456
L- and T-Type Ca2+ Channels in Canine Cardiac Purkinje Cells Single-Channel Demonstration of L-Type Ca2+ Window Current Stephen R. Shorofsky and Craig T. January Canine cardiac Purkinje cells contain both L- and T-type calcium currents, yet the single Ca2' channels have not been characterized from these cells. Additionally, previous studies have shown an overlap between the steady-state inactivation and activation curves for L-type Ca2' currents, suggesting the presence of L-type Ca2' "window" current. We used the on-cell, patch-clamp technique to study Ca'+ channels from isolated cardiac Purkinje cells. Patches contained one or more Ca2' channels 75% of the time. L-type channels were seen in 69%1 and T-type channels in 73% of these patches. With 110 mM Ba2` as the charge carrier, the conductances of the L- and T-type Ca2' channels were 24.2±0.8 pS (n=9) and 9.0±0.5 pS (n=8), respectively (mean±SEM). With 110 mM Ca2' as the charge carrier, the conductance of the L-type Ca2' channel decreased to 9.7± 1.2 pS (n =4), whereas the T-type Ca2' channel conductance was unchanged. Voltage-dependent inactivation was shown for both L- and T-type Ca2' channels, although for L-type Ca21 channel with Ba2` as the charge carrier, inactivation took at least 30 seconds at a potential of +40 mV. After channel inactivation was complete, L-type Ca2+ channel reopenings were observed following repolarizing steps into the window voltage range. Thus, our data identify both L- and T-type Ca2+ channels in cardiac Purkinje cells and demonstrate, at the single-channel level, L-type channel transitions expected for a window current. Window current may play an important role in shaping the action potential and in arrhythmogenesis. (Circulation Research 1992;70:456-464) KEY WoRDs * Ca2' channels window current Purkinje cells * heart inactivation .
C ardiac tissues contain both L- and T-type Ca2' channel currents.'-7 Studies of the voltagegated properties of these channels have demonstrated overlap between their steady-state inactivation and activation relations, particularly for the L-type Ca'+ channel, suggesting the possibility that steady-state Ca'+ ("window") currents exist.5,7-11 L-type Ca'+ window current has been proposed to have an important role in controlling the action potential shape,12"3 in some types of triggered arrhythmias,1415 and possibly in the regulation of contraction.3'16 A window current is usually defined as a steady-state current that arises from voltage-gated channels within the narrow voltage range where the steady-state inactivation and activation relations overlap (see Figure 1). At potentials negative to the window, channels distribute between closed and inactivated states. For sufficiently long duration voltage steps positive to the window, the channels preferentially distribute into an inactivated state. Only within the window voltage range can a channel theoretically cycle between closed, open, From the Cardiac Electrophysiology Laboratories, Department of Medicine (Cardiology), University of Chicago (Ill.). Supported by National Heart, Lung, and Blood Institute grants HL-20592 and HL-38927. Address for correspondence: Stephen R. Shorofsky, MD, PhD, Department of Medicine, Box 304, University of Chicago, 5841 South Maryland Avenue, Chicago, IL 60637. Received June 18, 1990; accepted October 15, 1991.
.
.
and inactivated states, thus generating, in cells, a steady current. If no voltage range exists that permits both recovery from inactivation and activation, then a true steady-state Ca'+ current should not exist. Previous attempts to demonstrate Ca'+ window current have focused on demonstrating a steady or persistent Ca2+ current under conditions when only timeindependent currents were expected. Cohen and Ledererll showed that the D600 sensitive L-type, steady Ca2+ current, measured 200 msec from the beginning of the depolarizing step, exhibited the voltage dependence predicted by the overlapping region of the steady-state inactivation and activation curves, supporting the concept of window current. McDonald and coworkers17 were able to record persistent L-type Ca'+ channel activity during 900-msec depolarizations. Cavalie and coworkers9 demonstrated persistent L-type Ca'2 channel activity, albeit rare, at holding potentials in the voltage range where the steady-state inactivation and activation curves overlapped, leading these authors to conclude that these channel openings represented window current. Similar whole-cell findings were shown for 1-second depolarizing steps by Hirano et al.5 Interpretation of these experiments, however, is complicated by the existence of a slowly inactivating component of the L-type Ca'+ current'8 and by the complex single-channel behavior that occur near threshold voltage (see References 17 and 19). Currents or channel activity noted on depolarization into the window voltage range
Downloaded from http://circres.ahajournals.org/ by guest on June 23, 2015
Shorofsky and January Single-Channel Demonstration of Ca2' Window Currents 1.0.
-o
c 0
0
>
Voltage+ FIGURE 1. Idealized plot of the relation between a steadystate inactivation and an activation versus voltage. The shaded area represents the window voltage range. Also shown are the predominant channel transitions that occur in the various voltage ranges. C, aggregate of closed states; 0, open states; I, inactivated states. See text for further discussion.
could represent a slowly inactivating Ca2+ current as well as true window current. The present study was designed to identify and characterize the types of Ca2' channels in cardiac Purkinje cells and to test the hypothesis that L-type Ca2' window current exists by analyzing the behavior of these Ca2' channels under voltage-clamped conditions in which window but not slowly inactivating currents should exist. Purkinje cells were chosen for this study because previous work from our laboratory had shown that these cells contain both L- and T-type Ca2' currents, with potential window voltage ranges for each.5 Because previous studies of single Ca2' channels have not been reported for this preparation, we first characterized some properties of the L- and T-type Ca2' channels. Subsequently, we showed that both channel types can be voltage inactivated. Once inactivated, we identify L-type Ca2' channel reopenings on repolarization into the window voltage ranges, demonstrating the single-channel transitions necessary for this window current to exist. Materials and Methods
Preparation Single canine cardiac Purkinje cells were isolated enzymatically as described previously.20 The cells were stored in a dispersion solution (see below for contents) and were studied within 8 hours of isolation. Small aliquots of cells were added to the bath solution (see below for contents) in a perfusion chamber constructed on a glass coverslip and mounted on the stage of an inverted microscope (Diaphot, Nikon). The chamber was treated with polylysine (0.1 mg/ml) before each experiment to facilitate cell adhesion to the glass coverslip. All experiments were performed at room temperature (20-22°C).
457
Electrical Recordings Single-channel recordings were made using the oncell patch-clamp method of Hamill and coworkers.21 Glass suction pipettes were fabricated from hematocrit capillary tubes (Drummond Scientific Co.). Pipettes were pulled with a two-stage microelectrode puller (model PP-83, Narishige), coated with Sylgard 184 (Corning Medical), and fire polished to a final tip diameter of 1-1.5 gtm by using a microforge (model MF-83, Narishige). When filled with pipette solutions (see below), the pipettes had resistances of 0.5-2 M[l. The bath solution was connected to ground via a 150 mM KCl/agar bridge and a Ag/AgCl half-cell electrode. Electrical contact with the pipette solution was via a chlorided silver wire. The electrode potential was adjusted to give a zero current between the pipette solution and the bath solution immediately before attempting to make a patch. Gigaohm seals between the pipette and single Purkinje cells were obtained by applying gentle suction to the pipette after contacting the cell membrane. Seal resistances for these experiments ranged between 10 and 100 Gfl. Single-channel currents were studied in the on-cell patch configuration by using a patch-clamp amplifier (List EP-7, Darmstadt, FRG). Voltage-clamp protocols were generated and single-channel currents recorded using a 12-bit, A/D board (DAS-16, Metrobyte), under computer control (Zenith 386). Single-channel currents were filtered at 1 kHz with an eight-pole Bessel filter (model 902, Frequency Devices) and digitized at 10 kHz. Two voltage-clamp protocols were used. The first consisted of either 100- or 200-msec voltage steps from the holding potential repeated every 2 seconds. The second consisted of recording 200 msec of channel activity (e.g., sampling period) every 2 seconds while holding the patch at fixed potentials for long periods of time. Partial electronic capacitance compensation was used in all experiments. All data were stored on a hard disk for subsequent analysis.
Data Analysis The data were capacity and leak corrected by subtracting an averaged null trace. By using the corrected tracings, an amplitude histogram was constructed and the open-channel amplitudes were estimated. The threshold to detect openings and closings was set at one half the estimated open-channel amplitude, and an open-closed table was constructed. These data were then used to create open-channel histograms, from which the actual open-channel current amplitudes were determined. Multiple simultaneous channel openings were not included in the analysis. To eliminate some of the error introduced by filtering, the current measured had to exceed threshold for at least three successive data points to be considered a valid opening. The standard deviation of the noise seen in most patches was less than or equal to 0.12 pA.
Solutions The dispersion solution in which the cells were stored after isolation contained (mM) potassium glutamate 130, MgC12 5.7, EGTA 0.1, and HEPES 10 (pH adjusted to 6.9 with KOH). The bathing solution contained (mM) potassium aspartate 140 and HEPES 10 (pH adjusted to
Downloaded from http://circres.ahajournals.org/ by guest on June 23, 2015
Circulation Research Vol 70, No 3 March 1992
458 A mV7 -79
B
1
11
-79 -- *
J~¶ZZ;
S^
..
LL
-1 --L-1
C
-
--i__ v
..
"'
D
21
v
FIGURE 2. L-type Ca2+ channel activity recorded with the voltage-clamp protocols shown above the tracings. The pipette contained 110 mM Ba2' as the charge carrier. Bay K 8644 (5 tiM) was present to prolong channel openings.
31
I
-79
-79
Ttrllrl7rr -q
.*.
P
-,
v,
.
f
IE_ 7~~~~~~~~b _R- I.
l- --
RP
-
.
A
.
-A
-
10 nis
7.4 with KOH). This solution depolarized the Purkinje cells to approximately 0 mV. The internal solution for the suction pipette contained (mM) either CaCl2 110 or BaCI2 110 with HEPES 10 (pH adjusted to 7.4 with tetraethylammonium hydroxide [TEA-OH]) and tetrodotoxin 0.012 (TTX; Sigma Chemical Co., St. Louis, Mo.). The TEA-OH and TTX were added to block K' and Na+ channels, respectively. When used, Bay K 8644 (1-5 ,M; Miles Pharmaceuticals) was added to the bathing solution (from a 10 mM stock of Bay K 8644 dissolved in absolute alcohol). The use of high divalent ion (Ca21 or Ba21) concentrations facilitates the identification of single-channel currents but also results in a shift to more positive voltages of the voltage-dependent properties of currents recorded from these cells.5
Where appropriate, data are reported
as
mean+±SEM.
Results Single Ca21 channels of both the L and T type could be demonstrated easily in isolated canine Purkinje cells.
Ca2' channels were identified in approximately 75% of the first 35 successful patches. Of the patches with channels, 73% contained at least one T-type Ca2+ channel, 69% had at least one L-type Ca2' channel, and 42% had both Ca2+ channel types. L-Type Ca2' Channels Typical openings of an L-type Ca21 channel during a range of voltage steps are shown in Figure 2. The pipette solution contained 110 mM BaCl2, and Bay K 8644 (5 ,uM) was added to the bath solution to prolong the channel openings. As the membrane patch was clamped to more positive potentials, the channel tended to open earlier and for longer durations. This is best demonstrated in Figure 3, where the probability that the channel shown in Figure 2 is open per sweep is plotted for consecutive voltage steps to the potentials shown. At the more positive potentials, the L-type Ca21 channel tended to either open or remain closed for the
Downloaded from http://circres.ahajournals.org/ by guest on June 23, 2015
Shorofsky and January Single-Channel Demonstration of Ca2' Window Currents 1 niV
PC
Il I
459
11 mV
1-
PO
.1
I
SWEEP NUMBER
IL
--
54
SWEEP
21 mV
NUMBERl
1-
FIGURE 3. The probability 45 that the L-type Ca2' channel
(from Figure 2) was open (PO) during successive 200-msec voltage-clamp steps to the indicated potentials.
pc
PC
SWEEP NUMBER
duration of the clamp step. The plot also shows that channel openings tended to occur in groups of sweeps. The open-channel amplitude histograms and the current-voltage (I-V) relation for the open channel is shown in Figure 4. In all experiments, the I-V relation appeared linear. With Ba2` (110 mM) as the charge carrier, the conductance of the L-type Ca2' channel was 24.2+0.8 pS and the extrapolated reversal potential (estimated by linear regression) was 60.8+1.7 mV (n=9). An estimate of the macroscopic currents resulting from a voltage step can be obtained by the ensemble average of single-channel recordings from many steps to the same voltage. The ensemble averages and their peak I-V relation for the channel from Figure 2 are shown in Figure 5. With Ba2' as the charge carrier, little inactivation occurred during the 100-msec voltage-clamp steps. The corresponding I-V relation revealed a peak at approximately +30 mV. Examples of L-type Ca2+ channel recordings, when Ca21 (110 mM) is used as the charge carrier in the pipette, are shown in Figure 6. The corresponding ensemble average currents are shown below the sample tracings. Bay K 8644 was not used in this experiment. Again, channel openings are more frequent at the more positive voltageclamp steps. In contrast to the experiments when Ba21 was the charge carrier, the ensemble currents demonstrate marked inactivation during the 200-msec voltage-clamp step. With Ca2+ in the pipette, the conductance of the L-type Ca2' channel was 9.7+1.2 pS and the extrapolated reversal potential was 70.2± 6.0 mV (n=4).
T-Type Ca2+ Channels Representative current recordings and ensemble averages obtained from T-type Ca21 channels with Ba21 (110
SWEEP NUMBER
mM) as the charge carrier are shown in Figure 7. Compared with the L-type Ca2' channels, the T-type Ca2' channels opened at more negative potentials, tended to open early, inactivated completely during the 200-msec voltage-clamp steps to positive potentials regardless of whether the charge carrier was Ca2+ or Ba2 , and were unaffected in either conductance or kinetics by Bay K 8644. The open-channel amplitude histograms and I-V relation are shown in Figure 8. The I-V relation was linear. The conductance of the T-type channels was 9.0+0.5 pS (n=8) with Ba21 (110 mM) as the charge carrier and 8.4 pS (n=2) with Ca2+ (110 mM) as the charge carrier. The extrapolated reversal potential with Ba2' as the charge carrier was 48.5±13.9 mV, which was not statistically different from that obtained from Ba2+ current through the L-type Ca21 channel. The large standard deviation of the reversal potential estimate is due in part to the fact that the open-channel conductance was determined over a relatively narrow voltage range that is far from the reversal potential, so the small variance in the slope (SEM= +0.5 pS) between patches yields a large variance in the extrapolated reversal potential. Inactivation T-type Ca2+ channels completely inactivate within 200 msec when clamped to positive potentials, as shown in Figure 7 (see also References 5, 6, 22, and 23), regardless of whether Ca2+ or Ba2+ is the charge carrier. In contrast, for the L-type Ca21 channel when Ca21 was the charge carrier, channel inactivation was incomplete by the end of a 200-msec voltage step, suggesting clear kinetic differences (see Figure 6). With Ca2' as the
Downloaded from http://circres.ahajournals.org/ by guest on June 23, 2015
Circulation Research Vol 70, No 3 March 1992
460
potentials shown and 200 msec of single-channel data was recorded every 2 seconds. Activity from only one L-type Ca21 channel was seen in this patch. The probability that the channel was open during each 200-msec sampling period is plotted. Channel activity developed after the potential across the patch was changed from -40 to + 13 mV, and within about 20 seconds it disappeared. The channel could be in either a closed or inactivated state. However, with further depolarization of the patch to +30 and +40 mV, at which channel openings could be easily recorded (see panel B), no further channel activity could be elicited, indicating inactivation of the channel.
1 mV
-9 mV
cc
z
CURRENT (pA) 21 mV 1200
z
0
..
-1.5
CURRENT (pA)
0.0
CJRRENT (pA)
31 mV
41
mV
2000
z
z
0-
0-
0°0
-15
CUR
0.0
-1.5
CURRENT (pA)
T (pM
1.OTpA
-30
_ T
mV
sol0
FIGURE 4. The open-channel amplitude histograms with their corresponding Gaussian fits and current -voltage relation for the L -type Ca2' channel shown in Figure 2. The conductance of this channel was 23.6 pS, and the extrapolated reversal potential was + 68.9 mV.
charge carrier, it is difficult to separate L-type from T-type Ca2+ channels because of the similarity of their conductances. With Ba2` as the charge carrier, L- and T-type Ca21 channel currents can be easily separated; however, the L-type Ca2+ channel revealed little inactivation during the 100-200-msec duration voltage-clamp steps we used (see Figure 3). The L-type Ca2+ channel can be voltage inactivated, however, if given sufficient time. An experiment demonstrating L-type Ca2+ channel inactivation with Ba2+ as the charge carrier is shown in Figure 9. In this experiment, the membrane patch was voltage clamped to the
L-Type Ca21 Window Current If a window current exists for the L-type Ca2' channel, then after inactivation of the channel at positive potentials, channel openings should return on repolarization into the window voltage range. The results of such an experiment are shown in Figure 10. This experiment is a continuation of the experiment shown in Figure 9. After inactivating the channel with a prolonged depolarization to +40 mV, the patch was repolarized to + 13 mV. Channel activity returned as shown by the sample tracings in panel A. The patch was again clamped to +40 mV, and after about 30 seconds channel activity again ceased. The membrane was then repolarized to + 25 mV, and no channel activity was noted. On further repolarization of the patch to +7 mV, channel activity again returned. Examples of the channel activity during this repolarization are shown in panel B. The difference in the pattern of channel reopenings seen after the repolarization steps to the two potentials (see panels A and B) does not imply a difference in channel behavior at the two potentials but is within the normal variability seen with a given L-type Ca2' channel. Protocols similar to that shown in Figure 10 were completed on nine patches containing L-type Ca2' channels. From these experiments, L-type Ca2' channel activity was identified on repolarization into the window voltage range in five patches. From these nine experiments, the probability of identifying an L-type Ca2' channel reopening per sampling period (200 msec of acquired data every 2 seconds) was 0.024. Because channel openings were brief (the probability that the channel was open within a sampling period in which reopenings were identified was about 0.1), the probability that the channel was open during the repolarizing steps was less than 0.002 (or less than 0.2%). Discussion Single-Channel Properties It is interesting that both L- and T-type Ca2' channels were observed frequently in these cells. Channels were seen in 75% of the successful patches. This is in sharp contrast to the results from guinea pig ventricular myocytes, in which L-type Ca2' channel activity was noted in only about 12% of the patches.24 Using the single-channel conductances measured in this report, with the whole-cell L- and T-type Ca2' currents reported by Hirano and coworkers5 under similar conditions, the minimum channel density for both channel types was calculated to be about six channels per square micrometer, a value comparable to that found for
Downloaded from http://circres.ahajournals.org/ by guest on June 23, 2015
Shorofsky and January Single-Channel Demonstration of Ca21 Window Currents
A
B F
461
0.1 -- PA t
i
_
- I
I
-_---- - --
---
-30
50
mY
1 1 mVy
21 m\/ _
31 mY
-0.4 - _ a
20 1"
FIGURE 5. Panel A: Ensemble averages for the L-type Ca2' channel shown in Figure 2. The averages from clamp steps to 1, 11, 21, and 31 mVsteps were constructed from 46, 40, 21, and 28 sweeps, respectively. Panel B: The peak current-voltage relation from the ensembles.
L-type Ca'+ channels in guinea pig ventricular myocytes.17 The fact that we observed T-type channels as frequently as L-type channels is consistent with the greater prevalence of T-type Ca'+ currents in Purkinje cells as opposed to ventricular cells.722 Neither N-type25 nor B-type26 Ca21 channels were identified in the isolated Purkinje cells. The general properties of both Ca21 channel types reported in this study (i.e., their conductance with Ba21 or Ca2' as the charge carrier, kinetics, and response to Bay K 8644) are in good agreement with those from A
B
-12
cultured neonatal rat ventricular cells,27 guinea pig ventricular myocytes,19,19,2228 calf sarcolemma membrane channels reconstituted in planar lipid bilayers,26 and neuronal cells.2529,30 It is unlikely that the T channel represents Ca2' transit through the Na+ channel because 1) the channels are not blocked by up to 100 ,M 1TX (unreported observation), 2) the single-channel kinetics differ from those of the Na+ channels seen in these cells,31 and 3) the T channels activated and inactivated at potentials more positive than would be expected if the current was carried through Na+ channels.
C
-2
a
mV -82
-82
-82
~ --' q
'A_
h
b%- % .0 M.
-
--
10 me
-
-
0I
cu
-~
20 ms
FIGURE 6. Sample recordings of L-type Ca2' channels and their ensemble averages with 110 mM Ca2' as the charge carrier. Voltage protocols are illustrated above the tracings. Note the decrease in channel current and more rapid decay of the ensemble currents compared with that seen when 110 mM Ba2` is the charge carrier. Downloaded from http://circres.ahajournals.org/ by guest on June 23, 2015
462
Circulation Research Vol 70, No 3 March 1992 -10
-30
j-s
mV j
W
-so
lI~ ~~~~~r -~ --
,-
-
I.
-.W. --- --.
1-
a~ -0 R-I, 1- M. a- offi o
---
-
--
--
---
-A-
--
'
-----
-1 be wo 110- ---
em . _
.A-
h-
1
-
-
-
lq .m
iw0 onwo aq ebo.m-11.
--
---
m-
-
---- - - -- --v -
---1.
~_
FIGURE 7. Sample recordings of T-type Ca2+ channels and the corresponding ensemble averages. Voltage protocols are illustrated above the tracings. Note the brief channel openings and rapid decay of the ensemble currents.
-
V_~ ~ ~ ~ ~ ~ C 20 ms
-AMMINA,
-AMMIAOW
20 ms
50-
z
-37
-47 mV
-
2
0-
t -05
-1.0
GURRENT
150-
mV
-27
(PM)
CGURREN
mV
0(
0
(PA)
-17 mV
.~~~~~~
FIGURE 8. The open-channel amplitude histogram with the Gaussian fits and current-voltage relation for a typical T-type Ca2' channel. In this patch, the T-type channel had a conductance of 9.2 pS and a reversal potential of +46 mV.
z
-0.5
Ns
0.0
Ue
CURRENT (pA) 11
ao
CURFENT (pA)
-7 mV
1.0-T pA
10
-80
mV
CURFENW (pA)
_3.0 l
Downloaded from http://circres.ahajournals.org/ by guest on June 23, 2015
Shorofsky and January Single-Channel Demonstration of Ca2' Window Currents A
40
30
13
13
my
mv
30
13
2
25
-40
-40
T
-40 mv
.27-
.090-
II
.07.p-
Po .0c .031-
1
.01 1-
.21-
13 mY
*'I'qS'" *rA
U'ivmw-i
m
---
PO
.15-
30 mv .09-
I
-1-i a -
A
7'
B
.03
20 ms
p
i a i t 4,9 SWEEP NLUMBE
R
a
1 -1 2i1
a
*
e
_t
30
m\@1
aBR^
1
'''U B 147 ' Sl-I S WE5No6s~~~~~~~~~~~~~~~~~~~~~ ie a
a
SVWEEP NUMBER A
B
0
4
463
B
-zt:j~---
0 -v Ar -~~~~Im .
A&L&ALAb-
n-~
my
~
_§---
-
1
A
.
__j
v-R---
-7~~ ~ ~ ~ ~ ~ cu
20
20 ms
FIGURE 9. Panel A: The probability that the L-type Ca' channel was open (PO) during the voltage protocol illustrated at the top of the figure. Inset are sample tracings at the various potentials. Note that the channel activity disappears with time after clamping the patch to +13 mV See text for further details. Panel B: Samples of L-type Ca' channel activity on clamping the patch from 0 to +30 mV, indicating that the lack of channel activity when clamped to +30 mIVin panel A was not due to an inability to resolve single-channel activity at +30 mV
Inactivation Results from experiments with a number of different cell types have consistently shown that the L-type Ca' current shows little inactivation over several hundred milliseconds when Ba` is the charge carrier."57'92532 The data presented here are consistent with those observations. Our results also show that under these conditions (e.g., Ba2+ as the charge carrier), L-type Ca2' channels can inactivate if given sufficient time. In our experiments, voltage-dependent inactivation required at least 30 seconds for a voltage step to +40 mV. These results are also in agreement with Yue and coworkers,32 who suggest that Ca2+-facilitated inactivation results from calcium favoring occupancy of a closed state from which the channel can reopen rather than accelerating inactivation into an absorbing state. With Ba2+ as the
charge carrier, depolarization favored channel inactivated state (see Figure 9).
pancy in an
occu-
ms
FIGURE 10. Probability that an L-type Ca' channel is open (PO) during the voltage protocol illustrated at the top of thefigure. Panels A and B: Single-channel activity that is seen on repolarization into the window voltage ranges correspond, in time, to the letters in the upperfigure. See the textforfurther details. In contrast, T-type Ca2' channels were completely inactivated within 200 msec at positive potentials regardless of whether the charge carrier was Ca2+ or Ba2 This agrees with results from whole-cell experiments with isolated canine Purkinje cells,6 rabbit sinoatrial cells,4 chick and rat sensory neurons,30 cultured 3T3 fibroblasts,23 and guinea pig ventricular cells.22 .
L-Type Ca>2 Window Current Hirano et al13 have studied L-type Ca>2 currents in whole-cell voltage-clamped isolated Purkinje cells with protocols similar to those used in this study. They were able to demonstrate recovery of an inward Ca>' current when the cell was repolarized into the L-type Ca21 window voltage range. This current responded to interventions that previously had been shown to affect L-type Ca21 current. They concluded that L-type Ca2+ window current could be shown directly and that it could be separated from other processes, such as slow inactivation. The data we present here support and extend their conclusions. For a window current to exist, a voltage-gated ion channel must be able to cycle from an inactivated state to an open state within the appro-
Downloaded from http://circres.ahajournals.org/ by guest on June 23, 2015
464
Circulation Research Vol 70, No 3 March 1992
priate voltage range. Our data demonstrate that L-type Ca'2 channels can undergo these transitions. Although our results do not demonstrate whether the channel must first recover to a closed state before reopening, the results of Hirano and coworkers13 suggest that L-type Ca'2 channels cycle from an inactivated state to a closed state before reopening within the window. In the present experiments we observed L-type Ca'+ channel reopenings when the patch was repolarized (from +40 mV) to the voltage range of 0 to +20 mV. This voltage range is close to the maximal overlap between the steady-state inactivation and activation curves for the L-type Ca2' current with similar concentrations of divalent ions (110 mM Ba'+, see Reference 5) and positive to the window voltage range found with lower divalent ion concentrations.579-11 In principle, it would have been advantageous to study the probability of L-type Ca'+ channel reopenings throughout the window voltage range; however, channel reopenings were rare, with the probability of identifying an L-type Ca2+ channel on repolarization into the window voltage range of less than 0.002. This value is in qualitative agreement with the results of Hirano et al,13 in which the whole-cell current measured on repolarization into the window voltage range was usually less than 20 pA. Thus, the delineation of the window voltage range is not easily suited to single-channel experiments but is better suited to whole-cell experiments in which the behavior of many channels is studied simultaneously. Our results do demonstrate clearly, however, that L-type Ca2+ channels can open on repolarization into the window voltage range. In summary, our results show that 1) L- and T-type Ca2+ channels are present in relatively high densities in canine cardiac Purkinje cells, 2) both channel types can be voltage inactivated although with differing kinetics and ion dependencies, and 3) once inactivated, at least L-type Ca2+ channels can reopen on repolarization into their window voltage ranges. Acknowledgment We would like to thank Dr. H.A. Fozzard for his helpful discussions of these data.
References 1. Nilius B, Hess P, Lansman JB, Tsien RW: A novel type of cardiac calcium channel in ventricular cells. Nature 1985;316:443-446 2. Bean BP: Two kinds of calcium channels in canine atrial cells:
Differences in kinetics, selectivity, and pharmacology. J Gen Physiol 1985;86:1-30 3. Mitra R, Morad M: Two types of calcium channels in guinea pig ventricular myocytes. Proc NatlAcad Sci USA 1986;83:5340-5344 4. Hagiwara N, Irisawa H, Kameyama M: Contribution of two types of calcium currents to the pacemaker potentials of rabbit sinoatrial node cells. J Physiol (Lond) 1988;395:233-253 5. Hirano Y, Fozzard HA, January CT: Characteristics of L- and T-type Ca2+ currents in canine cardiac Purkinje cells. Am J Physiol 1989;256:H1478-H1492 6. Hirano Y, Fozzard HA, January CT: Inactivation properties of T-type calcium current in canine cardiac Purkinje cells. Biophys J 1989;56:1007-1016
7. Tseng G-N, Boyden PA: Multiple types of Ca'+ currents in single canine Purkinje cells. Circ Res 1989;65:1735-1750 8. Reuter H, Scholz H: A study of the ion selectivity and the kinetic properties of the calcium dependent slow inward current in mammalian cardiac muscle. J Physiol (Lond) 1977;264:17-47 9. Cavalie A, Ochi R, Pelzer D, Trautwein W: Elementary currents through Ca'+ channels in guinea pig myocytes. Pflugers Arch
1983;398:284-297
10. Josephson IR, Sanchez-Chapula J, Brown AM: A comparison of calcium currents in rat and guinea pig single ventricular cells. Circ Res 1984;54:144-156 11. Cohen NM, Lederer WJ: Calcium current in isolated neonatal rat ventricular myocytes. J Physiol (Lond) 1987;391:169-191 12. McAllister RE, Nobel D, Tsien RW: Reconstruction of the electrical activity of cardiac Purkinje fibres. J Physiol (Lond) 1975;251:1-59 13. Hirano Y, Moscucci A, January CT: Direct measurement of L-type Ca2' window current in heart cells. Circ Res 1992;70: 445-455 14. January CT, Riddle JM: Early afterdepolarizations: Mechanism of induction and block: A role for L-type Ca'+ currents. Circ Res 1989;64:977-990 15. January CT, Shorofsky SR: Early afterdepolarizations: Newer insights into cellular mechanisms. J Cardiovasc Electrophysiol 1990; 1:161-169 16. Fabiato A: Simulated calcium current can both cause calcium loading in and trigger calcium release from the sarcoplasmic reticulum of a skinned canine Purkinje cell. J Gen Physiol 1985;85: 291-320 17. McDonald T, Cavalie A, Trautwein W, Pelzer D: Voltagedependent properties of macroscopic and elementary calcium channel currents in guinea-pig ventricular myocytes. Pflugers Arch 1986;406:437-448 18. Kass RS, Scheuer T: Slow inactivation of calcium channels in the cardiac Purkinje fiber. J Mol Cell Cardiol 1982;14:615-618 19. Cavalie A, Pelzer D, Trautwein W: Fast and slow gating behavior of single calcium channels in cardiac cells: Relation to activation and inactivation of calcium-channel current. Pflugers Arch 1986; 406:241-258 20. Sheets MF, January CT, Fozzard HA: Isolation and characterization of single canine Purkinje cells. Circ Res 1983;53:544-548 21. Hamill OP, Neher ME, Sakmann B, Sigworth FJ: Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pfiugers Arch 1981;391: 85-100 22. Droogmans G, Nilius B: Kinetic properties of the cardiac T-type calcium channel in the guinea-pig. J Physiol (Lond) 1989;419: 627-650 23. Chen C, Hess P: Mechanism of gating of T-type calcium channels. J Gen Physiol 1990;96:603-630 24. Pelzer D, Hescheler J, Cavalie A, Trautwein W: Electrical estimate of calcium channel density in single ventricular cells from hearts of adult guinea pigs (abstract). Pflugers Arch 1985;403:R48 25. Fox AP, Nowycky MC, Tsien RW: Single-channel recordings of three types of calcium channels in chick sensory neurones. I Physiol (Lond) 1987;394:173-200 26. Rosenberg RL, Hess P, Tsien RW: Cardiac calcium channels in planar lipid bilayers. J Gen Physiol 1988;92:27-54 27. Reuter H, Stevens CF, Tsien RW, Yellen G: Properties of single calcium channels in cardiac cell culture. Nature 1982;297:501-503 28. Hess P, Lansman JB, Tsien RW: Calcium channel selectivity for divalent and monovalent cations: Voltage and concentration dependence of single channel current in ventricular heart cells. J Gen Physiol 1986;88:293-319 29. Nowycky MC, Fox AP, Tsien RW: Long-opening mode of gating of neuronal calcium channels and its promotion by the dihydropyridine calcium agonist Bay K 8644. Proc Nati Acad Sci U S A 1985;82:2178-2182 30. Carbone E, Lux HD: Single low-voltage-activated channels in chick and rat sensory neurones. J Physiol (Lond) 1987;386:571-601 31. Scanley BE, Hanck DA, Chay T, Fozzard HA: Kinetic analysis of single sodium channels from canine cardiac Purkinje cells. J Gen Physiol 1990;95:411-437 32. Yue DT, Backx PH, Imredy JP: Calcium-sensitive inactivation in the gating of single calcium channels. Science 1990;250:1735-1738
Downloaded from http://circres.ahajournals.org/ by guest on June 23, 2015
L- and T-type Ca2+ channels in canine cardiac Purkinje cells. Single-channel demonstration of L-type Ca2+ window current. S R Shorofsky and C T January Circ Res. 1992;70:456-464 doi: 10.1161/01.RES.70.3.456 Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 1992 American Heart Association, Inc. All rights reserved. Print ISSN: 0009-7330. Online ISSN: 1524-4571
The online version of this article, along with updated information and services, is located on the World Wide Web at: http://circres.ahajournals.org/content/70/3/456
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Circulation Research can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for which permission is being requested is located, click Request Permissions in the middle column of the Web page under Services. Further information about this process is available in the Permissions and Rights Question and Answer document. Reprints: Information about reprints can be found online at: http://www.lww.com/reprints Subscriptions: Information about subscribing to Circulation Research is online at: http://circres.ahajournals.org//subscriptions/
Downloaded from http://circres.ahajournals.org/ by guest on June 23, 2015
L- and T-Type Ca2+ Channels in Canine Cardiac Purkinje Cells Single-Channel Demonstration of L-Type Ca2+ Window Current Stephen R. Shorofsky and Craig T. January Canine cardiac Purkinje cells contain both L- and T-type calcium currents, yet the single Ca2' channels have not been characterized from these cells. Additionally, previous studies have shown an overlap between the steady-state inactivation and activation curves for L-type Ca2' currents, suggesting the presence of L-type Ca2' "window" current. We used the on-cell, patch-clamp technique to study Ca'+ channels from isolated cardiac Purkinje cells. Patches contained one or more Ca2' channels 75% of the time. L-type channels were seen in 69%1 and T-type channels in 73% of these patches. With 110 mM Ba2` as the charge carrier, the conductances of the L- and T-type Ca2' channels were 24.2±0.8 pS (n=9) and 9.0±0.5 pS (n=8), respectively (mean±SEM). With 110 mM Ca2' as the charge carrier, the conductance of the L-type Ca2' channel decreased to 9.7± 1.2 pS (n =4), whereas the T-type Ca2' channel conductance was unchanged. Voltage-dependent inactivation was shown for both L- and T-type Ca2' channels, although for L-type Ca21 channel with Ba2` as the charge carrier, inactivation took at least 30 seconds at a potential of +40 mV. After channel inactivation was complete, L-type Ca2+ channel reopenings were observed following repolarizing steps into the window voltage range. Thus, our data identify both L- and T-type Ca2+ channels in cardiac Purkinje cells and demonstrate, at the single-channel level, L-type channel transitions expected for a window current. Window current may play an important role in shaping the action potential and in arrhythmogenesis. (Circulation Research 1992;70:456-464) KEY WoRDs * Ca2' channels window current Purkinje cells * heart inactivation .
C ardiac tissues contain both L- and T-type Ca2' channel currents.'-7 Studies of the voltagegated properties of these channels have demonstrated overlap between their steady-state inactivation and activation relations, particularly for the L-type Ca'+ channel, suggesting the possibility that steady-state Ca'+ ("window") currents exist.5,7-11 L-type Ca'+ window current has been proposed to have an important role in controlling the action potential shape,12"3 in some types of triggered arrhythmias,1415 and possibly in the regulation of contraction.3'16 A window current is usually defined as a steady-state current that arises from voltage-gated channels within the narrow voltage range where the steady-state inactivation and activation relations overlap (see Figure 1). At potentials negative to the window, channels distribute between closed and inactivated states. For sufficiently long duration voltage steps positive to the window, the channels preferentially distribute into an inactivated state. Only within the window voltage range can a channel theoretically cycle between closed, open, From the Cardiac Electrophysiology Laboratories, Department of Medicine (Cardiology), University of Chicago (Ill.). Supported by National Heart, Lung, and Blood Institute grants HL-20592 and HL-38927. Address for correspondence: Stephen R. Shorofsky, MD, PhD, Department of Medicine, Box 304, University of Chicago, 5841 South Maryland Avenue, Chicago, IL 60637. Received June 18, 1990; accepted October 15, 1991.
.
.
and inactivated states, thus generating, in cells, a steady current. If no voltage range exists that permits both recovery from inactivation and activation, then a true steady-state Ca'+ current should not exist. Previous attempts to demonstrate Ca'+ window current have focused on demonstrating a steady or persistent Ca2+ current under conditions when only timeindependent currents were expected. Cohen and Ledererll showed that the D600 sensitive L-type, steady Ca2+ current, measured 200 msec from the beginning of the depolarizing step, exhibited the voltage dependence predicted by the overlapping region of the steady-state inactivation and activation curves, supporting the concept of window current. McDonald and coworkers17 were able to record persistent L-type Ca'+ channel activity during 900-msec depolarizations. Cavalie and coworkers9 demonstrated persistent L-type Ca'2 channel activity, albeit rare, at holding potentials in the voltage range where the steady-state inactivation and activation curves overlapped, leading these authors to conclude that these channel openings represented window current. Similar whole-cell findings were shown for 1-second depolarizing steps by Hirano et al.5 Interpretation of these experiments, however, is complicated by the existence of a slowly inactivating component of the L-type Ca'+ current'8 and by the complex single-channel behavior that occur near threshold voltage (see References 17 and 19). Currents or channel activity noted on depolarization into the window voltage range
Downloaded from http://circres.ahajournals.org/ by guest on June 23, 2015
Shorofsky and January Single-Channel Demonstration of Ca2' Window Currents 1.0.
-o
c 0
0
>
Voltage+ FIGURE 1. Idealized plot of the relation between a steadystate inactivation and an activation versus voltage. The shaded area represents the window voltage range. Also shown are the predominant channel transitions that occur in the various voltage ranges. C, aggregate of closed states; 0, open states; I, inactivated states. See text for further discussion.
could represent a slowly inactivating Ca2+ current as well as true window current. The present study was designed to identify and characterize the types of Ca2' channels in cardiac Purkinje cells and to test the hypothesis that L-type Ca2' window current exists by analyzing the behavior of these Ca2' channels under voltage-clamped conditions in which window but not slowly inactivating currents should exist. Purkinje cells were chosen for this study because previous work from our laboratory had shown that these cells contain both L- and T-type Ca2' currents, with potential window voltage ranges for each.5 Because previous studies of single Ca2' channels have not been reported for this preparation, we first characterized some properties of the L- and T-type Ca2' channels. Subsequently, we showed that both channel types can be voltage inactivated. Once inactivated, we identify L-type Ca2' channel reopenings on repolarization into the window voltage ranges, demonstrating the single-channel transitions necessary for this window current to exist. Materials and Methods
Preparation Single canine cardiac Purkinje cells were isolated enzymatically as described previously.20 The cells were stored in a dispersion solution (see below for contents) and were studied within 8 hours of isolation. Small aliquots of cells were added to the bath solution (see below for contents) in a perfusion chamber constructed on a glass coverslip and mounted on the stage of an inverted microscope (Diaphot, Nikon). The chamber was treated with polylysine (0.1 mg/ml) before each experiment to facilitate cell adhesion to the glass coverslip. All experiments were performed at room temperature (20-22°C).
457
Electrical Recordings Single-channel recordings were made using the oncell patch-clamp method of Hamill and coworkers.21 Glass suction pipettes were fabricated from hematocrit capillary tubes (Drummond Scientific Co.). Pipettes were pulled with a two-stage microelectrode puller (model PP-83, Narishige), coated with Sylgard 184 (Corning Medical), and fire polished to a final tip diameter of 1-1.5 gtm by using a microforge (model MF-83, Narishige). When filled with pipette solutions (see below), the pipettes had resistances of 0.5-2 M[l. The bath solution was connected to ground via a 150 mM KCl/agar bridge and a Ag/AgCl half-cell electrode. Electrical contact with the pipette solution was via a chlorided silver wire. The electrode potential was adjusted to give a zero current between the pipette solution and the bath solution immediately before attempting to make a patch. Gigaohm seals between the pipette and single Purkinje cells were obtained by applying gentle suction to the pipette after contacting the cell membrane. Seal resistances for these experiments ranged between 10 and 100 Gfl. Single-channel currents were studied in the on-cell patch configuration by using a patch-clamp amplifier (List EP-7, Darmstadt, FRG). Voltage-clamp protocols were generated and single-channel currents recorded using a 12-bit, A/D board (DAS-16, Metrobyte), under computer control (Zenith 386). Single-channel currents were filtered at 1 kHz with an eight-pole Bessel filter (model 902, Frequency Devices) and digitized at 10 kHz. Two voltage-clamp protocols were used. The first consisted of either 100- or 200-msec voltage steps from the holding potential repeated every 2 seconds. The second consisted of recording 200 msec of channel activity (e.g., sampling period) every 2 seconds while holding the patch at fixed potentials for long periods of time. Partial electronic capacitance compensation was used in all experiments. All data were stored on a hard disk for subsequent analysis.
Data Analysis The data were capacity and leak corrected by subtracting an averaged null trace. By using the corrected tracings, an amplitude histogram was constructed and the open-channel amplitudes were estimated. The threshold to detect openings and closings was set at one half the estimated open-channel amplitude, and an open-closed table was constructed. These data were then used to create open-channel histograms, from which the actual open-channel current amplitudes were determined. Multiple simultaneous channel openings were not included in the analysis. To eliminate some of the error introduced by filtering, the current measured had to exceed threshold for at least three successive data points to be considered a valid opening. The standard deviation of the noise seen in most patches was less than or equal to 0.12 pA.
Solutions The dispersion solution in which the cells were stored after isolation contained (mM) potassium glutamate 130, MgC12 5.7, EGTA 0.1, and HEPES 10 (pH adjusted to 6.9 with KOH). The bathing solution contained (mM) potassium aspartate 140 and HEPES 10 (pH adjusted to
Downloaded from http://circres.ahajournals.org/ by guest on June 23, 2015
Circulation Research Vol 70, No 3 March 1992
458 A mV7 -79
B
1
11
-79 -- *
J~¶ZZ;
S^
..
LL
-1 --L-1
C
-
--i__ v
..
"'
D
21
v
FIGURE 2. L-type Ca2+ channel activity recorded with the voltage-clamp protocols shown above the tracings. The pipette contained 110 mM Ba2' as the charge carrier. Bay K 8644 (5 tiM) was present to prolong channel openings.
31
I
-79
-79
Ttrllrl7rr -q
.*.
P
-,
v,
.
f
IE_ 7~~~~~~~~b _R- I.
l- --
RP
-
.
A
.
-A
-
10 nis
7.4 with KOH). This solution depolarized the Purkinje cells to approximately 0 mV. The internal solution for the suction pipette contained (mM) either CaCl2 110 or BaCI2 110 with HEPES 10 (pH adjusted to 7.4 with tetraethylammonium hydroxide [TEA-OH]) and tetrodotoxin 0.012 (TTX; Sigma Chemical Co., St. Louis, Mo.). The TEA-OH and TTX were added to block K' and Na+ channels, respectively. When used, Bay K 8644 (1-5 ,M; Miles Pharmaceuticals) was added to the bathing solution (from a 10 mM stock of Bay K 8644 dissolved in absolute alcohol). The use of high divalent ion (Ca21 or Ba21) concentrations facilitates the identification of single-channel currents but also results in a shift to more positive voltages of the voltage-dependent properties of currents recorded from these cells.5
Where appropriate, data are reported
as
mean+±SEM.
Results Single Ca21 channels of both the L and T type could be demonstrated easily in isolated canine Purkinje cells.
Ca2' channels were identified in approximately 75% of the first 35 successful patches. Of the patches with channels, 73% contained at least one T-type Ca2+ channel, 69% had at least one L-type Ca2' channel, and 42% had both Ca2+ channel types. L-Type Ca2' Channels Typical openings of an L-type Ca21 channel during a range of voltage steps are shown in Figure 2. The pipette solution contained 110 mM BaCl2, and Bay K 8644 (5 ,uM) was added to the bath solution to prolong the channel openings. As the membrane patch was clamped to more positive potentials, the channel tended to open earlier and for longer durations. This is best demonstrated in Figure 3, where the probability that the channel shown in Figure 2 is open per sweep is plotted for consecutive voltage steps to the potentials shown. At the more positive potentials, the L-type Ca21 channel tended to either open or remain closed for the
Downloaded from http://circres.ahajournals.org/ by guest on June 23, 2015
Shorofsky and January Single-Channel Demonstration of Ca2' Window Currents 1 niV
PC
Il I
459
11 mV
1-
PO
.1
I
SWEEP NUMBER
IL
--
54
SWEEP
21 mV
NUMBERl
1-
FIGURE 3. The probability 45 that the L-type Ca2' channel
(from Figure 2) was open (PO) during successive 200-msec voltage-clamp steps to the indicated potentials.
pc
PC
SWEEP NUMBER
duration of the clamp step. The plot also shows that channel openings tended to occur in groups of sweeps. The open-channel amplitude histograms and the current-voltage (I-V) relation for the open channel is shown in Figure 4. In all experiments, the I-V relation appeared linear. With Ba2` (110 mM) as the charge carrier, the conductance of the L-type Ca2' channel was 24.2+0.8 pS and the extrapolated reversal potential (estimated by linear regression) was 60.8+1.7 mV (n=9). An estimate of the macroscopic currents resulting from a voltage step can be obtained by the ensemble average of single-channel recordings from many steps to the same voltage. The ensemble averages and their peak I-V relation for the channel from Figure 2 are shown in Figure 5. With Ba2' as the charge carrier, little inactivation occurred during the 100-msec voltage-clamp steps. The corresponding I-V relation revealed a peak at approximately +30 mV. Examples of L-type Ca2+ channel recordings, when Ca21 (110 mM) is used as the charge carrier in the pipette, are shown in Figure 6. The corresponding ensemble average currents are shown below the sample tracings. Bay K 8644 was not used in this experiment. Again, channel openings are more frequent at the more positive voltageclamp steps. In contrast to the experiments when Ba21 was the charge carrier, the ensemble currents demonstrate marked inactivation during the 200-msec voltage-clamp step. With Ca2+ in the pipette, the conductance of the L-type Ca2' channel was 9.7+1.2 pS and the extrapolated reversal potential was 70.2± 6.0 mV (n=4).
T-Type Ca2+ Channels Representative current recordings and ensemble averages obtained from T-type Ca21 channels with Ba21 (110
SWEEP NUMBER
mM) as the charge carrier are shown in Figure 7. Compared with the L-type Ca2' channels, the T-type Ca2' channels opened at more negative potentials, tended to open early, inactivated completely during the 200-msec voltage-clamp steps to positive potentials regardless of whether the charge carrier was Ca2+ or Ba2 , and were unaffected in either conductance or kinetics by Bay K 8644. The open-channel amplitude histograms and I-V relation are shown in Figure 8. The I-V relation was linear. The conductance of the T-type channels was 9.0+0.5 pS (n=8) with Ba21 (110 mM) as the charge carrier and 8.4 pS (n=2) with Ca2+ (110 mM) as the charge carrier. The extrapolated reversal potential with Ba2' as the charge carrier was 48.5±13.9 mV, which was not statistically different from that obtained from Ba2+ current through the L-type Ca21 channel. The large standard deviation of the reversal potential estimate is due in part to the fact that the open-channel conductance was determined over a relatively narrow voltage range that is far from the reversal potential, so the small variance in the slope (SEM= +0.5 pS) between patches yields a large variance in the extrapolated reversal potential. Inactivation T-type Ca2+ channels completely inactivate within 200 msec when clamped to positive potentials, as shown in Figure 7 (see also References 5, 6, 22, and 23), regardless of whether Ca2+ or Ba2+ is the charge carrier. In contrast, for the L-type Ca21 channel when Ca21 was the charge carrier, channel inactivation was incomplete by the end of a 200-msec voltage step, suggesting clear kinetic differences (see Figure 6). With Ca2' as the
Downloaded from http://circres.ahajournals.org/ by guest on June 23, 2015
Circulation Research Vol 70, No 3 March 1992
460
potentials shown and 200 msec of single-channel data was recorded every 2 seconds. Activity from only one L-type Ca21 channel was seen in this patch. The probability that the channel was open during each 200-msec sampling period is plotted. Channel activity developed after the potential across the patch was changed from -40 to + 13 mV, and within about 20 seconds it disappeared. The channel could be in either a closed or inactivated state. However, with further depolarization of the patch to +30 and +40 mV, at which channel openings could be easily recorded (see panel B), no further channel activity could be elicited, indicating inactivation of the channel.
1 mV
-9 mV
cc
z
CURRENT (pA) 21 mV 1200
z
0
..
-1.5
CURRENT (pA)
0.0
CJRRENT (pA)
31 mV
41
mV
2000
z
z
0-
0-
0°0
-15
CUR
0.0
-1.5
CURRENT (pA)
T (pM
1.OTpA
-30
_ T
mV
sol0
FIGURE 4. The open-channel amplitude histograms with their corresponding Gaussian fits and current -voltage relation for the L -type Ca2' channel shown in Figure 2. The conductance of this channel was 23.6 pS, and the extrapolated reversal potential was + 68.9 mV.
charge carrier, it is difficult to separate L-type from T-type Ca2+ channels because of the similarity of their conductances. With Ba2` as the charge carrier, L- and T-type Ca21 channel currents can be easily separated; however, the L-type Ca2+ channel revealed little inactivation during the 100-200-msec duration voltage-clamp steps we used (see Figure 3). The L-type Ca2+ channel can be voltage inactivated, however, if given sufficient time. An experiment demonstrating L-type Ca2+ channel inactivation with Ba2+ as the charge carrier is shown in Figure 9. In this experiment, the membrane patch was voltage clamped to the
L-Type Ca21 Window Current If a window current exists for the L-type Ca2' channel, then after inactivation of the channel at positive potentials, channel openings should return on repolarization into the window voltage range. The results of such an experiment are shown in Figure 10. This experiment is a continuation of the experiment shown in Figure 9. After inactivating the channel with a prolonged depolarization to +40 mV, the patch was repolarized to + 13 mV. Channel activity returned as shown by the sample tracings in panel A. The patch was again clamped to +40 mV, and after about 30 seconds channel activity again ceased. The membrane was then repolarized to + 25 mV, and no channel activity was noted. On further repolarization of the patch to +7 mV, channel activity again returned. Examples of the channel activity during this repolarization are shown in panel B. The difference in the pattern of channel reopenings seen after the repolarization steps to the two potentials (see panels A and B) does not imply a difference in channel behavior at the two potentials but is within the normal variability seen with a given L-type Ca2' channel. Protocols similar to that shown in Figure 10 were completed on nine patches containing L-type Ca2' channels. From these experiments, L-type Ca2' channel activity was identified on repolarization into the window voltage range in five patches. From these nine experiments, the probability of identifying an L-type Ca2' channel reopening per sampling period (200 msec of acquired data every 2 seconds) was 0.024. Because channel openings were brief (the probability that the channel was open within a sampling period in which reopenings were identified was about 0.1), the probability that the channel was open during the repolarizing steps was less than 0.002 (or less than 0.2%). Discussion Single-Channel Properties It is interesting that both L- and T-type Ca2' channels were observed frequently in these cells. Channels were seen in 75% of the successful patches. This is in sharp contrast to the results from guinea pig ventricular myocytes, in which L-type Ca2' channel activity was noted in only about 12% of the patches.24 Using the single-channel conductances measured in this report, with the whole-cell L- and T-type Ca2' currents reported by Hirano and coworkers5 under similar conditions, the minimum channel density for both channel types was calculated to be about six channels per square micrometer, a value comparable to that found for
Downloaded from http://circres.ahajournals.org/ by guest on June 23, 2015
Shorofsky and January Single-Channel Demonstration of Ca21 Window Currents
A
B F
461
0.1 -- PA t
i
_
- I
I
-_---- - --
---
-30
50
mY
1 1 mVy
21 m\/ _
31 mY
-0.4 - _ a
20 1"
FIGURE 5. Panel A: Ensemble averages for the L-type Ca2' channel shown in Figure 2. The averages from clamp steps to 1, 11, 21, and 31 mVsteps were constructed from 46, 40, 21, and 28 sweeps, respectively. Panel B: The peak current-voltage relation from the ensembles.
L-type Ca'+ channels in guinea pig ventricular myocytes.17 The fact that we observed T-type channels as frequently as L-type channels is consistent with the greater prevalence of T-type Ca'+ currents in Purkinje cells as opposed to ventricular cells.722 Neither N-type25 nor B-type26 Ca21 channels were identified in the isolated Purkinje cells. The general properties of both Ca21 channel types reported in this study (i.e., their conductance with Ba21 or Ca2' as the charge carrier, kinetics, and response to Bay K 8644) are in good agreement with those from A
B
-12
cultured neonatal rat ventricular cells,27 guinea pig ventricular myocytes,19,19,2228 calf sarcolemma membrane channels reconstituted in planar lipid bilayers,26 and neuronal cells.2529,30 It is unlikely that the T channel represents Ca2' transit through the Na+ channel because 1) the channels are not blocked by up to 100 ,M 1TX (unreported observation), 2) the single-channel kinetics differ from those of the Na+ channels seen in these cells,31 and 3) the T channels activated and inactivated at potentials more positive than would be expected if the current was carried through Na+ channels.
C
-2
a
mV -82
-82
-82
~ --' q
'A_
h
b%- % .0 M.
-
--
10 me
-
-
0I
cu
-~
20 ms
FIGURE 6. Sample recordings of L-type Ca2' channels and their ensemble averages with 110 mM Ca2' as the charge carrier. Voltage protocols are illustrated above the tracings. Note the decrease in channel current and more rapid decay of the ensemble currents compared with that seen when 110 mM Ba2` is the charge carrier. Downloaded from http://circres.ahajournals.org/ by guest on June 23, 2015
462
Circulation Research Vol 70, No 3 March 1992 -10
-30
j-s
mV j
W
-so
lI~ ~~~~~r -~ --
,-
-
I.
-.W. --- --.
1-
a~ -0 R-I, 1- M. a- offi o
---
-
--
--
---
-A-
--
'
-----
-1 be wo 110- ---
em . _
.A-
h-
1
-
-
-
lq .m
iw0 onwo aq ebo.m-11.
--
---
m-
-
---- - - -- --v -
---1.
~_
FIGURE 7. Sample recordings of T-type Ca2+ channels and the corresponding ensemble averages. Voltage protocols are illustrated above the tracings. Note the brief channel openings and rapid decay of the ensemble currents.
-
V_~ ~ ~ ~ ~ ~ C 20 ms
-AMMINA,
-AMMIAOW
20 ms
50-
z
-37
-47 mV
-
2
0-
t -05
-1.0
GURRENT
150-
mV
-27
(PM)
CGURREN
mV
0(
0
(PA)
-17 mV
.~~~~~~
FIGURE 8. The open-channel amplitude histogram with the Gaussian fits and current-voltage relation for a typical T-type Ca2' channel. In this patch, the T-type channel had a conductance of 9.2 pS and a reversal potential of +46 mV.
z
-0.5
Ns
0.0
Ue
CURRENT (pA) 11
ao
CURFENT (pA)
-7 mV
1.0-T pA
10
-80
mV
CURFENW (pA)
_3.0 l
Downloaded from http://circres.ahajournals.org/ by guest on June 23, 2015
Shorofsky and January Single-Channel Demonstration of Ca2' Window Currents A
40
30
13
13
my
mv
30
13
2
25
-40
-40
T
-40 mv
.27-
.090-
II
.07.p-
Po .0c .031-
1
.01 1-
.21-
13 mY
*'I'qS'" *rA
U'ivmw-i
m
---
PO
.15-
30 mv .09-
I
-1-i a -
A
7'
B
.03
20 ms
p
i a i t 4,9 SWEEP NLUMBE
R
a
1 -1 2i1
a
*
e
_t
30
m\@1
aBR^
1
'''U B 147 ' Sl-I S WE5No6s~~~~~~~~~~~~~~~~~~~~~ ie a
a
SVWEEP NUMBER A
B
0
4
463
B
-zt:j~---
0 -v Ar -~~~~Im .
A&L&ALAb-
n-~
my
~
_§---
-
1
A
.
__j
v-R---
-7~~ ~ ~ ~ ~ ~ cu
20
20 ms
FIGURE 9. Panel A: The probability that the L-type Ca' channel was open (PO) during the voltage protocol illustrated at the top of the figure. Inset are sample tracings at the various potentials. Note that the channel activity disappears with time after clamping the patch to +13 mV See text for further details. Panel B: Samples of L-type Ca' channel activity on clamping the patch from 0 to +30 mV, indicating that the lack of channel activity when clamped to +30 mIVin panel A was not due to an inability to resolve single-channel activity at +30 mV
Inactivation Results from experiments with a number of different cell types have consistently shown that the L-type Ca' current shows little inactivation over several hundred milliseconds when Ba` is the charge carrier."57'92532 The data presented here are consistent with those observations. Our results also show that under these conditions (e.g., Ba2+ as the charge carrier), L-type Ca2' channels can inactivate if given sufficient time. In our experiments, voltage-dependent inactivation required at least 30 seconds for a voltage step to +40 mV. These results are also in agreement with Yue and coworkers,32 who suggest that Ca2+-facilitated inactivation results from calcium favoring occupancy of a closed state from which the channel can reopen rather than accelerating inactivation into an absorbing state. With Ba2+ as the
charge carrier, depolarization favored channel inactivated state (see Figure 9).
pancy in an
occu-
ms
FIGURE 10. Probability that an L-type Ca' channel is open (PO) during the voltage protocol illustrated at the top of thefigure. Panels A and B: Single-channel activity that is seen on repolarization into the window voltage ranges correspond, in time, to the letters in the upperfigure. See the textforfurther details. In contrast, T-type Ca2' channels were completely inactivated within 200 msec at positive potentials regardless of whether the charge carrier was Ca2+ or Ba2 This agrees with results from whole-cell experiments with isolated canine Purkinje cells,6 rabbit sinoatrial cells,4 chick and rat sensory neurons,30 cultured 3T3 fibroblasts,23 and guinea pig ventricular cells.22 .
L-Type Ca>2 Window Current Hirano et al13 have studied L-type Ca>2 currents in whole-cell voltage-clamped isolated Purkinje cells with protocols similar to those used in this study. They were able to demonstrate recovery of an inward Ca>' current when the cell was repolarized into the L-type Ca21 window voltage range. This current responded to interventions that previously had been shown to affect L-type Ca21 current. They concluded that L-type Ca2+ window current could be shown directly and that it could be separated from other processes, such as slow inactivation. The data we present here support and extend their conclusions. For a window current to exist, a voltage-gated ion channel must be able to cycle from an inactivated state to an open state within the appro-
Downloaded from http://circres.ahajournals.org/ by guest on June 23, 2015
464
Circulation Research Vol 70, No 3 March 1992
priate voltage range. Our data demonstrate that L-type Ca'2 channels can undergo these transitions. Although our results do not demonstrate whether the channel must first recover to a closed state before reopening, the results of Hirano and coworkers13 suggest that L-type Ca'2 channels cycle from an inactivated state to a closed state before reopening within the window. In the present experiments we observed L-type Ca'+ channel reopenings when the patch was repolarized (from +40 mV) to the voltage range of 0 to +20 mV. This voltage range is close to the maximal overlap between the steady-state inactivation and activation curves for the L-type Ca2' current with similar concentrations of divalent ions (110 mM Ba'+, see Reference 5) and positive to the window voltage range found with lower divalent ion concentrations.579-11 In principle, it would have been advantageous to study the probability of L-type Ca'+ channel reopenings throughout the window voltage range; however, channel reopenings were rare, with the probability of identifying an L-type Ca2+ channel on repolarization into the window voltage range of less than 0.002. This value is in qualitative agreement with the results of Hirano et al,13 in which the whole-cell current measured on repolarization into the window voltage range was usually less than 20 pA. Thus, the delineation of the window voltage range is not easily suited to single-channel experiments but is better suited to whole-cell experiments in which the behavior of many channels is studied simultaneously. Our results do demonstrate clearly, however, that L-type Ca2+ channels can open on repolarization into the window voltage range. In summary, our results show that 1) L- and T-type Ca2+ channels are present in relatively high densities in canine cardiac Purkinje cells, 2) both channel types can be voltage inactivated although with differing kinetics and ion dependencies, and 3) once inactivated, at least L-type Ca2+ channels can reopen on repolarization into their window voltage ranges. Acknowledgment We would like to thank Dr. H.A. Fozzard for his helpful discussions of these data.
References 1. Nilius B, Hess P, Lansman JB, Tsien RW: A novel type of cardiac calcium channel in ventricular cells. Nature 1985;316:443-446 2. Bean BP: Two kinds of calcium channels in canine atrial cells:
Differences in kinetics, selectivity, and pharmacology. J Gen Physiol 1985;86:1-30 3. Mitra R, Morad M: Two types of calcium channels in guinea pig ventricular myocytes. Proc NatlAcad Sci USA 1986;83:5340-5344 4. Hagiwara N, Irisawa H, Kameyama M: Contribution of two types of calcium currents to the pacemaker potentials of rabbit sinoatrial node cells. J Physiol (Lond) 1988;395:233-253 5. Hirano Y, Fozzard HA, January CT: Characteristics of L- and T-type Ca2+ currents in canine cardiac Purkinje cells. Am J Physiol 1989;256:H1478-H1492 6. Hirano Y, Fozzard HA, January CT: Inactivation properties of T-type calcium current in canine cardiac Purkinje cells. Biophys J 1989;56:1007-1016
7. Tseng G-N, Boyden PA: Multiple types of Ca'+ currents in single canine Purkinje cells. Circ Res 1989;65:1735-1750 8. Reuter H, Scholz H: A study of the ion selectivity and the kinetic properties of the calcium dependent slow inward current in mammalian cardiac muscle. J Physiol (Lond) 1977;264:17-47 9. Cavalie A, Ochi R, Pelzer D, Trautwein W: Elementary currents through Ca'+ channels in guinea pig myocytes. Pflugers Arch
1983;398:284-297
10. Josephson IR, Sanchez-Chapula J, Brown AM: A comparison of calcium currents in rat and guinea pig single ventricular cells. Circ Res 1984;54:144-156 11. Cohen NM, Lederer WJ: Calcium current in isolated neonatal rat ventricular myocytes. J Physiol (Lond) 1987;391:169-191 12. McAllister RE, Nobel D, Tsien RW: Reconstruction of the electrical activity of cardiac Purkinje fibres. J Physiol (Lond) 1975;251:1-59 13. Hirano Y, Moscucci A, January CT: Direct measurement of L-type Ca2' window current in heart cells. Circ Res 1992;70: 445-455 14. January CT, Riddle JM: Early afterdepolarizations: Mechanism of induction and block: A role for L-type Ca'+ currents. Circ Res 1989;64:977-990 15. January CT, Shorofsky SR: Early afterdepolarizations: Newer insights into cellular mechanisms. J Cardiovasc Electrophysiol 1990; 1:161-169 16. Fabiato A: Simulated calcium current can both cause calcium loading in and trigger calcium release from the sarcoplasmic reticulum of a skinned canine Purkinje cell. J Gen Physiol 1985;85: 291-320 17. McDonald T, Cavalie A, Trautwein W, Pelzer D: Voltagedependent properties of macroscopic and elementary calcium channel currents in guinea-pig ventricular myocytes. Pflugers Arch 1986;406:437-448 18. Kass RS, Scheuer T: Slow inactivation of calcium channels in the cardiac Purkinje fiber. J Mol Cell Cardiol 1982;14:615-618 19. Cavalie A, Pelzer D, Trautwein W: Fast and slow gating behavior of single calcium channels in cardiac cells: Relation to activation and inactivation of calcium-channel current. Pflugers Arch 1986; 406:241-258 20. Sheets MF, January CT, Fozzard HA: Isolation and characterization of single canine Purkinje cells. Circ Res 1983;53:544-548 21. Hamill OP, Neher ME, Sakmann B, Sigworth FJ: Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pfiugers Arch 1981;391: 85-100 22. Droogmans G, Nilius B: Kinetic properties of the cardiac T-type calcium channel in the guinea-pig. J Physiol (Lond) 1989;419: 627-650 23. Chen C, Hess P: Mechanism of gating of T-type calcium channels. J Gen Physiol 1990;96:603-630 24. Pelzer D, Hescheler J, Cavalie A, Trautwein W: Electrical estimate of calcium channel density in single ventricular cells from hearts of adult guinea pigs (abstract). Pflugers Arch 1985;403:R48 25. Fox AP, Nowycky MC, Tsien RW: Single-channel recordings of three types of calcium channels in chick sensory neurones. I Physiol (Lond) 1987;394:173-200 26. Rosenberg RL, Hess P, Tsien RW: Cardiac calcium channels in planar lipid bilayers. J Gen Physiol 1988;92:27-54 27. Reuter H, Stevens CF, Tsien RW, Yellen G: Properties of single calcium channels in cardiac cell culture. Nature 1982;297:501-503 28. Hess P, Lansman JB, Tsien RW: Calcium channel selectivity for divalent and monovalent cations: Voltage and concentration dependence of single channel current in ventricular heart cells. J Gen Physiol 1986;88:293-319 29. Nowycky MC, Fox AP, Tsien RW: Long-opening mode of gating of neuronal calcium channels and its promotion by the dihydropyridine calcium agonist Bay K 8644. Proc Nati Acad Sci U S A 1985;82:2178-2182 30. Carbone E, Lux HD: Single low-voltage-activated channels in chick and rat sensory neurones. J Physiol (Lond) 1987;386:571-601 31. Scanley BE, Hanck DA, Chay T, Fozzard HA: Kinetic analysis of single sodium channels from canine cardiac Purkinje cells. J Gen Physiol 1990;95:411-437 32. Yue DT, Backx PH, Imredy JP: Calcium-sensitive inactivation in the gating of single calcium channels. Science 1990;250:1735-1738
Downloaded from http://circres.ahajournals.org/ by guest on June 23, 2015
L- and T-type Ca2+ channels in canine cardiac Purkinje cells. Single-channel demonstration of L-type Ca2+ window current. S R Shorofsky and C T January Circ Res. 1992;70:456-464 doi: 10.1161/01.RES.70.3.456 Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 1992 American Heart Association, Inc. All rights reserved. Print ISSN: 0009-7330. Online ISSN: 1524-4571
The online version of this article, along with updated information and services, is located on the World Wide Web at: http://circres.ahajournals.org/content/70/3/456
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Circulation Research can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for which permission is being requested is located, click Request Permissions in the middle column of the Web page under Services. Further information about this process is available in the Permissions and Rights Question and Answer document. Reprints: Information about reprints can be found online at: http://www.lww.com/reprints Subscriptions: Information about subscribing to Circulation Research is online at: http://circres.ahajournals.org//subscriptions/
Downloaded from http://circres.ahajournals.org/ by guest on June 23, 2015
