FPL-64176 modifies pore properties of L-type Ca2+
channels
Jing-Song
Fan1,
Yuhui
Yuan1, and
Philip
Palade1,2
Departments of 1 Physiology and Biophysics and
2 Pharmacology and Toxicology, University of Texas Medical
Branch, Galveston, Texas 77555-0641
 |
ABSTRACT |
In addition to its known effects on Ca2+
and Ba2+ currents, the L-type Ca2+ channel
agonist FPL-64176 was found to affect channel function in isolated rat
ventricular myocytes in the absence of Ca2+, with other
ions as current carriers through the channel. FPL-64176 induced
Cd2+ current through the L-type Ca2+
channel, suggesting that certain selectivity properties had changed, perhaps indicative of a small change in pore structure. FPL-64176 slightly but significantly decreased the effectiveness of
Co2+ as a blocker of the channel. FPL-64176 also increased
conductance through single L-type Ca2+ channels recorded in
the cell-attached configuration, from 71.9 ± 11.6 to 94.1 ± 8.3 pS, with Na+ carrying the current at pH 9.0. At present
it is uncertain whether FPL-64176 produces small alterations of a sole
open state of the channel or whether it increases the prevalence of a
second, higher conductance open state. These changes, particularly the
conversion of Cd2+ from a pure blocker to a permeant ion,
may be of eventual help in discriminating among different models for
Ca2+ channel selectivity.
cardiac myocytes; Cd2+; Co2+; conductance; selectivity
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INTRODUCTION |
THE CALCIUM CHANNEL
AGONIST FPL-64176 has been used by several different groups in
studies of cardiac excitation-contraction coupling because it reduces
the rate of decay of L-type Ca2+ currents and enables
identification of a separate adaptation or inactivation process that
turns off Ca2+ release mediated by ryanodine receptors
(28, 30). In the process of determining its effects on
Ca2+ channel gating currents and the Ca2+
dependence of its actions on inactivation, we examined the effects of
FPL-64176 on the channel with other ions carrying the current.
Most studies of FPL-64176 have concentrated on alterations in the time
course of macroscopic or single-channel currents. By comparison, far
less has been reported of effects on pore properties of the channel.
One report of the effect on single L-type channels in failing human
heart suggested that single-channel conductance might be increased
(11). In the process of studying the effects of FPL-64176
on L-type gating currents (8), we determined that Cd2+, used by others for gating current measurements, is
capable of carrying current through the channel in the presence of
FPL-64176. Some of these results have been reported previously in
abstract form (7).
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MATERIALS AND METHODS |
Myocyte dissociation.
Rat ventricular myocytes were prepared from 200- to 300-g male
Sprague-Dawley rats by dissociation with collagenase (Yakult Pharmaceuticals, Tokyo, Japan) as previously described (5, 6,
32). Myocytes were stored at 4°C in a high-potassium, low-sodium, Kraftbrühe-like solution (33) until use.
Measurement of current through L-type
Ca2+ channels.
Patch-clamp (List EPC-7) measurements were performed with a
perforated-patch version of the whole cell recording configuration with
the use of 50 µM
-escin in the pipette solution (5)
to circumvent Ca2+ channel rundown. The compositions of
experimental external solutions utilized in this study are
given in Tables 1 and
2. The internal (pipette)
solution consisted of 120 mM Cs-aspartate supplemented with 20 mM CsCl,
3 mM Na2ATP, 3.5 mM MgCl2, 5 mM EGTA, and 5 mM HEPES, pH 7.3. Holding potential was
40 or
50 mV in most
experiments on selectivity. Leak and capacity compensation was
generally performed manually with the patch-clamp controls by using
10-mV hyperpolarizations from the holding potential. For the more
critical experiments shown in Figs. 3 and 4, leak and linear
capacitance corrections were carried out digitally by utilizing 25-mV
hyperpolarizing pulses from a holding potential of
100 mV. All
experiments were performed at room temperature. FPL-64176 and
S(
)-BAY K 8644 were obtained from RBI (Natick, MA).
Single-channel measurements were performed in the cell-attached
recording configuration by using a Dagan 3900 patch clamp. Normal
Tyrode solution contained 1 mM Ca2+, with Cs+
substituted for K+ for purposes of blocking inward
rectifier currents in most experiments. Under these conditions, the
resting potential of the cells approximated
65 mV. Two pipette
solutions were used. The first consisted of Ca2+- and
Cs+-free Tyrode solution with pH adjusted to 9.0 (12). The second consisted of 70 mM BaCl2 and
110 mM sucrose, pH 7.4 (3, 11). FPL-64176 was added to the
bath solution in all cases. All experiments were performed at room
temperature with filtering at 1 kHz and sampling every 0.5 ms. Analysis
of records was performed by using pCLAMP 6, with cursors set at 50% of
the full current openings for kinetic analysis.
Statistical analysis.
Statistical significance was assigned at the P < 0.05 level, using the unpaired Student t-test.
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RESULTS |
The effects of FPL-64176 are apparent with other ions carrying
current through the channel. In some cases the effects of the standard
1 µM FPL-64176 employed in L-type Ca2+ current studies
(8) were so marked that lower FPL-64176
concentrations needed to be employed to avoid inducing unacceptably
large increases in holding current. As shown in Fig.
1 and Table
3, Sr2+, Ba2+,
and Na+ currents through the L-type channel all were
affected by FPL-64176. The traces in Fig. 1 reveal that channel
activation continued for a longer time than normal, resulting in less
decay of the current during the pulse. Furthermore, tail currents were
enhanced, and a portion of the tail exhibited a very slow decay. This
finding is reflected in the analysis provided in Table 3, where it is shown that the vast majority of the tail current declined with a fast
time constant of ~2 ms in control records in each ion, but both fast
and slow time constants were significantly increased in the presence of
FPL-64176, and the proportion of the tail exhibiting the slow time
constant, now in excess of 100 ms, was also increased.

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Fig. 1.
FPL-64176 affects the channel if Sr2+,
Ba2+, or Na+ carries current through the L-type
channel instead of Ca2+. Top: currents carried
by 1 mM Sr2+ substituted for Ca2+ in the
extracellular solution under control conditions (left) and
in the presence of 0.1 µM FPL-64176 (right).
Middle: currents carried by 1 mM Ba2+
substituted for Ca2+ in the external solution under control
conditions and in the presence of 0.1 µM FPL-64176.
Bottom: currents carried by Na+ in the
extracellular solution in the absence of Ca2+ (with 0.5 mM
EGTA added to nominally Ca2+-free external solution) under
control conditions and in the presence of 0.1 µM FPL-64176. All
currents were measured in response to stimulations to 0 mV from a
holding potential of 40 mV. Results are representative of 3 experiments each with Ba2+ or Sr2+ as current
carrier and 2 experiments with Na+ as current carrier.
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Peak currents in Ba2+ remained slightly larger than those
in Ca2+ solutions, as evidenced by the traces shown in Fig.
2, left. However, the
difference is not statistically significant. The current-voltage
relationships for currents carried by the two charge carriers were
different in the presence of FPL-64176 (Fig. 2,
right). The shifts observed in the presence of
Ba2+ were accompanied by a small but significant increase
in inward holding current, and the reversal potential for current flow
through the channel was shifted in the negative direction, consistent with a possible buildup of intracellular Ba2+. With these
observations taken into consideration, there do not appear to be major
changes in the relative permeabilities of Ba2+ and
Ca2+ through the channel.

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Fig. 2.
Ba2+ continues to pass slightly more current than
Ca2+ through FPL-64176-modified channels. Left:
traces of currents carried by 1 mM Ca2+ (top)
and 1 mM Ba2+ (bottom) in response to
depolarizations from 30 to +80 mV from a holding potential of 40 mV
are shown in ventricular myocytes exposed to 0.1 µM FPL-64176.
Right: current-voltage plots of the relationships in 4 such
FPL-64176-treated cells each successively in 1 mM Ca2+ and
1 mM Ba2+ solutions. Vt, test potential.
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FPL-64176 renders Cd2+ permeable
through the channel.
Unlike the highly permeant ions Sr2+, Ba2+, and
Na+, Co2+ and Cd2+ are normally
impermeant through the channel. As shown in Fig.
3, the combinations of either
Co2+ and triethanolamine (Tri) or Co2+ and
tetraethylammonium (TEA) yielded no increase in current in the presence
of FPL-64176. TEA solution without divalents also failed to pass inward
tail current in the presence of FPL-64176 (not shown). Thus neither
Co2+, Tri, nor TEA is permeant in the presence of
FPL-64176. In contrast, Cd2+/Tri and Cd2+/TEA
carried inward current through the channel. The effects were most
apparent during the tails that followed repolarization. The enhanced
tail currents suggest that Cd2+ is permeant through the
FPL-64176-modified channel.

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Fig. 3.
Altered channel selectivity in the presence of FPL-64176.
Tail currents in response to repolarization from +10 to 50 mV are
shown in the presence of only normally impermeant cations in the
extracellular solution. Top to bottom: Co2+ + triethanolamine (Tri) (n = 3); Co2+ + tetraethylammonium (TEA) (n = 4);
Cd2+ + Tri (n = 3); and
Cd2+ + TEA (n = 3). Numbers in
parentheses indicate no. of representative experiments performed.
Left: control conditions; right: corresponding
records in the same solutions in the presence of 1 µM FPL-64176. Note
that there was no FPL-64176-induced increase in inward current in
Co2+-containing solutions, but current clearly increased in
solutions containing Cd2+.
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Further Cd2+ tail current measurements are shown in Fig.
4, left, where the pulse
protocol involved pulses to different potentials, followed by a return
to the
50-mV holding potential. Under control conditions, essentially
only gating currents were observed. In the presence of FPL-64176,
significant inward current was observed during the pulse, but much
larger tail currents were observed during repolarization. In Fig. 4,
right, peak inward tail currents are plotted, with
approximate compensation for gating currents achieved by subtraction of
the corresponding "on" gating current from the tail. In the absence
of FPL-64176, entry of Cd2+ was clearly insignificant,
whereas in the presence of FPL-64176, the current reached very large
amplitudes. As with Ca2+ current in the presence of
FPL-64176 when the same protocol was used (not shown), Cd2+
tail currents continued to increase as the prepulse potential was made
quite positive.

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Fig. 4.
Cd2+ tail current in response to
depolarizations to different potentials. Left: current
traces in Cd2+ solution in response to depolarizations from
30 to +40 mV from a holding potential of 50 mV under control
conditions (top) and in the presence of 1 µM FPL-64176
(bottom). Right: current-voltage plot of
instantaneous tail current (Itail) elicited on
repolarization to 50 mV, with "on" gating current amplitudes
subtracted from the "off" tail responses. Leak and capacity
compensation was performed by subtracting scaled responses to 25-mV
hyperpolarizations from a holding potential of 100 mV. Results are
pooled from 3 cells in Cd2+-Tri solution and 3 cells in
Cd2+-TEA solution (for compositions, see Table 1) before
and after treatment with FPL-64176. *P < 0.05.
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Additional experiments were conducted to examine possible channel
rectification of Cd2+ current in the presence of FPL-64176.
A prepulse to near the normal Ca2+ equilibrium potential
was applied for a period long enough to open most channels.
Subsequently, the membrane was repolarized to different potentials to
elicit Ca2+, Cd2+, or Co2+ inward
currents. Traces in the presence of Co2+ were then
subtracted from those in the presence of Ca2+ or
Cd2+, resulting in the families of traces shown in Fig.
5, left. Currents carried by 3 mM Cd2+ were half the size of those observed in the
presence of 1 mM Ca2+, in the presence of FPL-64176. In
Fig. 5, top right, it may be seen that there was no
equivalent detectable Cd2+ current in the absence of
FPL-64176. There is rectification of the curve in the presence of
Ca2+ because the tail currents became very brief and
attenuated at potentials below
20 mV. In contrast, in the
presence of FPL-64176, this attenuation was shifted to more negative
potentials (not shown) such that the driving force increases
nearly linearly with voltage over the range from +20 to
40 mV, for both Cd2+ and Ca2+ (Fig. 5,
bottom right).

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Fig. 5.
Cd2+ current through the channel in the
presence (+) of FPL-64176 is significant even compared with
Ca2+ current. Left: current traces in response
to a strong depolarization to +80 mV, followed by repolarization to
different potentials from +70 (smallest tails) to 40 mV (largest,
most swiftly declining tails). Individual cells were exposed to
Ca2+/TEA (top) or Cd2+/TEA solution
(bottom), followed by Co2+/TEA solution. Traces
in Co2+/TEA were subtracted from traces in other solutions
to provide as much leak and capacity compensation as possible.
Top right: current voltage plots of traces under control
conditions, in the absence ( ) of FPL-64176, demonstrating an absence
of Cd2+ tail current (n = 3). Bottom
right: current-voltage plot of traces in the presence of 1 µM
FPL-64176 (n = 4). Vr, repolarization potential.
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FPL-64176 reduced the effectiveness of
Co2+ as a blocker.
Figure 6 demonstrates that the efficacy
of Co2+ as a blocker was reduced in the presence of
FPL-64176. In this case, Ca2+ was the current carrier. In
these experiments, progressively higher concentrations of
Co2+ were added to the external solution.
As shown in Fig. 6, top, there was generally even a small
increase in current (run-up) at the beginning of the experiments,
perhaps because of slow enhancement caused by the FPL added earlier. At
higher concentrations, blockers always inhibited the current, and the
effects were quite reversible. Plots in Fig. 6, bottom, show
the results from several such experiments with Co2+.

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Fig. 6.
Co2+ is a less effective blocker in the
presence of FPL-64176. Top: Ca2+ currents
(ICa) in the presence of 1 µM FPL-64176 were
progressively blocked by increasing concentrations of Co2+
added cumulatively. Bottom: block by Co2+ of
normalized ICa elicited under control conditions
(n = 5) or in the presence of 1 µM FPL-64176
(n = 4), with Co2+ added cumulatively to
the bathing solution. *P < 0.05 compared with
control.
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FPL-64176 affects single-channel conductance.
We also wanted to determine whether single-channel conductance
was increased by FPL-64176. In the presence of 1 µM FPL-64176 in 70 mM BaCl2 and 110 mM sucrose, three cells gave values of 28.2, 23.2, and 27.1 pS (not shown). We encountered difficulty in
identifying Ca2+ channel openings and measuring current
amplitudes in the absence of drug, but conductance in the presence of
drug was not significantly different from that reported in rat
ventricular myocytes in the same solution without FPL-64176
(27.7 ± 0.7 pS; Ref. 3).
To pursue this matter further, we employed other solutions. Recordings
of single L-type Ca2+ channel activity in the absence of
Ca2+ channel agonist were facilitated by experimental
conditions that yielded large-amplitude currents (monovalent
Na+ passing through the channel at pH 9.0; Refs.
21-23). A typical determination is shown in Fig.
7, and all determinations included data
points at four or more potentials. In the absence of agonist, the
conductance of the channel with current carried by Na+ at
pH 9.0 was 77.4 ± 18.2 pS (n = 8). In the
presence of 1 µM FPL-64176, openings were resolved over a wider range
of potentials, including responses obtained on repolarization to the
resting potential. The conductance measured under the same conditions in the presence of FPL-64176 was 94.1 ± 8.3 pS (n = 9, P = 0.025 vs. control; i.e., significantly
different statistically). We found only occasional long openings in the
absence of FPL-64176. Because short openings in the controls might have
resulted in underestimates of the true conductance, we were uncertain
whether to utilize long opening data under control conditions. The
above analysis included two such long openings. When these two long openings were discarded from the analysis, the control conductance was
decreased, the data became less variable (73.3 ± 12.2 pS, n = 8), and the statistical significance of the
differences between control and FPL-64176 data improved to
P = 0.00086.

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Fig. 7.
Single-channel activity due to L-type Ca2+
channels and its modification by FPL-64176 in Na+ solution,
pH 9.0. Top: single sweeps of activity are shown for
depolarizations of 10 and 20 mV from the estimated resting potential of
65 mV under control conditions (left) and in the presence
of 1 µM FPL-64176 (right). Note the increased open
probability both during and after the 20-mV stimulus. Records were
obtained in control Na+ solution, pH 9.0, from a patch with
2 channels present. In certain cases the background subtraction of leak
and capacity was imperfect. All channel openings are downward.
Bottom: FPL-64176 increased the single-channel conductance
( ) of L-type Ca2+ channels. Records such as those shown
at top were used to determine the single-channel
conductance. Potentials indicate excursions from the resting
potential.
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 |
DISCUSSION |
Our characterization of effects of FPL-64176 on the pore
properties of the L-type Ca2+ channel includes changes in
which ions can pass through the channel (a measure of selectivity),
single-channel conductance, and block by ions thought to interact with
binding sites in the pore.
Selectivity.
FPL-64176 exerted its characteristic effects with ions other than
Ca2+ carrying the current. Not only were the currents
carried by divalents like Ba2+ and Sr2+
affected, but so were currents carried by Na+. In our
limited experiments with these ions carrying the current, it did not
appear that currents carried by one of these ions were increased much
more than currents carried by the others. Thus, to a first
approximation, FPL-64176 did not seem to affect the ability of the
channel to discriminate among the principal known current carriers
through the channel.
Because FPL-64176 exerted more dramatic effects on amplitude of
Cd2+ currents compared with those carried by
Ca2+ or Ba2+, effects of the drug on currents
carried by other divalents were also examined (with TEA as the other
extracellular cation). Zn2+ was found to carry marginal
current through the channel in the absence of drug and much greater
current in the presence of drug (n = 4, not shown). In
contrast, Mg2+ (n = 2), Ni2+
(n = 3), and Be2+ (n = 2)
carried no current through the channel in either the absence of drug or
the presence of FPL-64176 (not shown). While most permeant divalents
have larger crystal radii than blockers like Cd2+, the
ability of the even smaller Mn2+ (1, 20) and
Zn2+ ions to pass through the channel normally suggests
that crystal radius is not an important determinant of selectivity.
Similar arguments suggest that hydrated ion size also is not a
determinant of selectivity and that FPL-64176 is not simply affecting
the diameter of the pore. We speculate instead that the affinity of Cd2+ for its high-affinity site in the pore
(29) could be reduced to the point that it becomes more
susceptible to being knocked through the channel by the presence of a
second Cd2+ ion in the pore (as for Ca2+ in
Refs. 13 and 18). Alternatively, either its high-affinity site or the postulated innermost lower affinity site in the "step" model of Dang and McCleskey (4) could be altered. In
principle, speculation about alteration of the higher affinity site
could be tested by performing equivalent Cd2+ block
experiments, as performed with Co2+ and shown in Fig. 6.
Unfortunately, Cd2+ block was much slower, progressive, and
poorly reversible than Co2+ block (not shown), and this
rendered equivalent assessment of IC50 for Cd2+ unfeasible.
These experiments also indicated that FPL-64176 did not require
Ca2+-bound states of the channel to exert its effects.
Replacement of Ca2+ in our solutions would have diminished
its influence, and while Sr2+ has been reported to be
capable of releasing Ca2+ from the sarcoplasmic reticulum
(25), Ba2+ and Na+ would not
(19). The finding that similar FPL-64176-induced kinetic
changes occurred with other ions substituting for Ca2+ as a
current carrier argues against an obligatory role for Ca2+
in mediating the effects of FPL-64176. Consequently, even
though certain models of the L-type channel include a
Ca2+-dependent inactivation or Ca2+-bound mode
of the channel (14), such states must not be required for
FPL-64176 to exert its effects.
The observation that Cd2+ passes through FPL-64176-modified
channels but not through untreated channels was unexpected and is entirely novel. Although this occurrence initially hindered experiments designed to measure the effects of the drug on L-type Ca2+
channel gating currents, it demonstrates that the pore properties of
the channel are changed by the drug. Either the pore region of
the channel is made slightly wider or the affinity of Cd2+
to a binding site within the pore is affected. Of these two
possibilities, we favor the latter because TEA, a marginal current
carrier through skeletal L-type channels (18), remained
impermeant even in the absence of divalent blockers (not shown).
Cd2+ is a well-known L-type channel blocker whose effects
are voltage dependent (16), indicating that it binds to a
site within the permeation pathway. Significantly, Cd2+ has
been reported to be permeable through insect muscle Ca2+
channels (9). In addition, all divalent current carriers
through this channel are also known to block movement of monovalents
through the channel (13). Thus the conversion of
Cd2+ from blocker to current carrier should not be so
surprising. Other drugs have been reported to induce changes in
selectivity of other cardiac channels (26).
Because most experiments assessing effects of BAY K 8644 on mammalian
myocyte Ca2+ channel gating currents have been carried out
with Cd2+ present (10), we also tested whether
BAY K 8644 caused an increase in current carried by Cd2+.
No increases in tail currents were noted when Co2+ was
present (n = 8, not shown). However, inward currents
were clearly apparent during the pulse, and tails were slowed in
Cd2+/Tri (n = 2, not shown) and slowed and
increased in Cd2+/TEA (n = 3, not shown).
Thus it appears that BAY K 8644 may increase channel permeability to
Cd2+, although not as much as FPL-64176.
Conductance.
Another potential pore property is the single-channel conductance.
Handrock et al. (11) recently reported that FPL-64176 increased single-channel conductance with Ba2+ as a charge
carrier in human ventricle. We tested for an increase with
Na+ as the charge carrier instead.
Large-amplitude currents carried by Na+ through L-type
Ca2+ channels were first reported by the late Peter Hess
and colleagues (12). Still larger amplitude events were
obtained with alkaline pH patch-pipette solutions
(21-23). Na+ also normally carries
current through sodium channels, and it can also carry current through
T-type channels in cardiac myocytes in the absence of divalents.
Nevertheless, Na+ single-channel conductance is much lower
(2) than that shown here, even at alkaline pH (31,
34), and channel activity dies off much faster (27)
than during the observations made here. T-type Ca2+
currents are much smaller than L-type currents in adult myocytes, and
single-channel conductances in T-type channels conducting Na+ are much lower than those of L-type channels, even at
alkaline pH (15). Thus we can be confident that our
recordings were from L-type Ca2+ channels.
The values we obtained under control conditions are generally less than
those obtained by Hess and coworkers (12) using another
Ca2+ channel agonist, BAY K 8644 (85 pS) (12),
but our recordings in the presence of FPL-64176 are generally greater
than those. Recordings in similar solutions with another agonist,
(+)-S-202-791, appear very similar (22) or were not
quantified in terms of single-channel conductance (23,
24). The increase in single-channel conductance that we observed
with FPL-64176 with Na+ as a charge carrier is nevertheless
in agreement with the increase from 16.6 ± 1.2 to 23.7 ± 2.8 pS observed in human ventricular myocytes in Ba2+
solution (11).
Three possibilities could be contemplated to explain the modest
increase in single-channel conductance that we observed in the presence
of FPL-64176. The first possibility is that short openings in our
control records caused us to selectively underestimate the true
single-channel conductance in the absence of drug. We regard this as
relatively unlikely because we always selected the longest openings for
these determinations, and the histograms from these experiments
indicate mean openings of ~2 ms in the absence of drug with 1-kHz
filtering (8). The second possibility is that the two
gating modes observed in the absence of drug (12, 21)
actually have two different single-channel conductances and that
FPL-64176 enhances mode 2-type gating. An argument in favor of such an
interpretation is that conductance measurements that included mode
2-like long openings on repolarization appeared to yield higher
conductance estimates than those that excluded such openings. An
argument against such an interpretation is that Hess et al.
(12) observed no difference in unitary current between control and BAY K 8644-treated patches. Furthermore, they found no
difference in amplitudes between mode 1 and mode 2 openings. The third
possibility is that FPL-64176 causes the channel to be modified into a
higher conductance state not found under control conditions. An
argument in favor of this interpretation is the finding that
Cd2+ was rendered permeant by FPL-64176.
Modification of channel block.
The block of the permeation pathway by Cd2+ was affected in
a striking fashion, converting it from a blocker of the channel to a
current carrier. While this conversion might be unique to Cd2+, the blocking action of Co2+ was also
affected by FPL-64176, albeit in a more subtle fashion. In previous
work, we used 3-4 mM Co2+ to block Ca2+
movement through the channel to separate ionic current from
capacitative current (6). In the experiments reported
here, the effective IC50 for Co2+ was shifted
from 0.50 ± 0.26 to 1.62 ± 0.73 mM (P < 0.05) in the presence of FPL-64176.
Overall effects on pore properties.
In summary, FPL-64176 affects L-type Ca2+ channels even
with ions other than Ca2+ carrying the current. It renders
Cd2+ permeant and reduces the effectiveness of
Co2+ as a blocker of the channel pore. Finally, it either
favors an open state of the channel with higher conductance than the
normal principal open state or actually causes the channel to open to a
higher conductance state than normal. The modification of
Ca2+ channel selectivity by FPL-64176 might prove useful in
discriminating among different models for Ca2+ channel
permeation (17).
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ACKNOWLEDGEMENTS |
This work was supported by National Institutes of Health Grants
AR-41526 and AR-43200 (to P. Palade).
 |
FOOTNOTES |
Present address of Y. Yuan: Dept. of Cell Biology, Baylor College of
Medicine, Houston, TX 77030.
Address for reprint requests and other correspondence: P. Palade, Dept. of Physiology and Biophysics, Univ. of Texas Medical Branch, Galveston, TX 77555-0641 (E-mail:
ppalade{at}utmb.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 28 March 2000; accepted in final form 20 September 2000.
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REFERENCES |
1.
Almers, W,
and
Palade PT.
Slow calcium and potassium currents across frog muscle membrane: measurements with a Vaseline-gap technique.
J Physiol
312:
159-176,
1981[Abstract].
2.
Cachelin, AB,
De Peyer JE,
Kokubun S,
and
Reuter H.
Sodium channels in cultured cardiac cells.
J Physiol
340:
389-401,
1983[Abstract].
3.
Chen, L,
El-Sherif N,
and
Boutjdir M.
1-Adrenergic activation inhibits
-adrenergic-stimulated unitary Ca2+ currents in cardiac ventricular myocytes.
Circ Res
79:
184-193,
1996[Abstract/Free Full Text].
4.
Dang, TX,
and
McCleskey EW.
Ion channel selectivity through stepwise changes in binding affinity.
J Gen Physiol
111:
185-193,
1998[Abstract/Free Full Text].
5.
Fan, J-S,
and
Palade P.
Perforated patch recording with
-escin.
Pflügers Arch
436:
1021-1023,
1998[ISI][Medline].
6.
Fan, J-S,
and
Palade P.
One calcium ion may suffice to open the tetrameric ryanodine receptor in cardiac myocytes.
J Physiol
516:
769-780,
1999[Abstract/Free Full Text].
7.
Fan, J-S,
and
Palade P.
FPL 64176-induced modification of cardiac L-type calcium channels (Abstract).
Biophys J
76:
A406,
1999[ISI].
8.
Fan, J-S,
Yuan Y,
and
Palade P.
Kinetic effects of FPL 64176 on L-type Ca2+ channels in cardiac myocytes.
Naunyn Schmiedebergs Arch Pharmacol
361:
465-476,
2000[ISI][Medline].
9.
Fukuda, J,
and
Kawa K.
Permeation of manganese, cadmium, zinc, and beryllium through calcium channels of an insect muscle membrane.
Science
196:
309-311,
1977[ISI][Medline].
10.
Hadley, RW,
and
Lederer WJ.
Comparison of the effects of BAY K 8644 on cardiac Ca2+ current and Ca2+ channel gating current.
Am J Physiol Heart Circ Physiol
262:
H472-H477,
1992[Abstract/Free Full Text].
11.
Handrock, R,
Schroder F,
Hirt S,
Haverich A,
Mittmann C,
and
Herzig S.
Single-channel properties of L-type calcium channels from failing human ventricle.
Cardiovasc Res
37:
445-455,
1998[ISI][Medline].
12.
Hess, P,
Lansman JB,
and
Tsien RW.
Calcium channel selectivity for divalent and monovalent cations.
J Gen Physiol
88:
293-319,
1986[Abstract].
13.
Hess, P,
and
Tsien RW.
Mechanism of ion permeation through calcium channels.
Nature
309:
453-456,
1984[ISI][Medline].
14.
Imredy, JP,
and
Yue DT.
Mechanism of Ca2+-sensitive inactivation of L-type Ca2+ channels.
Neuron
12:
1301-1318,
1994[ISI][Medline].
15.
Kawano, S,
and
DeHaan RL.
Analysis of the T-type calcium channel in embryonic chick ventricular myocytes.
J Membr Biol
116:
9-17,
1990[ISI][Medline].
16.
Lansman, JB,
Hess P,
and
Tsien RW.
Blockade of current through single calcium channels by Cd2+, Mg2+, and Ca2+.
J Gen Physiol
88:
321-347,
1986[Abstract].
17.
McCleskey, EW.
Calcium channel permeation: a field in flux.
J Gen Physiol
113:
765-772,
1999[Free Full Text].
18.
McCleskey, EW,
and
Almers W.
The Ca channel in skeletal muscle is a large pore.
Proc Natl Acad Sci USA
82:
7149-7153,
1985[Abstract].
19.
Nabauer, M,
Callewaert G,
Cleemann L,
and
Morad M.
Regulation of calcium release is gated by calcium current, not gating charge, in cardiac myocytes.
Science
244:
800-803,
1989[ISI][Medline].
20.
Ochi, R.
Manganese-dependent propagated action potentials and their depression by electrical stimulation in guinea-pig myocardium perfused by sodium-free medium.
J Physiol
263:
139-156,
1976[ISI][Medline].
21.
Pietrobon, D,
and
Hess P.
Novel mechanism of voltage-dependent gating in L-type calcium channels.
Nature
346:
651-655,
1990[ISI][Medline].
22.
Pietrobon, D,
Prud'hom B,
and
Hess P.
Conformational change associated with ion permeation in L-type calcium channels.
Nature
333:
373-376,
1988[ISI][Medline].
23.
Pietrobon, D,
Prud'hom B,
and
Hess P.
Interaction of protons with single open L-type calcium channels.
J Gen Physiol
94:
1-21,
1989[Abstract].
24.
Prud'hom, B,
Pietrobon D,
and
Hess P.
Direct measurement of proton transfer rates to a group controlling the dihydropyridine-sensitive Ca2+ channel.
Nature
329:
243-246,
1987[ISI][Medline].
25.
Rousseau, E,
Pinkos J,
and
Savaria D.
Functional sensitivity of the native skeletal Ca2+ release channel to divalent cations and the Mg-ATP complex.
Can J Physiol Pharmacol
70:
394-402,
1992[ISI][Medline].
26.
Santana, LF,
Gomez AM,
and
Lederer WJ.
Ca2+ flux through promiscuous cardiac Na+ channels: slip-mode conductance.
Science
279:
1027-1033,
1998[Abstract/Free Full Text].
27.
Scanley, BE,
Hanck DA,
Chay T,
and
Fozzard HA.
Kinetic analysis of single sodium channels in canine cardiac Purkinje cells.
J Gen Physiol
95:
411-437,
1990[Abstract].
28.
Sham, JSK,
Song L-S,
Chen Y,
Deng L-H,
Stern MD,
Lakatta EG,
and
Cheng H.
Termination of Ca2+ release by a local inactivation of ryanodine receptors in cardiac myocytes.
Proc Natl Acad Sci USA
95:
15096-15101,
1998[Abstract/Free Full Text].
29.
Yang, J,
Ellinor PT,
Sather WA,
Zhang J-F,
and
Tsien RW.
Molecular determinants of Ca2+ selectivity and ion permeation in L-type Ca2+ channels.
Nature
366:
158-161,
1993[ISI][Medline].
30.
Yasui, K,
Palade P,
and
Gyorke S.
Negative control mechanism with features of adaptation controls Ca2+ release in cardiac myocytes.
Biophys J
67:
457-460,
1994[Abstract].
31.
Yatani, A,
Brown AM,
and
Akaike N.
Effect of extracellular pH on sodium current in isolated, single rat ventricular cells.
J Membr Biol
78:
163-168,
1990.
32.
Yazawa, K,
Kaibara M,
Ohara M,
and
Kameyama M.
An improved method for isolating cardiac myocytes useful for patch clamp studies.
Jpn J Pharmacol
40:
157-163,
1990.
33.
Zahradnik, I,
and
Palade P.
Multiple effects of caffeine on calcium currents in rat ventricular myocytes.
Pflügers Arch
424:
129-136,
1993[ISI][Medline].
34.
Zhang, J-F,
and
Siegelbaum SA.
Effects of external protons on single cardiac sodium channels from guinea pig ventricular myocytes.
J Gen Physiol
98:
1065-1083,
1991[Abstract].
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