cells in which T current was a regular observation, in some ventricular cells (4 out of 12), no T current was detectable at a V h of -80 or -70 mV. In the other eight ventricular cells, T current was seen, but its amplitude relative to that of the L current was much smaller than in Purkinje cells. Two examples of Ca 2+ currents recorded from ventricular cells are given in Figure 3 : one with a T current (panels labeled "a") and the other without a detectable T current (panels labeled "b"). The densities of T and L currents were estimated for five ventricular cells that displayed a detectable T current, by use of the same method as for Purkinje cells. The density of T current in ventricular cells was 1.4±1.1 pA/pF and that of L current was 7.5 ±1.7 pA/pF. The ratio of maximal T to maximal L current was 0.19±0.14. Thus, in ventricular cells the density of L current is higher, but the density of T current is lower, than that in Purkinje cells. Kinetics of Decay of T and L Currents During Depolarization In various tissues, it has been shown that Ba 2+ substitution for Ca 2+ in the extracellular solution changes the L current markedly without significantly affecting the T current. 2 -29 Figure 4 shows selected original current tracings and peak I-V relations recorded from a canine Purkinje cell in 5 mM Ca 2+ and then after substituting extracellular Ca 2+ with equimolar Ba 2+ . Similar to the observations made in other tissues, Ba 2+ substitution greatly augmented the L current and slowed its decay. Moreover, there was a negative shift in theyoltage of th&maximal L current due to a change in external surface potential. 2 -29 The inward currents at -30 mV or more negative voltages-, on the xrther hand, were not significantly altered by Ba 2 * substitution. For the current tracing at -30 mV ( Figure 4A ), the late current was more inward in Ba -containing solution (probably due to an enhancement and a negative shift of L current by Ba 2+ substitution). Therefore, the transient current component (T current) was actually smaller after Ba 2+ substitution. The time course of decay of T and. L currents during depolarization was studied by fitting the current change between the inward peak and current level 100 msec (for T current) or 300 msec (for L current) after depolarization with a single-or double-exponential function. Figure 5 illustrates representative data from Purkinje cells. The left two panels in Figure 5A give examples of the time course of decay of T current carried by Ca 2+ or Ba 2+ . It is clear that the decay of T current during Figure 5A illustrate the time course of decay of L current carried by Ca 2+ or Ba 2+ . In both cases, double-exponential functions fit the data very well. The T, and T 2 of Ca 2+ -carried current were 9.2 and 47.1 msec with the fast component being 68% of depolarization followed a single-exponential time total. The T X and T 2 of Ba 2+ -carricd current were 33.7 Values are mean±SD. The T current was induced by a step from a holding voltage of -70 mV (in 5 mM Ca 2+ ) or -80 mV (in 5 mM Ba 2+ ) to the voltage marked on the left. The time constant was obtained by fitting the current change between the inward peak and current level 100 msec after depolarization with a single-exponential function. The L current was induced by a step from a holding voltage of -30 mV (in 5 mM Ca 2+ ) or -40 mV (in 5 mM Ba 2+ ) to the voltage marked on the left. The time course of current change between the inward peak and cun-ent level 300 msec after depolarization was fitted with a double-exponential function to obtain the fast (TI) and the slow (r^ time constants. For Ca 2+ -carried L current, the fast component accounted for 60-70% of the total amplitude. For Ba 2+ -carricd L current, the amplitude of the fast component was 20-30% of the total. and 226.6 msec with the fast component being 30% of total. Complete results from these experiments are shown in Figure 5B . The voltage range for T current is limited to -20 mV or more negative voltages, to avoid uncertainty in isolating T current from L current. The L current was isolated from the T current by using a V h of -30 or -40 mV; the voltage range is limited to -10 mV or more positive voltages because the decay of L current was very slow at threshold depolarization; thus, curve fitting was made difficult. The decay T of T current decreased monotonically as the voltage became less negative. Ba 2+ substitution affected neither the time constant nor its voltage dependence. On the other hand, the decay r t of Ca 2+ -carried L current displayed a U-shaped voltage dependence with the minimal T J occurring at +5 to +10 mV. The T 2 of Ca 2+ -carried L current also displayed a U-shaped voltage dependence with the minimal2+ T 2 occurring at + 15 to +20 mV (see Table 1 ). Ba 2+ substitution not only prolonged the T ] and r 2 of L current but also changed their voltage dependences: in Bacontaining solutions, T, decreased monotonically as the voltage became more positive, and T 2 did not show a clear voltage-dependence (see Table 1 ). When Ca was the charge carrier, the decay of T current was slower in Purkinje cells than in ventricular cells, but the reverse was true for L current in these two cell types (Table 1) . Thus, in comparing the T and L currents between Purkinje and ventricular cells, not only the difference in current density (as described above) but also the variation in the current time course will contribute to a difference in the importance of these two currents in affecting membrane electrical activity in the two cell types. As described for other tissues, Ba 2 +* substitution for Ca slowed the decay of L current; this finding indicated a Ca*-dependent inactivatien process dar-ing depolarization for L current. The time course of T current was not affected by Ba 2+ substitution. Moreover, the time constant of decay of the T current showed a monotonic voltage dependence. These results indicate a predominantly voltagedependent inactrvation process during depolarization for T current in Purkinje cells. Kinetics of Decay of T and L Currents During Depolarization Since the T current could be readily recorded in every Purkinje cell studied, the following results of experiments designed to determine the properties of the two Ca 2+ channel currents are based solely on data from single canine Purkinje cells. Restitution of T and L Currents The time course of recovery from inactivation (restitution) of T and L currents was studied by using a double-pulse protocol. Figure 6 illustrates results from a representative experiment. The voltage-clamp protocol is shown schematically on top of panel A. From a V h of -80 mV, double pulses each for 100 msec to -40 mV (for T current) or to 0 mV (for L current) were applied every 7-10 seconds with the interpulse interval varying between 10 and 4,000 msec. The degree of restitution was estimated by dividing the peak current amplitude during the second pulse (I 2 ) by that during the first pulse (I,). In the experiment shown in Figure 6 , restitution of L current followed a singleexponential time course (T=30.6 msec) whereas restitution of T current followed a doubleexponential time course (^=83 msec and T 2 =444 msec, with the fast component accounting for 67% of total). Similar results were obtained in two other cells and when Ba 2 * was the charge carrier (n=2). Therefore, the restitution of L current was faster than that of the Tcu««nt. The restitution of L current was voltage dependent: time constant of 0 -40 V c (mV) FIGURE JO. Tseng and Boyden Multiple Ca 2+ Currents In Canine PurklnJe Cells Graph showing voltage dependence of availability of T (left) and L (right) Can currents in Puridnje cells. The voltage-clamp protocol and the calculation of the availability are described in the text. Different symbols represent data from different cells. V^ are voltages of depolarizing conditioning pulses. For each current, data from individual cells were fitted by a Boltzmann function to obtain the half-maximal voltage and slope factor. The average values of the half-maximal voltage and slope factor were used for calculating the availability curve using the Boltzmann junction (curves superimposed on the data points). The horizontal bars denote the standard deviations of the average half-maximal voltage (10 mV for T current and 8 mVfor L current). For each current, the voltage dependence of the availability of the individual cell has been shifted along the voltage axis so that the midpoint coincided with the average halfmaximal voltage. In each experiment the charge carrier was 5 mM Co 2 *, and the temperature was 36? C. Cells: 8712713, 87128, 87803/1. restitution was 26±8, 76±23, and 155 ±60 msec at -80, -40, and -30 mV, respectively (n = 3). Voltage Dependence of Availability of T and L Currents To study the voltage dependence of availability of T and L currents, a 500 -msec depolarizing conditioning pulse to different voltages (V c ) was followed by a test pulse to the voltage of maximal T (-30 fo -20 mV) or maximal L (+15 to +25 mV) current. The holding voltage was -80 mV for T current and -40 mV for L current (5 mM [Ca 2+ ] 0 ). The availability of each current after the conditioning pulse was determined by dividing the current during the test pulse (I t ) by the maximal current obtained without a conditioning pulse (!"«). The data were fitted with the simple Boltzmann function: IJ I mu ={l+exp[(V c -V 05 )/k;]}-1 . In this way, the halfmaximum voltage (V 0J ) and slope factor (k) were obtained for each cell. Figure 7 illustrates data summarized from three cells, all with 5 mM [Ca 2+ ] 0 and at 35-37° C. For T current, the average V os was -48±10 mV (n=3), and the average k was 4.0±1.6 160 ai FIGURE Figure 7 . The activation variable ofL current was estimated by normalizing the peak chord conductance with respect to the maximal peak chord conductance; the chord conductance at each depolarization voltage was calculated by dividing the peak current amplitude by the driving force (with the reversal potential estimated to be +75 mV from Figures 1 and 2) . 31 Superimposed on the data points are curves calculated from the Boltzmann function with the following values ofhalf-maximal voltage and slope factor that best fit the data points (mV): 2.5 and -6.9 for the activation ofL current, -10 and 4.6 for the availability ofL current, -47 and 3.1 for the availability of T current. The activation variable of T current was approximated by dividing the peak T current amplitude by the maximal peak T current that occurred at -30 mV. The dashed curve connecting the data points was drawn by eye. Cell: 8712713. mV. For L current, the average V o 5 was -10±8 mV (n=3), and the average k was 5.3+0.7 mV. The availability of T and L currents fell into two distinct voltage ranges. The availability of T current displayed a slightly steeper voltage dependence than that of the L current. Schematic diagram, tracings, and graph showing relations between the voltage dependence of activation and that of availability for T and L currents in a Purkinje cell [Ca 2+ ] 0 was 5 mM, and temperature was 36° C. Panel A: Voltage-clamp protocol is shown schematically in upper section. A 500-msec depolarizing conditioning pulse (VJ was followed by a test pulse (VJ for 300 msec; for T current, holding voltage (V^ was -70mV, and V, was -20 mV; for L current, V h was -30 mV, and V, was +15 mV. Lower section of panel A shows selected original current tracings during the conditioning {with V c marked above) and test pulses, for T (left) and L (right) currents. Panel B: Voltage dependences of activation and availability for Tand L currents from the experiment shown in panel A. The availability of each current was calculated as described for A comparison of the voltage dependence of activation and availability of both T and L currents reveals that, for each current, there is a voltage range in which the activation and availability curves overlap. Figure 8 illustrates a representative example. Shown in Figure 8A are selected original current tracings during both the conditioning and test pulses for T (left column) and L (right column) currents. The voltage-clamp protocols are shown schematically above the current tracings in Figure 8A . The V c is marked above the corresponding tracing of the current during the conditioning pulse. For both T and L currents, at certain-voltages the conditioning pulse induced a sizable inward current without .totally inactivating the inward current induced by the following test-pulse (e.g., -45 mV for T current and -7 mV for L current as shown in Figure 8A ). Figure 8B depicts the activation and availability curves of both T and L currents from this cell. The availabilities and the activation variables for both T and L currents were calculated as described in the figure legend. In this cell, the availability and activation curves for T current overlap between -50 and -30 mV, with a peak overlap occurring at about -45 mV. For L current the overlap is between -30 and +10 mV, with a peak at about -5 mV. Similar findings were obtained in three other Purkinje cells. These observations indicate that there is a Sustained or slowly decaying Ca 2+ influx in two voltage ranges: one around -50 or -40 mV through the T channels and the other near the action potential plateau voltages through the L channels. Responses of Ca 2+ Channel Currents to Channel Blockers Ni 2+ has been shown to block the T channel more potently than the L channel. 5 -8 ' 32 Figure 9A trates that this is also the case in Purkinje cells. Ni 2+ (50 fiM) decreased the maximal T current recorded at -20 mV by 47% whereas Ni 2+ only decreased the maximal L current recorded at +20 mV by 11%. Elevating the concentration of Ni 2+ to 500 FIM caused a further reduction in T current (by 72%) but still a much smaller decrease in the L current (28%) (data not shown). Similar observations were obtained in a total of three experiments. In rabbit sinoatrial nodal cell 5 and frog atrial cell, 8 40 pM Ni 2+ totally abolished T current without affecting the L current. By comparison, in Purkinje cells the sensitivity of T current to Ni 2+ is less, and the sensitivity of L current seems more, than that determined for atrial cells. Responses of Ca 2+ Channel Currents to Channel Blockers In cardiac tissues, many have claimed that dihydropyridine antagonists are selective for the L channel 5 -7 (however, see . We tested this by studying the effects of nisoldipine on T and L currents in Purkinje cells. In the experiment shown in Figure 9B (5 mM [Ba 2+ ] 0 ), under control conditions the T current was induced by a step from -80 to -30 mV. This was not contaminated by the L current, because a step from -50 to -30 mV did not induce any inward current (see peak I-V of Figure 9B ). The L current was induced by a step from -80 to +20 mV. This was negligibly contaminated by T current because +20 mV was close to the apparent reversal potential of T current in this cell; that is, the difference current between V h of -80 and of -50 mV was flat at +20 mV (see peak I-V in Figure 9B ). Nisoldipine (1 pM) was then FiGU RE 9. Tracings and graph showing effects of nickel (Ni 2 *) and nisoldipine on T and L currents. Panel A: Maximal T current induced by a step from -80 mV to -20 mV before (control) and after the addition of 50 fjM Mi 2+ ; maximal L current recorded from the same cell induced by a step from -40 mV to +20 mV before and after the addition of Ni 2 +. lCa 2 +J o was 5 mM, and temperature was 35° C. Cell: 88O26/1. Panel B: Upper, maximal T current induced by a step from -80 to -30 mV before (control) and after the addition of1 fiM nisoldipine; L current induced by a step from -80 to +20 mVfrom the same cell before and after the addition of nisoldipine. Lower, peak currentvoltage relations at the two levels of holding voltage (VJ and the difference current under control conditions and at V h of -80 mV after the addition of nisoldipine. In the presence of drug, only a negligible inward (L) current was activated at V h of -50 mV. [Ba 2+ ] o was 5 mM, and temperature was 34° C. Horizontal lines denote the zero current level Celt 89201/1. added while the cell was held at -50 mV. After 5 minutes, a step from -80 to +20 mV did not induce any inward current; thus, the L current was apparently abolished by 1 fiM nisoldipine under these experimental conditions. However, the T current induced by the same voltage step as in control was also decreased by 47%. In a second cell studied (5 mM [Ba 2+ ] o ), 0.1 /xM nisoldipine was used. It decreased the L current by 68% and the T current by 25%. In a third cell (5 mM [Ca 2+ ] 0 , at 37° C), 0.1 jiM nisoldipine decreased the maximal L (induced by a step from -70 to +20 mV) by 35% and the maximal T (induced by a step from -70 to -20 mV) by 11%. In this last cell, elevating the concentration of nisoldipine to 1 pM caused more blockade of both currents (the maximal L decreased by 79%, and the maximal T decreased by 18%). Therefore, nisoldipine is a potent blocker of L channel in Purkinje cells, but it can also decrease the T current. This is similar to the blockade of both L and T channels by dihydropyridine antagonists reported for guinea pig atrial cells,34 33 rat hypothalamic neurons,35 34 and smooth muscle cells from rat portal vein. 35 However, the mechanism of channel blockade by dihydropyridine antagonists may be different between T and L channels. In all three experiments studying the effects of nisoldipine, the availability curves of T current were not significantly shifted, whereas in the two experiments using 0.1 FIM nisoldipine, the availability curve of L current was shifted in the negative direction by about 35 mV (data not shown). A similar difference in the voltage dependence of channel blockade by a dihydropyridine antagonist has already been described. 35 The reason for this difference is not known at this time. Therefore, the differential pharmacological properties of T and L channels in Purkinje cells are similar to what has been described for other tissues. 3 -7 -8 - 29 However, in our experiments, neither Ni 2+ nor nisoldipine could totally block one type of current without affecting the other. Effects of Catecholamines on T and L Currents We studied the effects of /3-adrenoceptor stimulation (by 1 /iM isoproterenol) or a-adrenoceptor stimulation (by 10 ^.M norepinephrine in the presence of 2 /xM propranolol) on the two types of Ca 2+ channel currents in Purkinje cells. In a representative experiment using Ca 2+ as the charge carrier, isoproterenol augmented the amplitude of L current (at +5 mV from 295 pA to 457 pA) and slowed its decay (at +5 mV, T, and T 2 were 7.8 and 70.8 msec for control and 13.6 and 106.0 msec in the presence of isoproterenol). On the other hand, the T current was not significantly affected (at -35 mV, amplitude and T of decay were 347 pA and 9.5 msec, for control, and 369 pA and 10.7 msec in the presence of isoproterenol) (data not shown). This is similar to what has been reported for canine atria] cells, 2 rabbit sinoatrial nodal cells,36 5 and guinea pig ventricular cells in which £-adrenoceptor stimulation augments the L but not the T current. Effects of Catecholamines on T and L Currents There has been a lack of agreement as to the effects of a-adrenoceptor stimulation on the L-type Ca 2+ current. 37 -39 Furthermore, the effects of a-adrenoceptor stimulation on the T current are largely unknown. In 10 cells in which the effects of a-adrenoceptor stimulation were tested, seven showed an increase in the T current. Figure 10A illustrates the results of one such experiment, which was carried out at 24° C and used Ba 2+ as the charge carrier. To totally eliminate the contamination of an L current in the measurement of the T current, the T current was measured at -50 mV from a V h of -80 mV. The T current showed "run-up" (a gradual increase in amplitude) but stabilized before the application of norepinephrine ( Figure 10A) . After the addition of norepinephrine for 5 minutes, the amplitude of the T current was transiently decreased, but then by 10 minutes, it had increased to a level above that seen before the application of norepinephrine. The augmentation in T current was reversed by 10 yM prazosin (n=2, one shown in Figure 10A ). In the cell shown in Figure 10A , the L current gradually decreased after the addition of norepinephrine; thus there was no 0-adrenoceptor stimulation under these conditions. The experiment shown in Figure 10A was the only one in which the changes in the T current caused by norepinephrine were biphasic. In the other six experiments, norepinephrine caused only a monophasic increase in the T current. Among the seven experiments, six were done at room temperature and one at 35° C. The stimulatory effect of a-adrenoceptor activation on the T current was quantified by calculating the percent increase in the amplitude of T current induced by a step from -80 to -50 or -40 mV after an exposure to 10 FIM norepinephrine (in the presence of 2 fjM propranolol) for 5-15 minutes. The increase was 68.7±33.6% (n=7). In the other three cells, there was only a decrease in T current in response to a-adrenoceptor stimulation. Figure 10B shows the results of one such experiment that was carried out at 35° C and used Ca 2+ as the charge carrier. This effect of norepinephrine was dose dependent in a concentration range from 1 to 50 pM (n=l, the same cell shown in Figure 10B } data not shown) and reversible after washout of the drug (n=2, one shown in Figure 10B ). In these three experiments, two were done at 35° C and one at room temperature. The average decrease in the amplitude of T current after an exposure to 10 norepinephrine (in the presence of 2 fiM propranolol) for 5-15 minutes was 43.6±20.0%. A clear determination of the effects of o-adrenoceptor stimulation on the L current was hindered by the run-down of this current during the course of our experiments. From these results we conclude that in canine Purkinje cells, T and L channels can be modulated by the stimulation of a-and fiadrenoceptors, respectively. The variability in the effects of a-adrenoceptor stimulation on the T current may be due to the complicated intracellular pathways mediating the effects of a-adrenoceptor stimulation (see "Discussion"). Discussion Ca 2+ Channel Current Measurement in Canine Purkinje Cells Studying Ca 2+ channel currents by using single Purkinje cells, especially with a dialyzing suction pipette, has advantages over using multicellular Purkinje fibers. First, the spatial homogeneity during the peak Ca 2+ channel conductance can undoubtedly be achieved. Second, currents other than those through Ca 2+ channels can be effectively suppressed by intracellular and extracellular ion substitution. Third, as a result of the suppression of other currents, a much wider range of holding voltages can be explored, and the findings can lead to knowledge of Ca 2+ channels not available before. However, although we could qualitatively describe the properties of Ca 2+ channel currents in Purkinje cells over a wide voltage range, we limited our quantitative discussion to the maximal T and L currents due to the problems of large cell capacitances and the nonlinear leak I-V relation. In this cell type, there is no pharmacological agent that can isolate one current component from the other. Therefore, we relied on the difference in the voltage dependence of availability between the two current components for isolation. In doing so, we made sure that the less negative holding voltage did not cause appreciable inactivation of the L current and that there was no appreciable run-down of L current between the runs using the two levels of holding voltage. Ca 2+ Channel Currents in Canine Purkinje Cells In canine Purkinje cells in addition to the conventional Ca 2+ current (L current) that remained available at a V h of -30 mV, we recorded another current through Ca 2+ channels that was available only at a V h negative to -30 mV, decayed rapidly even when Ba 2+ was the charge carrier, and did not show run-down after prolonged intracellular dialysis. This current reached a maximal peak amplitude at -30 to -20 mV and declined at more positive voltages; thus, the "shoulder" morphology in the descending limb of the peak I-V relation was created. These properties of this Ca 2+ channel current are similar to those of the T current described for many
{'paper_id': '45717464', 'title': 'Multiple types of Ca2+ currents in single canine Purkinje cells.', 's2_url': 'https://api.semanticscholar.org/CorpusID:45717464', 'source': 'S2ORC'}