From the Department of Molecular Biophysics and Physiology, Rush University, Chicago, Illinois 60612
It is widely believed that Ba2+ currents carried through L-type Ca2+ channels inactivate by a voltage-
dependent mechanism similar to that described for other voltage-dependent channels. Studying ionic and gating
currents of rabbit cardiac Ca2+ channels expressed in different subunit combinations in tsA201 cells, we found a
phase of Ba2+ current decay with characteristics of ion-dependent inactivation. Upon a long duration (20 s) depolarizing pulse, IBa decayed as the sum of two exponentials. The slow phase (
6 s, 21°C) was parallel to a reduction of gating charge mobile at positive voltages, which was determined in the same cells. The fast phase of current decay (
600 ms), involving about 50% of total decay, was not accompanied by decrease of gating currents.
Its amplitude depended on voltage with a characteristic U-shape, reflecting reduction of inactivation at positive
voltages. When Na+ was used as the charge carrier, decay of ionic current followed a single exponential, of rate
similar to that of the slow decay of Ba2+ current. The reduction of Ba2+ current during a depolarizing pulse was
not due to changes in the concentration gradients driving ion movement, because Ba2+ entry during the pulse did
not change the reversal potential for Ba2+. A simple model of Ca2+-dependent inactivation (Shirokov, R., R. Levis,
N. Shirokova, and E. Ríos. 1993. J. Gen. Physiol. 102:1005-1030) robustly accounts for fast Ba2+ current decay assuming the affinity of the inactivation site on the
1 subunit to be 100 times lower for Ba2+ than Ca2+.
Brief openings of L-type Ca2+ channels cause the increases in intracellular Ca2+ concentration required to
effect rapid metabolic switching in a variety of cellular
processes (Tsien and Tsien, 1990). The Ca2+ signalling
process is controlled and limited by multi-layered inactivation mechanisms, that affect the plasmalemmal as
well as the intracellular release Ca2+ channels. Channel
inactivation sharpens the kinetics and temporal precision of the signals, and prevents longer term increases
in [Ca2+]i, which, among other problems, lead to stimulation of intracellular proteolysis (Turner et al., 1988
).
In L-type and other Ca2+ channels (Tareilus et al.,
1994; Cox and Dunlap, 1994
), inactivation is produced
by two mechanisms. One is mediated by the calcium
ions that carry the current (Brehm and Eckert, 1978
;
Eckert and Chad, 1984
), and it appears to involve binding to a site highly specific for Ca2+. In L-type channels
the site is located near the channel mouth (Imredy and
Yue, 1992
, 1994
; Shirokov et al., 1993
), on the main (
1) channel protein (Neely et al., 1994
), and has been
equated with an EF hand-like portion of the cytoplasmic COOH terminus (De León et al., 1995).
The other mechanism of inactivation does not require the passage of current (Fox, 1981). It is linked to
the change in transmembrane potential, and is accompanied by changes in gating currents, which are
broadly termed inactivation of charge (Shirokov et al.,
1992
). The two mechanisms function independently, as
demonstrated by the fact that changes in [Ca2+]i (Hadley and Lederer, 1991
) and Ca2+ current through the
channels (Shirokov et al., 1993
) do not affect gating
currents.
Parallel recording of gating currents and ion currents helped elucidate voltage-dependent aspects of
Ca2+ channel gating (Field et al., 1988; Bean and Ríos,
1989
; Hadley and Lederer, 1989
) but is complicated in
native cells by the presence of substantial Na+ channel
gating current. The present work overcomes this problem using tsA201 cells, an expression system virtually
free of endogenous channels. With Ba2+ as the permeant ion, we compared inactivation of gating charge with decay of ion currents, under the prevailing hypothesis that inactivation of L-type channel current is
exclusively voltage-dependent in the absence of Ca2+
(e.g., Kass and Sanguinetti, 1984
). The results disproved this hypothesis, favoring instead a dual mechanism of inactivation.
Preparation of Transfected and Native Cells
Experiments were performed in the large T antigen-transformed
human embryonic kidney cells (tsA201 line) grown in DME medium (Sigma Chemical Co., St. Louis, MO) supplemented with
10% FBS (BioWhittaker, Walkersville, MD) and 1% penicillin/
streptomycin (Sigma) in 5% CO2. Cardiac rabbit 1C, rat brain
2a, and rabbit skeletal muscle
2
cDNAs were cloned in pCR3,
pCMV, and pMT2 plasmid vectors respectively. High purity
(A260/A280
1.95) large-scale plasmid preparations were obtained using standard protocols (Qiagen Inc., Chadworth, CA).
Transfections were done with 30 µg of each expression plasmid
in different combinations (
1C,
1C +
2a, and
1C +
2a +
2
)
using a modified calcium phosphate precipitation method (Chien
et al., 1995
) in 100-mm tissue culture dishes. Cells were incubated with DNA for 6-8 h and then were shocked with 10% DMSO for 8 min. After the shock, cells were transferred onto
glass coverslips, fed with fresh media and incubated until further
evaluation. Electrophysiological recordings were made within
24-48 h post-transfection on round nonclustered cells. No sizable ionic or gating currents were observed in tsA201 cells in the
absence of transfection. After transfection, the fraction of cells
selected and patched that had Ca2+ currents was 60-80%. Recording of native Ca2+ currents was carried out in single rabbit
cardiac ventricular myocytes, which were obtained by an enzymatic dissociation method (modified from Mitra and Morad,
1985
) from young (1,500 g) male mutt rabbits.
Electrophysiological Recording
Cells were placed in small chambers (<200 µl) and perfused by
continuous flow of external solutions. Records were obtained by
a standard whole-cell patch clamp procedure using an Axopatch 200A amplifier and a 16-bit A/D-D/A converter card (HSDAS 16; Analogic Corp., Peabody, MA) under a 486 PC computer. Patch
electrodes had resistances of 1.5-4 M. Whole-cell capacitance,
calculated as the area under a linear capacitive transient elicited
by a
10 mV pulse, was 6-40 pF. Capacitance and resistance compensations were routinely done, and the charging time constant
was typically 100 µs. Ionic currents were sampled at 0.15-1 kHz
and gating currents at 10-40 kHz, depending on pulse duration.
All the currents represented are asymmetric, obtained after
subtraction of scaled control currents, and not corrected for baselines. The control currents were elicited by pulses from 90 to
110 mV before each depolarization and after holding the
cell at
90 mV for 30 s. All experiments were carried out at room
temperature (~20°C).
Exponential Fitting and Statistics
Data are presented as averages ± SEM. Signficance of differences
between mean values was evaluated by Student's t test. Time course of current decay was fitted (using a nonlinear least-squares routine) by the function Y1 = b + a exp (t/
) or the function Y2 = b + aslow exp (
t/
slow) + afast exp (
t/
fast). Afast, Aslow, and B
are the best fit amplitude parameters, afast, aslow, and b, normalized to their sum. A and B symbolize the normalized amplitudes in the single exponential fit. In all cases in which both fits converged, the statistical significance of the fit improvement provided by the two- exponential function Y2 was evaluated. The test
was based on the likelihood ratio statistic, LRS = (ssr1
ssr2)/
2
(Hoel, 1971
; Hui and Chandler, 1990
), in which ssr1 and ssr2 are
the sums of squares of the residuals for the two fits and
2 is the
variance of the record. LRS has a
2 distribution with 2 degrees of
freedom (the difference between the number of free parameters
of Y2 and Y1). If the statistic exceeds 6.0, the improvement in fit
provided by the two-exponential function is significant to better
than 5%.
2 was estimated by ssr2/(N
5), which, being probably
in excess, gave an underestimate of LRS.
Solutions
The internal solution contained (in mM): 150 CsOH (or NMG), 110 glutamate, 20 HCl, 10 HEPES, 5 MgATP, and 10 EGTA (pH adjusted to 7.6 by addition of CsOH). Osmolality of the internal solution was routinely measured and adjusted to 300-320 mosmole/kg.
External recording solutions contained (in mM): 160 TEA-Cl, 10 Tris, and either 10 CaCl2 or 10 BaCl2. Sodium external solution contained (in mM): 150 NaCl, 10 HEPES. In some experiments about 0.5 µM CaCl2 was added to this solution to stabilize the patch seal. In gating current experiments, 10-20 µM GdCl3 was added to the external solution with 10 mM Ca2+. All external solutions were adjusted to pH 7.2 and 300-320 mosmole/kg.
Inactivation of Ba2+ Currents Had Two Distinct Kinetic Phases
The lack of substantial endogenous voltage-dependent
ion currents in tsA201 cells allowed us to study the time
course of decay of Ba2+ currents (IBa) during very long
pulses (up to 40 s). Ca2+ and Ba2+ currents in a cell
transfected with 1C and
2a cDNAs are illustrated in
Fig. 1, A and B. As shown in panel B, and as described
often for ICa, the time course of IBa decay was well fitted
by a sum of two exponentials plus a constant (Y2 in
METHODS). The record and the best fit line are shown
to overlap in the inset, where they are plotted semilogarithmically. The single exponential fit (dashed line) was
poor.
Families of records from 33 cells were fitted with one
and two-exponential functions. At voltages positive to
+20 mV, 12 cells were discarded because they had a
small outward steady current. Two other cells, which
had unusually slow activation kinetics, were discarded
because the two-exponential fit was not significantly better than the single exponential fit, as judged by the
LRS. The averages of parameters obtained from the remaining 19 cells are in Fig. 1 C. On average, the time
constant of the fast component of IBa decay (fast) was
minimal at +20 to +40 mV and increased (at a P < 0.08) at +60 mV. The amplitudes of the two exponentials (Aslow and Afast) were comparable. Afast went
through a maximum at +20 mV, decaying at higher
voltages. The slow component became faster and its
amplitude greater at increasing depolarization. B decreased from 0.3 at
20 mV to 0.05 at +20 mV and increased to 0.15 at higher voltages. Because in the selected cells there was little if any outward current, even
after 40 s pulses to +40 mV, and because neither current rundown nor substitution of NMG+ for intracellular Cs+ affected the decay parameters, these appear to
represent kinetics of the expressed channels. The crisp
separation of the two decay phases and the observed
differences in the dependencies of their parameters on
voltage suggest the presence of two independent mechanisms of inactivation.
Only the Slow Phase of IBa Decay Was Accompanied by Reduction of Gating Charge
Using ionic current-blocking solutions we studied the
effects of prolonged depolarization on the availability
of gating currents associated with channel opening.
The asymmetric (ON) charge transferred after the depolarizing transition of a 0.1 s pulse was equal to the
(OFF) charge transferred upon repolarization. As expected from studies in native cells, prolonged depolarization reduced gating currents. This was demonstrated
with one- and two-pulse protocols, to rule out errors
due to ionic currents or fast recovery from inactivation
of gating charge. Fig. 2 A shows the OFF transients obtained upon repolarization from +20 mV, after periods
of depolarization indicated near the traces. The larger
transient in each pair is the reference OFF after a 45-ms
pulse (in every case preceding the prolonged depolarization). Surprisingly, reduction of charge transfer took
much more time than decay of IBa. Fig. 2 B plots reduction of the OFF charge vs. pulse duration, averaged in
nine cells. The curve is a single exponential fit (
8 s).
The reduction of the available charge after depolarization was also monitored by a double pulse protocol
illustrated at top of Fig. 2 C. The membrane was conditioned at +20 mV, then it was repolarized to 60 mV
for 7 ms and a test pulse to +50 mV was applied to
move the charge available at voltages of channel opening. During conditioning depolarization currents were
recorded at a low acquisition rate, which was increased
at the repolarization swing. As in Fig. 2 A, the larger
transient in each pair is the reference obtained before
the corresponding conditioning pulse. The reduction
in available charge is plotted vs. conditioning duration
in 2 D. The reduction of gating charge at positive voltages had the same time course as the reduction of the
OFF charge in the single pulse experiment.
Fig. 3 compares the time course of IBa decay and that
of gating charge reduction. In the figure we superimposed data and fit of charge availability (from Fig. 2 B)
on a record of IBa elicited by a 16.5 s depolarization.
The exponential decay of charge availability (solid curve)
has a time constant similar to that of the slow decay of
IBa, suggesting that these are due to a slow inactivation mechanism, distinct and voltage-dependent. On the
other hand, the figure clearly shows that about half of
the decay of IBa occurred much more rapidly than the
process causing charge reduction. Indeed, as shown in
Fig. 2 A, ON and OFF charges remained equal in single pulses of up to 1 s, a time at which ionic current had inactivated by ~40%.
Fast Decay of IBa Is a Current-dependent Process
Fast inactivation of IBa was studied with brief (0.3 s) test
pulses to +20 mV, following a 2 s conditioning pulse to
different voltages (which should cause fast inactivation
almost exclusively) and a 1 s interpulse to 60 mV (to
ensure the return of all intramembrane charge). Each
pair of pulses was preceded by a single test pulse for reference, and the sets were applied at 1-min intervals.
Reference and conditioned currents are shown in pairs
in Fig. 4 A, normalized to correct for rundown. A conditioning pulse to 0 mV (eliciting substantial IBa) reduced
the magnitude and the rate of decay of the following
test current. Conditioning at +80 mV evoked no inward current and had less effect on the test current. Averages in seven cells are plotted vs. conditioning voltage
in Fig. 4 B. Circles represent peak conditioning current
relative to the maximal value at +10 mV and squares
represent peak test current (measuring channel availability after conditioning). IBa appeared at
30 mV,
peaked at +10 mV, and decayed at higher voltages.
The availability had a U-shaped voltage dependence
that mimicked the voltage dependence of the conditioning current, as befits a current- (rather than voltage-) dependent inactivation process (Tillotson, 1979
).
This inactivation was not due to Ca2+ contamination, as
[Ca2+] was measured in the external solutions with a
Ca2+ electrode and found to be negligible (<5 µM).
A U-shaped voltage dependence of the current availability could also be a manifestation of potentiation of
channel activity by large positive pulses, as shown in
snail neurons (Heyer and Lux, 1976), chromaffin cells
(Hoshi et al., 1984
), skeletal muscle (Sculptoreanu et
al., 1993
) and cardiac cells (Lee, 1987
). However, the
extent of current decay during the test pulses (triangles in Fig. 4 B) was minimal after conditioning at +10 mV
and increased at more positive conditioning voltages.
This implies that conditioning to a high voltage had a
modest accelerating effect on the decay kinetics, which
is against a major role of voltage-dependent potentiation, because in native cardiomyocytes potentiated currents had a much slower rate of decay (Lee, 1987
). Also
against voltage-dependent potentiation is the observation that the conditioned Ba2+ currents were never
greater than reference currents, even after conditioning to +100 mV.
Fast Current Decay Was Absent when Na+ Was the Permeant Ion
Fig. 5 shows representative records of Na+ current (INa)
through the 1C/
2a channels at submicromolar concentrations of extracellular divalent cations. The voltage dependence of INa (diamonds in Fig. 5 B) and its
sensitivity to Ca2+ channel blockers were as described
in native channels (Kostyuk et al., 1983
; Hess et al.,
1986
), which proves that INa passed through the expressed L-type channels. In agreement with previous reports in isolated cardiac myocytes (Imoto et al., 1985
;
Hadley and Hume, 1987
), INa activated at potentials
about 30 mV more negative than IBa (circles). At all voltages INa decayed much more slowly than IBa. A scaled IBa
trace plotted together with INa (Fig. 5 C) illustrates that
the fast decay was absent when Ba2+ was removed. In
fact, a fast component of decay was not resolvable even
at very high densities of Na+ current (up to 54 A/F).
Including Ca2+ in the external solution at a concentration of 20 µM led to the appearance of a phase of INa
decay with kinetics similar to those of the fast inactivation of IBa (Fig. 5 C).
Table I. |
Absence of Bulk Accumulation Effects
It is believed that the level of Ca2+ accumulation produced by typical membrane Ca2+ currents cannot significantly change the driving force that determines Ca2+ flux in a cell buffered with several mM of EGTA
(Neher, 1986). The buffering capacity of such intracellular solutions will be lower for Ba2+ (because the affinity of EGTA for Ba2+ is about 100 times less than for
Ca2+; Harafuji and Ogawa, 1980
). It is therefore conceivable that the current dependent reduction of IBa is
due to a reduction of driving force on the ion upon accumulation of Ba2+ inside the cell. A simple single pool
model of accumulation of Ba2+ in the volume of the
cell, in which unidirectional Ba2+ fluxes are assumed to
be independent, predicts first order decay of current,
with rate constant proportional to initial current amplitude and to the reciprocal of cell volume. The volume
of round cells (a good approximation for transfected
tsA201 cells) is proportional to Cm3/2, where Cm is membrane capacitance. Fig. 6 A plots
fast
1 vs. (peak IBa/
Cm3/2) at +20 mV for 21 cells, demonstrating no correlation between these variables.
The above test does not rule out depletion of Ba2+
ions outside the cell, or their accumulation in restricted
spaces close to the conduction pathway. To test these
possibilities we sought to demonstrate changes in the
driving force for IBa, in experiments similar to those described in a study of Na+ accumulation (Hadley and
Hume, 1987) and illustrated in Fig. 6 B. IBa was elicited
with a double pulse protocol similar to that in Fig. 4.
The test pulse took the membrane to the reversal potential and elicited no net ionic current. If a large current preceding the test determined substantial accumulation
and a change in driving force for Ba2+, the conditioned
pulse should elicit an outward current. Conditioning at
0 mV elicited near maximal IBa but did not result in the
appearance of outward current during the test pulse.
The decay in current can therefore be safely attributed
to channel gating (inactivation) rather than a change
in driving force.
Fast Decay Was Not due to Acidification
Ca2+ channel activity decreases at low intracellular pH
(Kaibara and Kameyama, 1988) and EGTA is deprotonated when it chelates metal cations, with consequent
acidification (Jong et al., 1993
). Therefore, one possible mechanism of fast inactivation was channel closure
due to acidification. Against this possibility we found that the fast phase of decay was present even when intracellular pH was buffered with 100 mM HEPES (data
not shown). We also compared rates of IBa decay in cells
dialyzed with 0.5, 1, 10, or 20 mM of EGTA. The rapidly
decaying component was present in all and its kinetics
changed only slightly (data not shown). These observations again rule out accumulation, as well as other possible mechanisms involving EGTA in the development
of fast current decay.
Fast Inactivation of IBa Is a Property of the 1 Subunit
To test whether fast inactivation depended on certain
auxiliary subunits or other properties of the expression
system, experiments were carried out with different
combinations of subunits 1C,
2a, and
2
, and with native rabbit ventricular myocytes. As illustrated in Fig. 7,
both phases of IBa decay were present in all cases, including cells transfected with
1 alone. The parameters of two-exponential fits and their averages are listed in
Table I.
Although cells co-transfected with 1C +
2a had much
higher current density than cells transfected with
1C
alone, the rates of inactivation were similar in both
groups, consistent with the previous observation that
2a, is the least potent among other cloned
subunits
in acceleration of inactivation (De Waard and Campbell, 1995
). Afast increased significantly with the number of subunits, being maximal for the native channels.
The kinetics of fast inactivation did not depend on the
composition of the expressed channels, being in all
cases about threefold slower than in native channels.
The rate of the slow component increased with the
number of subunits. This change was marginally significant between
1C and
1C/
2a/
2
cells (at a level P < 0.09) and not significant between
1C/
2a and
1C/
2a/
2
cells. (The histogram of slow time constants of the
1C/
2a/
2
cells was bimodal, with approximately one
quarter of the
1C/
2a/
2
cells centered at about the
same modal value as the
1C/
2a group. It is possible that a group of
1C/
2a/
2
cells expressed little of the
2
subunit or that the subunit did not incorporate stoi-chiometrically to the complex [Gurnett et al., 1996
].
Therefore, the
2
subunit may have had a greater
effect than that reflected by our averaged measurements.)
The presence of both decay phases with every combination of subunits indicates that both inactivation
mechanisms are determined by the 1C subunit. That
the relative amplitudes and kinetics of the two phases varied with the subunit composition is consistent with a modulatory role of the auxiliary subunits (Catterall, 1995
).
The present study was designed to compare inactivation of ionic and gating currents of L-type Ca2+ channels in the simplest possible situation. The tsA201 cells provided a system essentially devoid of other channels.
Use of Ba2+ as current carrier was expected to eliminate the current-dependent inactivation mechanism.
In spite of these conditions, inactivation of ionic current was found to follow two kinetic phases, of which only the slow one was co-temporal with the observed
single exponential reduction of gating charge. The fast
phase was current-dependent and did not correlate
with the reduction of the gating charge. Because the
two kinetic phases of inactivation of IBa were seen in
cells transfected with the 1C subunit, alone, in combination with
2a or with
2a and
2
, as well as in rabbit
ventricular myocytes, they must be a property of the
1
subunit, depending on neither the auxiliary subunits
nor the expression system. In this section we analyze the
present findings, compare them with published observations and infer that the fast phase of decay is caused
by Ba2+ binding to the channel's Ca2+-inactivation site.
The Population of Ca2+ Channels Was Homogeneous
Ba2+ currents through L-type Ca2+ channels have been
observed to decay in two phases in many different preparations. In neurons, two components of decay were
observed, but the significance of the observation was
obscured by the existence of multiple types of high voltage-activated Ca2+ channels (Boland and Digledine,
1990; Kay, 1991). In cardiac myocytes, the presence of
two components of inactivation of IBa was interpreted
as due to two different processes of voltage-dependent inactivation, because inactivation of IBa was believed to
be purely voltage dependent (Kass and Sanguinetti,
1984
; Lee et al., 1985
; Boyett et al., 1994
). This view
could not be tested rigorously because in cardiac myocytes it is difficult to correlate inactivation of IBa with inactivation of gating currents, due to the contribution of
other channels to charge movement (Bean and Ríos,
1989
; Shirokov et al., 1992
). These problems may have
been circumvented in the present work, provided that
the Ca2+ channels expressed in a mammalian cell line
after transfection were a single population, homogeneous in its biophysical properties. This seems to have
been the case, for the three following reasons:
No sizable Ca2+ or Ba2+ currents were recorded in mock-transfected cells. In our experiments, amplitudes of endogenous currents (<0.5 A/F) were always one or two orders of magnitude smaller than those in transfected cells. Therefore, the bulk of the Ba2+ or Ca2+ current in transfected tsA201 cells was probably conducted through recombinant channels, and endogenous currents could not have been the reason for the multiplicity of inactivation phases.
In agreement with Kamp et al. (1996) and Josephson
and Varadi (1996)
, we found a consistent correlation
between the amount of intramembranous mobile
charge and the ionic current density, suggesting that a
major fraction of expressed channels function to carry
current.
When rundown reduced ion current amplitude, the relative magnitudes of the two inactivation phases were conserved. Had the processes taken place in two different sets of channels, their proportions would not have been likely to remain constant during rundown.
Assuming that the population of channels was homogeneous, the question that then arises is if the kinetic phases of inactivation correspond to two different mechanisms.
Ba2+-dependent Inactivation Caused the Fast Decay of Current
The fast decay of current depended on the current through the channel, rather than the applied voltage. This was demonstrated by three observations:
In experiments with a double pulse protocol, current
availability depended on the voltage of the conditioning pulse in a U-shaped manner, and inactivation reached
a maximum at voltages that made inward current greatest, much as has been described for Ca2+-dependent inactivation (Eckert and Chad, 1984). In the present experiments the observation was free from many errors
that may have obscured this property in previous studies with native cardiomyocytes. Although a U-shaped
voltage dependence is expected in current-dependent processes, it could also arise from (voltage-dependent)
potentiation, that offsets the inactivation effect at high
voltages (Lee, 1987
). Voltage-dependent potentiation
cannot explain the present observations because current elicited by the test after a high voltage conditioning pulse was never greater than reference, and its kinetics remained the same. Our failure to observe voltage-dependent potentiation agrees with the report of
Cens et al. (1996)
that
2a failed to promote voltage-dependent potentiation, at variance with the effects of
other types of
subunit.
Recovery of IBa from inactivation was slower after a
conditioning to +20 mV than to +60 mV (data not
shown). Much more Ba2+ enters the cell during a pulse
to +20 than at +60 mV. The longer lasting inactivation
induced at +20 mV may simply be a consequence of
the persistence of inactivating concentrations of Ba2+
near the channels for a longer time (as argued by Kay,
1991, for Ca2+ currents in neurons).
There was no immobilization or voltage shift of gating current with kinetics corresponding to those of the fast decay of ionic current. In the current view, inactivation of Na+ and K+ channels acquires its voltage dependence indirectly, through a link to channel opening. In Na+ and K+ channels inactivation is more likely to happen when gating particles are in the activating position. In a microscopically reversible system, this implies that inactivation stabilizes the voltage sensor in its activating position, negatively shifting the transition voltage of gating currents and reducing the availability of charge mobile in the range of voltages that normally cause activation. This was not found in our case for the fast component of inactivation of IBa.
The observed decay of IBa could not have been due to bulk accumulation or cell loading by the permeating Ba2+, as the effect failed to correlate with the geometry of the cell (Fig. 6 A), and Ba2+ entry during long-lasting depolarization failed to change the reversal potential of the current (Fig. 6 B). This conclusion is also consistent with the finding (Cavalié et al., 1983) that the amplitude of single channel current remains the same during prolonged depolarization. The decay can therefore be attributed to an inactivation gating process, dependent on the local increase of [Ba2+], at levels that do not significantly change the driving force for IBa.
The evidence strongly favors a mechanism that requires binding of Ba2+ to a site on the channel molecule. Most importantly, the fast decay of Ca2+ channel
current was absent when Na+ carried the current, even
at high current densities. This fact is further evidence
against an accumulation phenomenon, which would
not be expected to work solely for Ba2+. Furthermore,
because IBa in 1 cells exhibits the fast decay, the putative Ba2+ site has to be on the
1 polypeptide. That the
fast decay was observed in currents carried by Ba2+, by
Ca2+, and by Na+ in the presence of extracellular Ca2+,
suggests that Ba2+ causes inactivation by binding to the
Ca2+-inactivation site. That fast inactivation of Ba2+ current occurred without correlative changes in gating
current is consistent with this hypothesis, considering
that the voltage-dependent and the Ca2+-dependent
mechanisms of inactivation do not interact (Hadley and Lederer, 1991
; Shirokov et al., 1993
).
Previous work has failed to clarify whether or not IBa
inactivates by an ion-dependent mechanism. McDonald et al. (1986) did not find U-shaped voltage dependence for inactivation of Ba2+ currents in on-cell
patches of guinea pig ventricular myocytes. On the
same preparation, Imredy and Yue (1994)
saw some reduction in the extent of decay of single channel IBa at
voltages above +20 mV, and tentatively interpreted it as
evidence of Ba2+-dependent inactivation. Mazzanti et
al. (1991)
found fast decay of Ba2+ current provided
that there were multiple channels in a patch, and proposed a Ba2+-dependent mechanism of inactivation enhanced by channel clustering. The interpretation of
single channel on-cell measurements is complicated by
the possibility of inactivation by Ca2+ released from intracellular stores (Galli et al., 1994
; Sham et al., 1995
).
Schneider et al. (1991)
found that entry of Ba2+ into
smooth muscle cells through ATP-activated channels
reduced whole-cell L-type channel Ba2+ currents by
about 20%. Perfusion of rabbit portal vein smooth muscle cells with 100 µM of Ba2+ in the patch pipette led to
a 10% reduction of ICa (Ohya et al., 1987
). The results
are consistent with a finding of Brown et al. (1981)
that
perfusion of Helix aspersa neurons with 1 mM Ba2+ in
the pipette reduced IBa by some 20%. In sum, the evidence with single channels is inconclusive, while in
whole cells there are modest effects of perfusion with
Ba2+. These results do not rule out inactivation by the
high concentrations of Ba2+ that are likely to occur at
the putative site, which is presumably located near the
channel mouth (De León et al., 1995).
The main characteristics of Ca2+-dependent inactivation of ICa were previously reproduced with a simple
three-gate model of the L-type channel (Shirokov et al.,
1993). We now show that this model also accounts for
the properties of IBa, assuming that Ba2+ substitutes for
Ca2+ at the inactivation site. The model includes voltage-dependent properties, activation, and inactivation,
which are independent of cation binding, and are represented in SCHEME I:kRI,RPkRP,RIRIRPAIAPkAP,AIkAI,APkAP,RPkRP,AP(SCHEME I)kAI,RIkRI,AI
Activation is the opening of a voltage-operated gate,
whose states are resting (R) and activated (A). Voltage-dependent inactivation involves a second gate, with a
permissive (P) and an inactivated state (I). The P I
transition is not intrinsically voltage-dependent, but is
favored from channel state AP.1
Ion-dependent inactivation is pictured by a third gate, the states of which are represented in SCHEME II:
kC,CMekCMe,CC + Me2+C:Me2+U + Me2+U:Me2+kUMe,UkU,UMekUme, CMekCMe, UMe(SCHEME II)ku,ckc,u
The ion-controlled gate can be "covering" (C, channel
closed) or "uncovering" (U). Me2+, normally Ca2+, inactivates the channel because its binding stabilizes state C, that is, the equilibrium between C:Me2+ and U:Me2+
is shifted toward C:Me2+. Accordingly, and to satisfy microscopic reversibility, the affinity of the site must be
greater in state C than in state U. The channel state is
described by the state of its three gates and is open only
in state APU.2 The ion-dependent inactivation process
does not affect the voltage-dependent processes in any
way (implying that the evolution of states depicted in
SCHEME I can be solved independently), while the states
in SCHEME II are influenced by the voltage-dependent
gates only because the concentration of the current-carrying ion changes when the channel opens. [Me2+]
at the site was assumed to be an instantaneous function
of the channel current i, defined as: [Me2+]bulk + i/
4FDr, where [Me2+]bulk is 50 nM, F is the faraday, the
diffusion constant D is 10
6 cm2s
1, and the distance r
from the channel mouth to the binding site is 1 nm.
In this model, dissociation constants (Kd) for Ca2+ are supposed to be 1 µM in state C and 100 µM in state U. IBa was simulated assuming 100-fold higher Kds in both states (which was achieved by reducing the binding rate constants). The only other difference between the two ions was a two-times greater single channel current for Ba2+ at any given extracellular [Me2+].3
The simulations4 are illustrated in Fig. 8, where current records, exponential fits, and fit parameters are
shown in a manner similar to Fig. 1. With this choice of
parameters the model reproduced well the main properties of IBa, including: the presence of two kinetic
phases of inactivation, the voltage dependence of slow, the amplitudes of all components, and the fact that
fast
was steeply voltage-dependent for ICa (Fig. 8 A) but not
IBa (Fig. 8 B).
Voltage-dependent Inactivation Underlies the Slow Phase of Current Decay
The present results include a reduction in the amount
of charge available to move above 60 mV, upon prolonged depolarization. This reduction, which depends
exponentially on duration of the depolarizing pulse, is
cotemporal with the slow phase of current decay, regardless of the ion that carries the current. This implies that the slow phase of decay of IBa is the result of voltage-dependent inactivation. The concomitant reduction in the availability of gating charge could be the
consequence of two not mutually exclusive mechanisms: charge immobilization associated with inactivation (Armstrong and Bezanilla, 1977
), or a negative
shift in the voltage distribution of intramembrane mobile charge, similar to that accompanying slow inactivation of squid Na+ channels (Bezanilla et al., 1982
), inactivation of the voltage sensor of skeletal muscle (Brum
et al., 1988
), and inactivation of L-type channels in
guinea pig cardiomyocytes (Shirokov et al., 1992
) and
embryonic chick ventricular myocytes (Josephson, 1996
).
Implications for Other Ca2+ Channel Studies
The present results define two kinetic components of decay of Ba2+ currents through Ca2+ channels, show that the slow phase, with characteristic times of seconds, is caused by voltage-dependent inactivation, and suggest binding of Ba2+ as probable cause for the fast phase. These findings call into question the widespread use of Ba2+ currents as tools to quantify purely voltage- dependent processes in Ca2+ channels. By showing the close association between gating current inactivation and a decay of current that takes place in seconds, it forces reconsideration of studies that attribute decay phases of hundreds of ms to voltage-dependent processes.
For example, Zong et al. (1994), in their study of L-type
Ca2+ channels in HEK 293 cells, used the difference between the "slow" time constant (~200 ms) in a two-
exponential fit to ICa elicited by 400-ms pulses, and the
time constant in a single exponential fit to IBa (~300
ms) to argue that Ca2+ may make voltage-dependent inactivation faster. Clearly the time constants were too
short (and so were the applied pulses) to significantly explore the voltage-dependent process, which we now
show to be much slower.
Zhang et al. (1994) analyzed the kinetics of IBa
through chimeric channels in Xenopus oocytes to localize determinants of voltage-dependent inactivation. In
this study, an
1A (a putatively P/Q-type channel from
brain) with relatively slow rate of IBa decay was chimerized with the rapidly inactivating
1E (doe-1, marine
ray). The work unquestionably localized the determinants of subunit-specific differences in decay of IBa to
segment IS6 and its vicinity. Because the inactivation of
IBa in doe-1 is believed to be exclusively voltage dependent (Ellinor et al., 1993
), the rates of decay found in
the rapidly inactivating chimeras, 10-30 s
1, were naturally attributed to voltage-dependent inactivation. However, P-type channels are susceptible to Ca2+-dependent inactivation (Tareilus et al., 1994
), and therefore the assertion that fast inactivation in the chimeras is
voltage-dependent should be supported with the use of
longer pulses or other tests.
Possible involvement of the first domain in determining the rate of IBa decay was also demonstrated by Parent et al. (1995) working with a chimera of
1C and
1S.
Even though the demonstration was clear, the conclusion that the first repeat affects voltage-dependent inactivation can be criticized on similar grounds, because it relies on the assumption that IBa inactivation is exclusively voltage-dependent.
In conclusion, the identification of structural determinants of voltage-dependent inactivation in Ca2+ channels can not be based solely on the analysis of IBa, since the ability to induce inactivation of the L-type Ca2+ channel appears not to be a unique property of Ca2+ ions. The main consequence of the present work is to require careful reconsideration of the mechanisms attributed to inactivation in previous and future studies.
Original version received 30 October 1996 and accepted version received 16 January 1997.
Address correspondence to Roman Shirokov, Department of Molecular Biophysics and Physiology, Rush University School of Medicine, 1750 W. Harrison St., Suite 1279JS, Chicago, IL 60612. Fax: 312-942-8711; E-mail: rshiroko{at}rush.edu
The tsA201 cell lines were a gift of Dr. M.M. Hosey (Northwestern University, Chicago, IL). The L-type Ca2+ channels cDNA sequences were gifts of Drs. E. Perez-Reyes (Loyola University, Maywood, IL) and M.M. Hosey. We thank Andy Chien, Tipu Puri and M.M. Hosey (Northwestern) for supplying the rabbit cardiomyocytes.This work was supported in part by National Institutes of Health Grant AR 43113 and a Grant-in-Aid from the American Heart Association (to E. Ríos) and by grants from the American Heart Association of Metropolitan Chicago (to R. Shirokov).