Institute of Pharmacology and Toxicology, Consejo Superior de
Investigaciones Científicas, School of Medicine, Universidad
Complutense, Madrid, Spain
We have studied and compared the effects of bupivacaine with those
induced by a new local anesthetic, IQB-9302, on human cardiac K+ channels hKv1.5, Kv2.1, Kv4.3, and HERG.
Both drugs have a close chemical structure, only differing in their
N-substituent (n-butyl and cyclopropylmethyl, for
bupivacaine and IQB-9302, respectively). Both drugs blocked Kv2.1,
Kv4.3, and HERG channels similarly. Bupivacaine
inhibited these channels by 48.6 ± 3.4, 45.4 ± 12.4, and
43.1 ± 9.1%, respectively, and IQB-9302 by 48.1 ± 3.3, 36.1 ± 3.7, and 50.3 ± 6.6%, respectively. However,
bupivacaine was 2.5 times more potent than IQB-9302 to block hKv1.5
channels (EC50 = 8.9 ± 1.4 versus 21.5 ± 4.7 µM). Both drugs induced a time- and voltage-dependent block of
hKv1.5 and Kv2.1 channels. Block of Kv4.3 channels induced by either
drug was time- and voltage-dependent at membrane potentials coinciding
with the activation of the channels. IQB-9302 produced an instantaneous
block of Kv4.3 and hKv1.5 channels at the beginning of the depolarizing
pulse that can be interpreted as a drug interaction with a
nonconducting state. Bupivacaine and IQB-9302 induced a similar degree
of block of HERG channels and induced a steep
voltage-dependent decrease of the relative current. These results
suggest that 1) bupivacaine and IQB-9302 block the open state of
hKv1.5, Kv2.1, Kv4.3, and HERG channels; and 2) small
differences at the N-substituent of these drugs do not affect the
drug-induced block of Kv2.1, Kv4.3, or HERG, but specifically modify block of hKv1.5 channels.
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Introduction |
Local
anesthetics block the generation and conduction of nerve impulses by
inhibiting the current through voltage-gated Na+
channels in the membrane of nerve cells (Hille, 1977
; Hondeghem and
Katzung, 1977; Strichartz, 1987). Bupivacaine is a potent amide local
anesthetic widely used for long-lasting epidural anesthesia (Strichartz, 1987). However, bupivacaine-like local anesthetics are not
selective Na+ channel blockers. In fact, at the
same range of concentrations used in the clinical practice to block the
generation and propagation of nerve action potentials, this type of
local anesthetic also blocks K+ channels
(Valenzuela et al., 1995a,b; Lipka et al., 1998). Moreover, at higher
concentrations, they also block the L-type Ca2+
channels, thus decreasing cardiac contractility and conduction velocity
through the atrioventricular node (Strichartz, 1987; Sanchez-Chapula,
1988). Bupivacaine-induced block of K+ channels
has been considered the molecular mechanism by which this drug induces
a prolongation of the QTc interval of the ECG in anesthetized dogs
(Kasten and Martin, 1985; Wheeler et al., 1988) and human volunteers
receiving high doses of this anesthetic (Scott et al., 1989).
hKv1.5, Kv2.1, Kv4.3, and HERG channels are involved in the
repolarization of the human cardiac action potential (Wang et al.,
1993; Curran et al., 1995; Firek and Giles, 1995; Mays et al., 1995;
Sanguinetti et al., 1995; Van Wagoner et al., 1997; Kaab et al., 1998).
In fact, Kv1.5, Kv4.3, and HERG are considered to be the
cloned counterparts of the IKur,
ITO, and
IKr, respectively (Fedida et al.,
1993; Wang et al., 1993, 1999; Sanguinetti et al., 1995; Dixon et al.,
1996; Feng et al., 1997). Moreover, the presence of mRNA encoding the
expression of Kv2.1 channels as well as the Kv2.1 protein in human
atria has been demonstrated, although there is not a direct
relationship of this channel with a native potassium current
(Van Wagoner et al., 1997). The pharmacological effects of bupivacaine
on different K+ channels, including
HERG, Kv1.4, Kv4.3, and hKvLQT1+minK have been already
studied after the expression of the corresponding cRNA in
Xenopus oocytes (Lipka et al., 1998). However, it has been
reported that when studying the effects of lipophilic drugs (such as
bupivacaine) that act from the inside of the cell membrane, the
apparent drug potency is approximately 10 to 100 times lower when using
whole oocyte recordings than cell-free patch recordings (free of yolk)
(Yatani et al., 1993; Ficker et al., 1998). Therefore, it would be of
interest to study the effects of bupivacaine on K+ channels expressed in mammalian cells, that
exhibit closer pharmacological responses to human cardiac myocytes than
Xenopus oocytes. IQB-9302 is a new amide type local
anesthetic, chemically related to bupivacaine (Fig.
1), synthesized as a less cardiotoxic
alternative to bupivacaine and with similar potency to block
Na+ channels (Gallego-Sandín et al.,
1999; Ruiz-Nuño et al., 1999). In the present study, we have
analyzed the electrophysiological effects of bupivacaine and IQB-9302
on hKv1.5, Kv2.1, Kv4.3, and HERG channels expressed in
mammalian cell lines. Preliminary results have been published in
abstract form (González et al., 2000).
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Materials and Methods |
Cell Culture.
Stably transfected
Ltk
cells with the gene encoding the
expression of hKv1.5 or Kv2.1 channels were cultured in Dulbecco's modified Eagle's medium with 10% horse serum and 0.25 mg/ml
G418 (GIBCO, Paisley, Scotland, UK) in a 5%
CO2 atmosphere as previously described
(Valenzuela et al., 1995a, 1996). Chinese hamster ovary cultures were
grown in Ham's F-12 medium with 10% fetal bovine serum and
transiently transfected with the cDNA encoding the Kv4.3 or
HERG channel (4 µg) and the cDNA encoding the CD8 antigen
(0.5 µg) by use of lipofectAMINE. Before experimental use, cells were incubated with polystyrene microbeads precoated with anti-CD8 antibody
(Dynabeads M450; Dynal, Oslo, Norway). Most of the cells beaded
also had channel expression.
Electrophysiological Recording.
The intracellular
pipette-filling solution contained 80 mM potassium aspartate, 50 mM
KCl, 3 mM phosphocreatine, 10 mM
KH2PO4, 3 mM MgATP, 10 mM
HEPES-K, and 5 mM EGTA and was adjusted to pH 7.25 with KOH. The bath
solution contained 130 mM NaCl, 4 mM KCl, 1.8 mM
CaCl2, 1 mM MgCl2, 10 mM
HEPES-Na, and 10 mM glucose, and was adjusted to pH 7.40 with NaOH.
IQB-9302 [1-(cyclopropylmethyl-2',6'-pipecoloxylidide] (gift from
Dr. A. Galiano, IQB-Inibsa S.A., Barcelona, Spain) and
bupivacaine (Sigma Chemical Co., St. Louis, MO) were dissolved in
distilled deionized water.
hKv1.5, Kv2.1, Kv4.3, and HERG currents were recorded at
room temperature (20-22°C) using the whole-cell patch-clamp
technique (Hamill et al., 1981) with an Axopatch 1C patch-clamp
amplifier (Axon Instruments, Foster City, CA). Kv1.5, Kv2.1, and Kv4.3
currents were filtered at 2 kHz (four-pole Bessel filter) and sampled
at 4 kHz; HERG currents were filtered at 100 Hz and sampled
at 200 Hz. Micropipettes were pulled from borosilicate glass capillary tubes (GD-1; Narishige, Tokyo, Japan) on a programmable horizontal puller (Sutter Instrument Co., San Rafael, CA) and heat-polished with a
microforge (Narishige). Micropipettes resistance were 1 to 3 M
.
Maximum hKv1.5 current amplitudes at +60 mV averaged 2.2 ± 0.3 nA, mean uncompensated access resistance was 2.4 ± 0.5 M, and
cell capacitance was 11.2 ± 0.8 pF (n = 10).
Thus, no significant voltage errors (<5 mV) were expected with the
electrodes used.
In all cases, cells were held at 
80 mV. After control data were
obtained, bath perfusion was switched to drug-containing solution. The
effects of drug infusion was monitored with test pulses to +60 mV or to
+30 mV (for HERG currents), applied every 30 s until
steady state was obtained (after ~12 min). Steady-state current-voltage relationships (IV) were obtained by averaging the
current over a small window at the end of 250-ms or 5-s (for HERG currents) depolarizing pulses. Between 80 and 40 mV
only passive linear leak was observed and least-squares fits to these data were used for passive leak correction. Deactivating "tail" currents of hKv1.5 and Kv2.1 channels were recorded at 40 mV and
HERG tail currents were recorded at 60 mV. The activation curve of hKv1.5, Kv2.1, and HERG channels were obtained from
the tail current amplitude measured just after the capacitive
transient. Block of Kv4.3 channels was calculated as the reduction of
the amount of charge crossing the membrane during the application of
250-ms depolarizing pulses from 80 mV to +60 mV (estimated from the
integral of the current). Command potentials, data acquisition, and
measurements were done using the Clampfit program of pClamp 6.0.1, origin 5.0 (Microcal Software, Northampton, MA) and by a custom-made
analysis program.
In the case of hKv1.5 channels, EC50 values were
obtained from f = 1/[1 + (EC50/[D])nH],
where [D] represents the drug concentration and
nH, the Hill coefficient. The apparent
rate constants for binding (k) and unbinding (l)
were obtained from the following equation
![k×[D]+l=1/&tgr;<SUB><UP>B</UP></SUB>=&lgr;](/content/vol296/issue2/fulltext/573/img001.gif)
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(1)
|
where 
B represents the time constant of
the drug-induced fast initial decline during depolarization to +60 mV.
Thus, k and l were calculated from the fit of
1/B versus different drug concentrations. For Kv2.1 and
Kv4.3 currents, B values were obtained from
the monoexponential fit of the
(IControl IDrug)/IControl ratio.
The dominant time constant of the activation process was analyzed
fitting it to a single exponential, following a procedure previously
described and used for the same purpose (Valenzuela et al., 1995a).
Deactivation and inactivation were fitted to a biexponential process as
follows:
|
(2)
|
where 1 and 2
are the system time constants, A1 and
A2 are the amplitudes of each
component of the exponential, and C is the baseline value.
Half-maximal voltages (Eh) and slope
factors (s) of activation of hKv1.5, Kv2.1, and
HERG channels were determined by fitting data with a
Boltzmann equation: y = 1/[1 + exp((E Eh)/s)]. The curve-fitting
procedure used a nonlinear least-squares (Gauss-Newton) algorithm;
results were displayed in linear and semilogarithmic format, together
with the difference plot. Goodness of fit was judged by the
2 criterion and by inspection for systematic
nonrandom trends in the difference plot.
Voltage dependence of block was determined as follows: leak-corrected
current in the presence of drug was normalized to matching control to
yield the fractional block at each voltage (f = 1 IDrug/IControl).
The voltage dependence of block was fitted to the following equation:
![f=[D]/([D]+K<SUB><UP>D</UP></SUB>*×<UP>exp</UP>(<UP>−</UP>&dgr;zFE/RT)),](/content/vol296/issue2/fulltext/573/img003.gif)
|
(3)
|
where z, F, R, and T
have their usual meaning, 
represents the fractional electrical
distance, i.e., the fraction of the transmembrane electrical field
sensed by a single charge at the receptor site and
KD* represents the apparent
dissociation constant at the reference potential (0 mV).
Statistical Methods.
Results are expressed as mean ± S.E.M. Direct comparisons between mean values in control conditions
versus mean values in the presence of drug for a single variable were
performed by a paired Student's t test. ANOVA was used to
compare more than two groups. Student's t test was also
used to compare two regression lines. Differences were considered
significant if the p value was less than 0.05.
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Results |
Effects of IQB-9302 and Bupivacaine on hKv1.5 Channels.
Figure
2A shows current records obtained in the
absence and in the presence of 20 µM bupivacaine or IQB-9302. The
holding potential was maintained at 80 mV and 250-ms depolarizing
pulses in duration to membrane potentials between 80 and +60 mV were applied. Tail currents were recorded upon repolarization to 40 mV. At
20 µM, both drugs inhibited hKv1.5 current, bupivacaine being more
potent than IQB-9302 (57.3 ± 2.9%, n = 4 versus
41.6 ± 2.3%, n = 10, p < 0.01, respectively). The effects of both drugs were reversible upon perfusion
of the cells with drug-free external solution (90 ± 3% of the
control values, n = 6, and 82 ± 4%,
n = 9, for bupivacaine and IQB-9302, respectively).
Both anesthetics significantly accelerated the activation kinetics of
the current at +60 mV (1.44 ± 0.08 versus 1.21 ± 0.09 ms,
n = 5, p < 0.05; and 1.28 ± 0.11 versus 0.81 ± 0.01 ms, n = 4, p < 0.01, for bupivacaine and IQB-9302, respectively). The degree of
inhibition of hKv1.5 current was measured at the end of depolarizing
pulses from 80 to +60 mV induced by different bupivacaine and
IQB-9302 concentrations (between 0.2 and 100 µM). Individual data
were fit with the Hill equation to estimate an
EC50 for hKv1.5 current inhibition and a Hill
coefficient. These fits yielded EC50 values of
8.9 ± 1.4 µM (n = 22) and 21.8 ± 4.7 µM
(n = 29, p < 0.01) for bupivacaine and
IQB-9302, and Hill coefficients of 0.88 ± 0.02 and 0.92 ± 0.01, respectively.
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Fig. 2.
Effects of bupivacaine and IQB-9302 on hKv1.5
currents. A, hKv1.5 current traces obtained after depolarizations from
a holding potential of 80 mV to voltages between 80 and +60 mV in
steps of 10 mV. Tail currents were recorded at 40 mV. The middle
panels show the effects of 20 µM bupivacaine or IQB-9302. Note the
time-dependent effects of bupivacaine and IQB-9302 on the maximum
activated current. B, tail current records recorded in the absence and
in the presence of bupivacaine or IQB-9302. In both cases, the
deactivation kinetics was slower than under control conditions and a
crossover (indicated by an arrow) between the control tail current and
the tail current recorded in the presence of drug was observed.
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Both drugs induced a fast initial decline of the current that was
superimposed to the slow inactivation (Fig. 2A). This time-dependent decay of the current was more evident in the presence of bupivacaine than in the presence of IQB-9302, reflecting a different kinetics of
block for each drug. The time constant of this drug-induced initial
decline of the current was faster at higher concentrations and thus, it
was considered as an index of the kinetics of binding of the drug
(B). Figure 3, A
and B, show the apparent rate of block (1/B)
versus bupivacaine and IQB-9302 concentration. The straight lines are
the least-squares fit to the relation 1/B = k × [D] + l which for
bupivacaine, yielded apparent association (k) and
dissociation (l) rate constants of 2.45 ± 0.19 µM1 s1 and 29.1 ± 8.8 s1 (n = 14),
respectively. The k value for IQB-9302 was similar to that
obtained for bupivacaine (2.07 ± 0.43 µM1 s1,
n = 8, p > 0.05), whereas l
was ~2 times faster than that obtained with bupivacaine (56.8 ± 9.2 s1, n = 8, p < 0.01). These results suggest a more stable
interaction between the receptor and bupivacaine than with IQB-9302 and
explain differences in potency between both drugs. To further analyze the time course of development of block, in Fig. 3, A and B (insets), we represented the ratio between the drug-sensitive current and the
current in control conditions
[(IControl IDrug)/IControl] during the first 100 ms in the presence of 20 µM bupivacaine or IQB-9302. In the presence of either drug, block developed following a
monoexponential function with time constants of 11.3 ± 0.8 ms (n = 7) and 12.6 ± 1.4 ms (n = 7), respectively. Whereas bupivacaine development of block only
appeared upon depolarization; an initial "instantaneous" component
of block was observed in the presence of IQB-9302 at the beginning of
the depolarizing pulse (t = 0 ms), which averaged
15.3 ± 2.0% (n = 7), and that was followed by
the previously described time-dependent increase in block. In fact,
IQB-9302 induced a higher degree of block measured at the maximum peak
current than that produced by bupivacaine. Thus, block at the maximum
peak current induced by bupivacaine and IQB-9302 contributed by
47.5 ± 4.2% (n = 4) and 77.4 ± 2.4%
(n = 10, p < 0.01), respectively, to
the total amount of block.
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Fig. 3.
Time- and voltage-dependent block of hKv1.5 channels
induced by bupivacaine and IQB-9302. A, rate of block as a function of
bupivacaine concentration. The time constant of bupivacaine-induced
fast component (B) was obtained from biexponential fits
to the initial falling phase. The inverse of B was
plotted versus bupivacaine concentration. For a first order blocking
scheme, a linear relation is expected: 1/B = k × [D] + l. The
solid line represents the linear fit, from which the apparent binding
and unbinding rate constants were obtained. B, rate of block as a
function of IQB-9302 concentration. The inverse of B was
plotted versus IQB-9302 concentration. For a first order blocking
scheme, a linear relation is expected: 1/B = k × [D] + l. The
solid line represents the linear fit, from which the apparent binding
and unbinding rate constants were obtained. A and B (insets),
representative traces of the time course of the development of block of
hKv1.5 channels induced by bupivacaine or IQB-9302 after a depolarizing
pulse to +60 mV from a holding potential of 80 mV. The reduction of
hKv1.5 current in the presence of drug is expressed as a proportion of
the control current at any given time after the start of the
depolarizing pulse. In the presence of bupivacaine and IQB-9302,
inhibition of the current increases exponentially during
depolarization. Note that in the presence of IQB-9302, an instantaneous
block at t = 0 is observed. C, current-voltage
relationship (250 ms) obtained under control conditions ( ), in the
presence of IQB-9302 ( ), after washout of the drug ( ) and in the
presence of bupivacaine ( ). All the experiments shown in the figure
were performed in the same cells. Panel D: Relative current expressed
as IDrug/IControl
from the data shown in A plotted versus membrane potential. The dotted
line represents the activation curve of hKv1.5 channels. Block
increased steeply between 20 and 0 mV, which corresponds to the
voltage range of activation of hKv1.5 channels. At membrane potentials
positive to 0 mV, a shallower increase in block was observed. This
voltage dependence was fitted (continuous line) following eq. 3 (under
Materials and Methods) and yielded -values of
0.20 ± 0.01 and 0.17 ± 0.02 for bupivacaine and IQB-9302,
respectively. Each point represents the mean ± S.E.M. of four
experiments.
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A time-dependent block was also observed in the deactivating process.
Figure 2B shows superimposed current traces obtained under control
conditions and in the presence of 20 µM bupivacaine or IQB-9302. In
the absence of bupivacaine, deactivation process of hKv1.5 current
exhibited a biexponential decay with a fast time constant
(f) of 22.8 ± 1.6 ms and a slow one
(s) that averaged 95.9 ± 22.6 ms.
Bupivacaine eliminated the fast component of deactivation, so that
deactivation became a monoexponential process with a time constant of
97.2 ± 19.2 ms (n = 5, p > 0.05 versus the slow time constant obtained under control conditions).
IQB-9302 increased the two time constants from 19.0 ± 3.4 and
57.9 ± 15.0 ms to 28.2 ± 2.9 ms (n = 5, p < 0.05) and 112.5 ± 33.0 ms (n = 5, p > 0.05). Moreover, in the presence of both
IQB-9302 or bupivacaine, an initial rising phase at the beginning of
the tail current was observed, thus indicating the dissociation of the
drug from the receptor at the channel. The slowing of the deactivating
process induced by both drugs produced a "crossover" phenomenon
when the tail currents obtained under control conditions and in the
presence of the drug were superimposed, suggestive of an open channel
block mechanism (Armstrong, 1971).
Figure 3C shows the IV relationships for hKv1.5 obtained in the absence
and in the presence of 20 µM bupivacaine or IQB-9302. The degree of
block induced by bupivacaine and IQB-9302 was higher at more positive
membrane potentials, suggesting an open channel block mechanism. To
quantitate this voltage dependence the relative current in the presence
of bupivacaine or IQB-9302 was plotted versus the membrane potential
(Fig. 3D). Block steeply increased in the activation range of hKv1.5
channels (dotted line) and it significantly increased in a shallower
way at membrane potentials positive to 0 mV. This voltage dependence
was explained as the consequence of the effects of the transmembrane
electrical field on the interaction between the charged form of the
drug and its receptor in the channel. Thus, following a Woodhull
formalism, we calculated the -values, that averaged 0.20 ± 0.02 (n = 14) for bupivacaine. For IQB-9302, averaged 0.17 ± 0.01 (n = 10, p > 0.05).
Effects of IQB-9302 and Bupivacaine on Kv2.1 Channels.
Figure
4A shows original records of Kv2.1
currents in the absence and in the presence of bupivacaine and
IQB-9302. At 20 µM, bupivacaine and IQB-9302 induced a percentage of
block of Kv2.1 channels that averaged 48.6 ± 3.4%
(n = 6) and 48.1 ± 3.3% (n = 6, p > 0.05), respectively. Both drugs produced a
decrease of the time constant of activation of the current [from
15.5 ± 2.3 to 11.7 ± 2.8 ms (n = 6, p < 0.01) in the absence and in the presence of
bupivacaine, and from 22.5 ± 2.2 to 19.7 ± 2.8 ms
(n = 6, p < 0.05), in the absence and
in the presence of IQB-9302]. In contrast to the effects observed in
hKv1.5 channels, neither drug induced a fast initial decline of the
maximum activated Kv2.1 current, i.e., block did not display time
dependence on the maximum activated outward current. However, as it can
be observed in Fig. 4B, both drugs slowed the time course of
deactivation of Kv2.1 channels. Under control conditions, the
deactivation process was fitted following a biexponential process, with
a fast and a slow time constant of 10.5 ± 0.7 and 54.2 ± 8.1 ms (n = 4), respectively. In the presence of
bupivacaine, the deactivation process was monoexponential with a time
constant of 37.1 ± 5.4 ms (n = 4, p > 0.05 compared with the control
s) and a crossover phenomenon was observed. IQB-9302 also slowed the deactivation process, but at the same concentration than bupivacaine this new local anesthetic slowed the
fast time constant of deactivation (14.8 ± 1.4 versus 18.5 ± 1.2 ms, n = 5, p < 0.05), without
modifying the slow one (77.7 ± 17.0 versus 80.0 ± 16.6 ms,
n = 5, p > 0.05). To calculate the time constant of development of block, in Fig.
5A we represented the ratio between the
drug-sensitive current and the current in control conditions
[(IControl IDrug)/IControl].
The fit of this ratio to a monoexponential function yielded the time
constant of development of block, which averaged 9.3 ± 0.6 ms
(n = 5) and 11.7 ± 1.3 ms (n = 6, p > 0.05) for bupivacaine and IQB-9302, respectively.
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Fig. 4.
Effects of bupivacaine and IQB-9302 on Kv2.1
currents. A, Kv2.1 current traces obtained after depolarizations from a
holding potential of 80 mV to voltages between 80 and +60 mV in
steps of 10 mV. Tail currents were recorded at 40 mV. The middle
panels show the effects of 20 µM bupivacaine or IQB-9302. B, tail
current recorded at 40 mV in the absence and in the presence of
bupivacaine or IQB-9302. In both cases, the deactivation kinetics was
slower than under control conditions.
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Fig. 5.
Time- and voltage-dependent block of Kv2.1 channels
induced by bupivacaine and IQB-9302. A, representative trace of the
time course of the development of block of Kv2.1 channels induced by
bupivacaine and IQB-9302 after a depolarizing pulse to +60 mV from a
holding potential of 80 mV. The reduction of Kv2.1 in the presence of
each drug is expressed as a proportion of the control current at any
given time after the start of the depolarizing pulse. Note that in the
presence of bupivacaine and IQB-9302, inhibition of the current
increases exponentially during depolarization. B, current-voltage
relationship (250 ms) obtained under control conditions (), and in
the presence () of bupivacaine (top) or IQB-9302 (bottom). C,
relative current expressed as
IDrug/IControl
from the data shown in the middle panel versus membrane potential. The
dashed line represents the activation curve of Kv2.1 channels. Block
increased steeply between 10 and 15 mV, which corresponds to the
voltage range of activation of Kv2.1 channels. At membrane potentials
positive to 15 mV, a shallower increase in block was observed. This
voltage dependence was fitted (continuous line) following eq. 3 (under
Materials and Methods) and yielded -values of
0.16 ± 0.01 and 0.20 ± 0.03 for bupivacaine and IQB-9302,
respectively. Each point represents the mean ± S.E.M. of four to
six experiments.
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Figure 5B shows the IV relationships for Kv2.1 obtained in the absence
and in the presence of 20 µM bupivacaine or IQB-9302. Block of Kv2.1
channels induced by bupivacaine and IQB-9302 was voltage-dependent
(Fig. 5C). Block steeply increased in the range of activation of Kv2.1
channels and it increased with a shallower slope at membrane potentials
positive to +15 mV. Using the same procedure described above (Woodhull,
1973), we calculated the -values, which averaged 0.16 ± 0.01 (n = 5) for bupivacaine and 0.20 ± 0.03 (n = 4, p > 0.05) for IQB-9302.
Effects of IQB-9302 and Bupivacaine on Kv4.3 Channels.
Figure
6A shows original records of Kv4.3
current after applying 250-ms depolarizing steps from a holding
potential of 80 mV to membrane potentials between 80 and +60 mV in
10-mV steps in the absence and in the presence of 20 µM bupivacaine
or IQB-9302. Both drugs decreased the peak current to a similar extent
[25.3 ± 2.8% (n = 6) and 32.6 ± 3.0%
(n = 7, p > 0.05), in the presence of
bupivacaine and IQB-9302, respectively]. However, their most prominent
effect was an acceleration of the time course of inactivation, which
was faster in the presence of bupivacaine than in the presence of
IQB-9302 (Fig. 6B). The fast time constants of inactivation decreased
from 27.4 ± 3.9 to 9.9 ± 1.4 ms (n = 5, p < 0.01) in the presence of bupivacaine and from
26.8 ± 2.9 to 13.2 ± 1.3 ms (n = 5, p < 0.05) in the presence of IQB-9302, with this
effect being reversible upon washout of the cells with drug-free
external solution. This accelerated decline of the current was
suggestive of an open channel block mechanism, indicating that the
reduction of peak current would be a nonequilibrium measure of block.
Therefore, block of Kv4.3 channels by bupivacaine and IQB-9302 was also
measured as the reduction of the amount of charge crossing the membrane (estimated from the integral of the current) during the application of
250-ms depolarizing pulses from 80 to +60 mV. Bupivacaine and
IQB-9302 produced similar levels of Kv4.3 inhibition, reducing the
integrated current by 45.4 ± 12.4% (n = 6) and
36.1 ± 3.7% (n = 7, p > 0.05),
respectively. Interestingly, the inhibition of the current induced by
bupivacaine measured at the maximum peak current was lower than that
obtained from the inhibition measurements of the integral of the
current (25.3 ± 2.8 versus 45.4 ± 12.4%, n = 5, p < 0.05), whereas IQB-9302 inhibited similarly the maximum peak current than the current integral (32.6 ± 3.0 versus 36.1 ± 3.7%, n = 7, p > 0.05), suggesting that IQB-9302 may block other state of Kv4.3 channels
previous to the open one.
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Fig. 6.
Effects of bupivacaine and IQB-9302 on Kv4.3 current.
A, Kv4.3 current traces obtained after depolarizations from a holding
potential of 80 mV to voltages between 80 and +60 mV in steps of 10 mV. The middle panels show the effects of 20 µM bupivacaine or
IQB-9302. B, superimposed Kv4.3 currents recorded in the absence and in
the presence of bupivacaine or IQB-9302. Traces were normalized to
match the maximum amplitude of the control record. In both cases, a
time-dependent inhibition of the current was observed.
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Using the procedure previously described, the
B values for bupivacaine and IQB-9302 on Kv4.3
channels were calculated from the fitting of the ratio between the
drug-sensitive current and the current in control conditions
[(IControl IDrug)/IControl] averaging 6.5 ± 0.7 ms (n = 4) and 7.5 ± 0.8 ms (n = 4, p > 0.05), respectively
(Fig. 7A). As it is observed in Fig. 7A,
although bupivacaine development of block began during the
depolarization, IQB-9302 development of block exhibited an initial
"instantaneous" component at the beginning of the depolarizing
pulse (t = 0 ms), which averaged 19.2 ± 1.0%
(n = 4) and that was followed by the previously
described time-dependent increase in block.
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Fig. 7.
Time- and voltage-dependent block of Kv4.3 channels
induced by bupivacaine and IQB-9302. A, representative trace of the
time course of the development of block of Kv4.3 channels induced by
bupivacaine and IQB-9302 after a depolarizing pulse to +60 mV from a
holding potential of 80 mV. The reduction of Kv4.3 current in the
presence of each drug is expressed as a proportion of the control
current at any given time after the start of the depolarizing pulse. In
the presence of bupivacaine and IQB-9302, inhibition of the current
increases exponentially during depolarization. Note that in the
presence of IQB-9302, an instantaneous block at t = 0 is observed. B, relationship between the integrated current and the
membrane potential obtained under control conditions (), and in the
presence () of bupivacaine (top) or IQB-9302 (bottom). C, relative
charge expressed as
IDrug/IControl
from the data shown in B. At membrane potentials positive to 0 mV, a
shallow increase in block was observed. This voltage dependence was
fitted (continuous line) following eq. 3 (under Materials and
Methods) and yielded -values of 0.11 ± 0.05 and
0.08 ± 0.04 for bupivacaine and IQB-9302, respectively. Each
point represents the mean ± S.E.M. of four to seven
experiments.
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Bupivacaine and IQB-9302 blocked Kv4.3 channels at all membrane
potentials tested (Fig. 7B). However, and in contrast to that observed
in hKv1.5 and Kv2.1 channels, block of Kv4.3 channels induced by
bupivacaine or IQB-9302 was weakly voltage-dependent at membrane
potentials positive to 0 mV, with -values measured from the inside
of the membrane that averaged 0.11 ± 0.05 (n = 4)
and 0.08 ± 0.04 (n = 4, p > 0.05) for bupivacaine and IQB-9302, respectively (Fig. 7C).
Effects of IQB-9302 and Bupivacaine on HERG
Channels.
Figure 8A shows original
records obtained after applying 5-s depolarizing steps from a holding
potential of 80 mV to membrane potentials between 80 and +50 mV in
10-mV steps in the absence and in the presence of bupivacaine or
IQB-9302 (20 µM). Under control conditions, outward HERG
currents during depolarization are reduced because channels inactivate
faster than they activate (Sanguinetti et al., 1995). Upon
repolarization, rapid recovery from inactivation preceded deactivation,
resulting in a hooked tail current (Sanguinetti et al., 1995).
Bupivacaine and IQB-9302 decreased to a similar extent the amplitude of
the current measured at 0 mV, 43.1 ± 9.1% (n = 5) and 50.3 ± 6.6% (n = 5, p > 0.05), respectively. Figure 8B shows tail HERG currents
obtained in the absence and in the presence of 20 µM bupivacaine or
IQB-9302. Deactivation kinetics of HERG channels at 60 mV
after depolarization of the cell membrane to +50 mV exhibited a
biexponential kinetics. Bupivacaine slowed the deactivation time course
from 168.3 ± 22.8 and 845.7 ± 89.8 ms to 260.0 ± 22.5 ms (n = 4, p < 0.01) and 979.0 ± 104.7 ms (n = 4, p < 0.05), without
modifying the contribution of the fast component to the total process
(0.47 ± 0.06 versus 0.43 ± 0.03, in the absence and in the
presence of bupivacaine, n = 4, p > 0.05). However, IQB-9302 did not modify the kinetics of the tail
currents. Figure 9A shows the IV
relationships obtained by plotting the magnitude of the HERG
current amplitude at the end of 5-s pulses as a function of the
membrane potential under control conditions and in the presence of
bupivacaine or IQB-9302 (20 µM). Both drugs inhibited to a similar
extent HERG current at membrane potentials ranging between
20 and +60 mV. Figure 9B shows the activation curves obtained in the
absence and in the presence of bupivacaine or IQB-9302. Both drugs
shifted the activation curve toward hyperpolarizing direction without
modifying its slope. In fact, the Eh
values in the absence and in the presence of bupivacaine averaged
3.1 ± 1.1 and 9.9 ± 1.7 mV, respectively (n = 4, p < 0.05). In the absence and
in the presence of IQB-9302, Eh
averaged 8.0 ± 1.8 and 14.0 ± 2.3 mV (n = 5, p < 0.05). Figure 9C shows the relative tail
current in the presence of bupivacaine or IQB-9302 versus membrane
potential. The relative current steeply decreased in the membrane
potential range coinciding with the HERG channels
activation, suggesting an open channel block mechanism. Similarly to
that observed in Kv4.3 channels, at membrane potentials positive to +15
mV, a slight increase in block was observed, consistent with -values
of 0.09 ± 0.02 (n = 4) and 0.08 ± 0.04 (n = 6, p > 0.05) in the presence of
bupivacaine and IQB-9302.
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Fig. 8.
Effects of bupivacaine and IQB-9302 on
HERG currents. A, HERG current traces
obtained after depolarizations from a holding potential of 80 mV to
voltages between 80 and +50 mV in steps of 10 mV. Tail currents were
recorded at 60 mV. The middle panels show the effects of 20 µM
bupivacaine or IQB-9302. B, tail current records recorded in the
absence and in the presence of bupivacaine or IQB-9302.
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Fig. 9.
Voltage-dependent effects of bupivacaine and IQB-9302
on HERG channels. A, IV relationships (5-s isochronal)
of HERG channels obtained in the absence () and in
the presence () of 20 µM bupivacaine (top) or IQB-9302 (bottom).
B, activation curves of HERG channels obtained under
control conditions () and in the presence of bupivacaine or IQB-9302
(). Note that both drugs shift toward hyperpolarizing potentials the
activation curve. C, relative current versus membrane potentials. Block
increases in the range of membrane potentials that coincides with the
activation of HERG channels. At membrane potentials
positive to +15 mV block slightly increased consistent with -values
of 0.09 ± 0.02 and 0.08 ± 0.04 for bupivacaine and
IQB-9302, respectively. Each point represents the mean ± S.E.M.
of four to six experiments.
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Discussion |
In the present article the effects of bupivacaine and IQB-9302 on
hKv1.5, Kv2.1, Kv4.3, and HERG channels expressed in
mammalian cells have been analyzed. Both drugs blocked Kv2.1, Kv4.3,
and HERG channels to a similar extent, whereas bupivacaine
resulted to be 2.5-fold more potent than IQB-9302 to block hKv1.5
channels. To our knowledge, this is the first study in which the
effects of bupivacaine have been analyzed on hKv1.5 and Kv2.1 channels.
Effects of Bupivacaine and IQB-9302 on hKv1.5, Kv2.1, Kv4.3, and
HERG Channels.
Block induced by bupivacaine and
IQB-9302 of hKv1.5 channels was time- and voltage-dependent. Both drugs
induced a fast initial decline of the current upon depolarization,
which reached steady state at the end of 250-ms depolarizing pulses.
Although bupivacaine block appeared only upon depolarization,
suggesting a pure open channel block mechanism, IQB-9302 block showed
an initial block previous to the development of block observed during
depolarization, which suggests that this drug also blocks a closed
state of the channel previous to the open one. The superposition of the
tail currents recorded under control conditions and in the presence of
either drug shows a "crossover" between them, indicating fast recovery from block during deactivation, consistent with an open channel block mechanism (Armstrong, 1971). Block induced by bupivacaine or IQB-9302 of hKv1.5 channels was voltage-dependent, in such a way
that block steeply increased in the range of membrane potentials coinciding with the range of activation of hKv1.5 channels, suggesting that these drugs need that the channels open before they can bind to
their receptor site and block K+ efflux. At
membrane potentials positive to 0 mV, block induced by bupivacaine and
IQB-9302 increased with a shallower slope, consistent with a -value
of ~0.17. This voltage-dependent block was interpreted to be the
consequence of the effects of the transmembrane electrical field on the
interaction between the cationic form of the drugs
(pKa of ~8.0) and their receptor site at the
channel level, as it has been proposed for bupivacaine enantiomers in hKv1.5 channels (Valenzuela et al., 1995a).
Time-dependent block of Kv2.1 channels was evident in the deactivation
process, but not at the maximum activated current during the
depolarization step. Possible explanations for these results would be
that bupivacaine and IQB-9302 bind to other state of Kv2.1 channels
different from the open state and/or that they bind to the open state
of Kv2.1 channels with a faster rate than the channel opening. The
later explanation seems to be more likely. In fact, the time constants
of block for bupivacaine and IQB-9302 (9 and 11 ms, respectively) were
faster than the dominant time constant of activation of Kv2.1 current
at +60 mV (15.5 ± 2.3 ms). As shown in Fig. 5A, both drugs
induced the development of block during the activation of the channel,
and this block increased exponentially until it reached steady-state
block after ~100 ms of the beginning of the depolarizing pulse.
Moreover, block of Kv2.1 channels induced a slowing of the tail
currents. These time-dependent effects may suggest an open channel
block mechanism. In fact, block induced by bupivacaine and IQB-9302 of
Kv2.1 channels was voltage-dependent consistent with a -value of
~0.17. All these results suggest that bupivacaine and IQB-9302 block
the open state of Kv2.1 channels.
Bupivacaine and IQB-9302 induced a faster inactivation process of the
Kv4.3 current and block increased in an monoexponential manner during
the activation of the channels, reaching a maximum value after ~50
ms, consistent with an open channel block mechanism. However, in the
presence of IQB-9302 an instantaneous block was observed at
t = 0 ms. This initial block, which appears before channel opening averaged 19.2 ± 1.0%, and could be attributed to
the drug interaction with a nonconducting state of the channel. Consistent with this hypothesis, the degree of inhibition of the maximum peak current and the charge by IQB-9302 were similar, whereas
for bupivacaine, both were significantly different, with the charge
inhibition being higher than that of the peak current. However, as
suggested by the further inhibition of the current observed during the
application of depolarizing pulses, it seems that IQB-9302 also binds
to the open state of the channel. Bupivacaine-induced block of Kv4.3
channels expressed in Xenopus oocytes exhibited an
EC50 value of ~33 µM (Lipka et al., 1998),
close to that found in the present study, in which, if we assume an
nH = 1, it would be ~24 µM.
Moreover, similarly to what has been previously described for
bupivacaine on Kv4.3 channels expressed in Xenopus oocytes (Lipka et al., 1998), we found that the blockade produced by
bupivacaine and IQB-9302 was weakly voltage-dependent at very positive
membrane potentials, consistent with -values of ~0.06.
HERG channels were also sensitive to the effects of
bupivacaine and IQB-9302. The inhibition produced by 20 µM
bupivacaine in the present study is similar to that reported for
HERG channels expressed in Xenopus oocytes (100 µM) (Lipka et al., 1998). These differences found in both expression
systems could be due to a different protein processing or membrane
environment between amphibian oocytes and mammalian cells. In fact,
differences between these two expression systems have been observed in
pharmacological studies examining quinidine block of hKv1.5 and hKv1.4
channels (Yatani et al., 1993; Deal et al., 1996; Yeola and Snyders,
1997; Franqueza et al., 1999). Block of HERG channels
induced by both drugs increased in the range of membrane potentials
coinciding with the activation of the current, thus suggesting an open
channel block mechanism. Similar to what was observed in Kv4.3
channels, block of HERG channels at positive membrane
potentials was weakly voltage-dependent ( of ~0.07). Therefore,
these results seem to suggest that both local anesthetics exhibit a
higher affinity for the open state than for the inactivated state of
the HERG channels.
Bupivacaine and IQB-9302 have the same chemical structure with the
exception that at position 1, bupivacaine exhibits a butyl group and
IQB-9302 a cyclopropylmethyl group (Fig. 1). Degree of block of Kv2.1,
Kv4.3, and HERG channels induced by bupivacaine and IQB-9302
was very close, thus suggesting that both drugs act as nonspecific
blockers of these K+ channels as previously found
for bupivacaine in Xenopus oocytes (Lipka et al., 1998).
However, in the present study, we found that the minor differences in
the chemical structure between bupivacaine and IQB-9302 were sufficient
to induce a 2.5-fold difference in potency to block hKv1.5 channels.
Therefore, the present results suggest that, in contrast to the results
obtained in Kv2.1, Kv4.3, and HERG channels, the affinity of
the receptor site for local anesthetics at hKv1.5 channels is very
sensitive to changes in the length of their N-substituent and/or to its
torsion availability. Moreover, block of Kv2.1 and Kv4.3 channels by
bupivacaine enantiomers is not stereoselective (Franqueza et al., 1997,
1999), whereas block of hKv1.5 channels induced by bupivacaine and
IQB-9302 is stereoselective (Valenzuela et al., 1995a; González
et al., 2001). Since stereoselectivity indicates a direct and specific
receptor-mediated action (Ariëns, 1993), these results may
suggest that hKv1.5 channels are more sensitive to changes in the
N-substituent than the other K+ channels studied.
Clinical Implications of the Present Study.
It has been
described that bupivacaine decreases intracardiac conduction velocity
and widens the QRS complex of the electrocardiogram, effects that were
attributed to the inhibition of INa
(Clarkson and Hondeghem, 1985; Wheeler et al., 1988). Both in animal
models and in humans, bupivacaine prolonged the duration of the cardiac action potential (Avery et al., 1984; Kasten and Martin, 1985; Scott et
al., 1989; Solomon et al., 1990) and the QTc interval of the ECG (Scott
et al., 1989), effects that can, eventually, result in the development
of a polymorphic ventricular tachycardia known as torsades de pointes
(Kasten and Martin, 1985). Accidental intravascular injection of
bupivacaine can result in transient plasma concentrations of its free
form of 4 to 12 µg/ml (12-36 µM) (Kotelko et al., 1984; Sage et
al., 1985). Since the concentrations of bupivacaine used in the present
study are within this range, it can be proposed that the prolongation
of the QTc interval observed in patients who have received an overdose
of bupivacaine can be explained by the blockade of several cardiac
K+ channels (Scott et al., 1989). In the present
experiments, IQB-9302 blocked human cloned potassium channels Kv2.1,
Kv4.3, and HERG similarly to bupivacaine. Therefore, it
would be expected that ventricular cardiotoxicity related to the
blockade of K+ channels would be similar for both
drugs. However, further clinical trials are needed to assess this possibility.
Conclusions.
The results shown in the present article indicate
that small differences at the N-substituent of bupivacaine-like local
anesthetics do not affect the degree of block of Kv2.1, Kv4.3, or
HERG channels, although specifically modify block of hKv1.5
channels, suggesting that hKv1.5 channels are more sensitive to changes
in the N-substituent than Kv2.1, Kv4.3, or HERG channels.
This study was supported by Comision Interministerial de
Ciencia y Tecnologia SAF98-0058 (to C.V.), Comision Interministerial de
Ciencia y Tecnologia SAF99-0069 (to J.T.), CAM 08.4/0016198 (to E.D.),
and U.S.-Spain Science and Technology Program 98131 (to C.V.) Grants.
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Abstract
Introduction
