Circulation Research. 1997;81:1053-1064
(Circulation Research. 1997;81:1053-1064.)
© 1997 American Heart Association, Inc.
Molecular Determinants of Stereoselective Bupivacaine Block of hKv1.5 Channels
Laura Franqueza,
Mónica Longobardo,
Javier Vicente,
Eva Delpón,
Michael M. Tamkun,
Juan Tamargo,
Dirk J. Snyders,
,
Carmen Valenzuela
From the Institute of Pharmacology and Toxicology (L.F., M.L., J.V.,
E.D., J.T., C.V.), CSIC School of Medicine, Universidad Complutense, Madrid,
Spain, and the Departments of Pharmacology, Molecular Physiology, and
Biophysics (M.M.T.) and the Departments of Medicine and Pharmacology (D.J.S.),
School of Medicine, Vanderbilt University, Nashville, Tenn.
Correspondence to Carmen Valenzuela, PhD, Institute of Pharmacology and Toxicology, CSIC, School of Medicine, Universidad Complutense, 28040 Madrid, Spain. E-mail carmenva{at}eucmax.sim.ucm.es
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Abstract
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Abstract Enantiomers of local anesthetics are useful probes
of
ion channel structure that can reveal three-dimensional relations
for
drug binding in the channel pore and may have important clinical
consequences.
Bupivacaine block of open hKv1.5 channels is
stereoselective,
with the
R(+)-enantiomer being 7-fold
more potent than the
S(-)-enantiomer
(
Kd=4.1
µmol/L versus 27.3
µmol/L). Using whole-cell voltage
clamp of hKv1.5 channels and
site-directed mutants stably expressed
in
Ltk- cells, we have identified a set of
amino acids that
determine the stereoselectivity of bupivacaine block.
Replacement
of threonine 505 by hydrophobic amino acids
(isoleucine, valine,
or alanine) abolished stereoselective
block, whereas a serine
substitution preserved it
[
Kd=60 µmol/L and 7.4 µmol/L
for
S(-)- and
R(+)-bupivacaine,
respectively]. A similar substitution
at the internal
tetraethylammonium binding site (T477S)
reduced
the affinity for both enantiomers similarly, thus preserving
the
stereoselectivity [
Kd=45.5
µmol/L and 7.8 µmol/L
for
S(-)- and
R(+)-bupivacaine, respectively]. Replacement of
L508 or
V512 by a methionine (L508M and V512M) abolished stereoselective
block,
whereas substitution of V512 by an alanine (V512A) preserved
it. Block
of Kv2.1 channels, which carry valine, leucine, and
isoleucine
residues at T505, L508, and V512 equivalent sites,
respectively, was
not stereoselective [
Kd=8.3 µmol/L
and
13 µmol/L for
S(-)- and
R(+)-bupivacaine, respectively].
These results suggest
that (1) the bupivacaine binding site
is located in the inner mouth of
the pore, (2) stereoselective
block displays subfamily selectivity, and
(3) a polar interaction
with T505 combined with hydrophobic
interactions with L508 and
V512 are required for stereoselective
block.
Key Words: local anesthetic K+ channel bupivacaine drug binding site enantiomer
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Introduction
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Local
anesthetics are useful probes of ion channel function
and structure.
Stereoselective interactions are especially interesting
because they
can reveal three-dimensional relationships between
drug and channel
with otherwise identical biophysical and physicochemical
properties.
Furthermore, stereoselectivity suggests direct and
specific
receptor-mediated action,
14 and identification
of such
stereospecific interactions may have important clinical
consequences.
1,5 Bupivacaine is a long-acting, potent, and
highly cardiotoxic
local anesthetic agent widely used for regional
anesthesia.
6,7 It contains one chiral carbon
atom, and it is clinically used
as a racemic mixture of
S(-)- and
R(+)-bupivacaine (Fig 1

).
In vivo
studies have demonstrated that the potency and duration
of local
anesthesia are equal or even larger for
S(-)-bupivacaine
than for the
R(+)-enantiomer.
5,8,9 More important, the
LD
50 was

30% to 40% lower for
R(+)-bupivacaine than for
S(-)-bupivacaine.
5,8 Although the higher
affinity of
R(+)-bupivacaine for cardiac
Na
+
channels
3 could explain its higher cardiotoxicity over
S(-)-bupivacaine,
several studies have also shown a
prolongation of the QT
c interval
of the ECG in
anesthetized dogs
1012 and human
volunteers
13 receiving high doses of bupivacaine.
Occasionally, this QT
c prolongation was accompanied by
torsades de pointes,
14 suggesting
that bupivacaine
cardiotoxicity also involves block of K
+ channels.
Furthermore,
it has been demonstrated that racemic bupivacaine inhibits
guinea
pig cardiac delayed rectifier K
+
current,
15,16 rat atrial transient
outward
current,
17 hKv1.5-like current,
4 and delayed
rectifier
K
+ current recorded in frog sensory ganglion
cells.
18 Inhibition
of cardiac transient outward current
and neuronal K
+ current
is not
stereoselective,
17,18 whereas open-channel block of a
human
Shaker-related channel, hKv1.5, by bupivacaine is highly
stereoselective,
with the
R(+)-enantiomer being 7-fold more
potent than
S(-)-bupivacaine.
4 The intrinsic
voltage dependence of hKv1.5 block was consistent
with a
fractional electrical distance referenced to the inner
surface of the
membrane (

) of 0.16 for both bupivacaine enantiomers.
This cloned
subunit most likely underlies the ultrarapid activating
delayed
rectifier described in human atrium and may contribute
to similar
currents in cardiac myocytes of other species.
1922 This
native hKv1.5-like current is involved in the control of
the cardiac
action potential duration
19 and, therefore,
represents
a potential molecular target for drugs that prolong
the cardiac
action potential duration as a therapeutic intervention or
a
proarrhythmic side effect. Kv1.5 is also expressed in pancreatic
beta
cells, vascular and visceral smooth muscle, GH
3 cells,
and
brain,
2327 indicating that it plays a role in the
excitability
of a variety of cell types. The hKv1.5 channels consist of
four
subunits each containing six transmembrane segments (S1 to S6).
The
segment between S5 and S6 forms the external entrance to and
part
of the ion conduction pathway (P loop). The flanking S5
and S6 segments
are considered to contribute to the presumably
wider intracellular
mouth of the ion channel,
28,29 which contains
binding sites
for quaternary ammonium open-channel blockers
and similarly acting
drugs such as quinidine.
3033 Both
electrostatic and
hydrophobic factors are apparently involved
in open-channel block by
clinically used drugs such as quinidine
and
bupivacaine.
4,33,34

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Figure 1. A, Alignment of S6 sequences for members of Kv1 to
Kv4 subfamilies and Shaker channels. Sequence is
one-letter code; sequence number for the first residue is indicated. B,
Chemical structure of bupivacaine. *Asymmetrical carbon.
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In the present study, we used site-directed mutagenesis to
examine the possible involvement in affinity and stereoselectivity of
bupivacaine binding to hKv1.5 channels of the internal TEA binding site
(T477 in Kv1.5)2830,35 and three residues located in the
midsection of S6: (1) T505, which has been implicated in the binding of
hydrophobic TEA derivatives in Shaker,30 and
quinidine, in Kv1.5,33 (2) L508, which has been involved in
the internal TEA binding in Kv3.1 channels (which contain a methionine
at the equivalent position), and (3) V512, which has been implicated in
the quinidine binding site in hKv1.5 channels.33 We found
that mutations at these sites affect both affinity and
stereoselectivity for bupivacaine binding, and the results suggest that
stereoselective binding of bupivacaine to hKv1.5 channels requires a
polar interaction at position 505 (T505) combined with hydrophobic
interactions at positions 508 and 512 (L508 and V512). Preliminary
reports of the present study have been published in abstract
form.36,37
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Materials and Methods
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Site-Directed Mutagenesis
The PCR-based method for site-directed mutagenesis in the S4
to
S6 region of hKv1.5 has been described in detail
elsewhere.
33 Briefly, PCR-based mutagenesis was used, and
all PCR-generated
sequences were verified directly by double-stranded
sequencing.
Once the desired sequence was confirmed, the complete
coding
sequence was ligated into the pMSVneo expression vector, used
for
stable transfection into
Ltk- cells as
described before.
19,38 For each mutation, at least six foci
were expanded into clonal
cell lines. In most cases, at least two cell
lines were examined.
For the mutations reported in the present
study, we found that
all Kv1.5 mRNApositive cell lines were
expressing current,
albeit at different levels. Of the expressing cell
lines, those
with currents between 1 and 4 nA at +40 mV were used
preferentially
to ensure adequate voltage-clamp control. Cell culture
and cell
preparation for experimental use were as reported
previously.
4,19
Solutions
The intracellular pipette filling solution contained
(mmol/L) potassium aspartate 80, KCl 50,
KH2PO4 10, MgATP 3, HEPES 10, and EGTA 5 and
was adjusted to pH 7.25 with KOH. The bath solution contained
(mmol/L) NaCl 130, KCl 4, CaCl2 1.8,
MgCl2 1, HEPES 10, and glucose 10 and was adjusted to pH
7.35 with NaOH. Both bupivacaine enantiomers (a gift from Chiroscience,
Cambridge, UK) were dissolved in distilled deionized water to yield
stock solutions of 10 mmol/L, from which further dilutions
were made to obtain the desired final concentration.
Electrical Recording
Experiments were performed in a small volume (0.5-mL) bath
mounted on the stage of an inverted microscope (model TMS, Nikon)
perfused continuously at a flow rate of 0.5 to 1.0 mL/min. The hKv1.5
currents were recorded at room temperature (20°C to 22°C) using
the whole-cell voltage-clamp configuration of the patch-clamp
technique39 with an Axopatch-1C patch-clamp amplifier (Axon
Instruments). Currents were filtered at 2 kHz (four-pole Bessel filter)
and sampled at 4 kHz. Data acquisition and command potentials were
controlled by the pClamp software (versions 5.5.1 and 6.01) (Axon
Instruments).
Micropipettes were pulled from borosilicate glass capillary tubes
(GD-1, Narishige) on a programmable horizontal puller (Sutter
Instrument Co) and heat-polished with a microforge (Narishige). When
filled with the intracellular solution and immersed into the bath
(external) solution, the pipette tip resistance ranged between 1 and 2
M
. After gigaohm (16±6 G
, n=21) seal formation, the membrane
patch was ruptured with brief additional suction. The capacitive
transients elicited by symmetrical 10-mV steps from -80 mV were
recorded at 50 kHz (filtered at 10 kHz) for subsequent calculation
of capacitive surface area, access resistance, and input impedance.
Capacitance and series resistance compensation were optimized, and 80%
compensation of the effective access resistance was usually obtained.
Pulse Protocol and Analysis
After control data were obtained, bath perfusion was switched to
drug-containing solution, and cells were pulsed from -80 to +60 mV
every 30 seconds to monitor drug effects. The holding potential was
maintained at -80 mV, and the cycle time for any protocol was 0.1 Hz
in order to avoid accumulation of block or incomplete recovery from
inactivation or slow deactivation. The protocol to obtain
I-V relationships and activation curves consisted of
250-millisecond pulses imposed in 10-mV increments between -80 and +60
mV. The "steady-state" I-V relationships were obtained
by measuring the current at the end of the 250-millisecond
depolarizations. These results were corrected for passive linear leak
obtained from fits to the data between -80 and -40 mV, which is below
the threshold for channel opening for WT hKv1.5 and most mutations. For
T505V, V512M, and V512A mutant channels, passive linear leak was
observed between -80 and -60 mV. Deactivating "tail" currents
were recorded at -40 mV. The activation curve was obtained from
the maximum tail current amplitude after the capacitive transient.
Measurements were performed using the clampfit program of pClamp and by
a custom-made analysis program. Block after starting the
perfusion with each enantiomer reached steady state after 12 to 15
minutes; therefore, 12 to 15 minutes of equilibration was allowed
before assessment of drug effects. Since the effects of bupivacaine
enantiomers on T477S, T505I, T505V, T505S, T505A, L508M, and V512M
mutant channels were voltage dependent and, in the case of T477S,
T505S, T505V, T505A, L508M, and V512M, also time dependent,
steady-state block of all these mutant channels by bupivacaine
enantiomers was measured at the end of 250-millisecond duration
depolarizing pulses to +60 mV.
Drug-channel interactions were described by one or two binding curves.
The apparent affinity constant, Kd, and the Hill
coefficient, nH, for Kv2.1 and T505V, T505A, L508M, and
V512M mutated hKv1.5 channels were obtained from fitting the fractional
block (f) at various drug concentrations ([D]) to one Hill curve:
 | (1) |
Experimental
data from T505I, T505S, and T477S mutated hKv1.5
channels were
fitted to the sum of two Hill equations:
 | (2) |
where
I1 and
I2
are the fractional current of each component
(
I1+
I2=1), and
Kd1 and
Kd2 are the
apparent dissociation constants. Apparent rate
constants for binding
(
k) and unbinding (
l) were obtained from
solving
the following equations, in which
B represents
the time
constant of the fast initial drug-induced current decay after
activation
from the holding potential to +60 mV:
 | (3A) |
 | (3B) |
In the
case of T505I channels, in which block induced by
bupivacaine
enantiomers was not time dependent,
k and
l were estimated as
follows. If one assumes that the abrupt
transition (compared
with control) from the rising phase of channel
opening into
the reduced steady-state level represents fast
block, ie, block
occurring on the time scale of channel opening, then
the time
constant for block with 20 µmol/L
S(-)- or
R(+)-bupivacaine
should be <2.5
milliseconds (or a binding rate of >400/s).
Combined with Equations 3a

, and 3b

, this allows an estimate
of the rate constants.
Activation curves were fitted with a Boltzmann equation:
 | (4) |
where
s represents the slope factor, and E
represents the membrane
potential. The time course of tail
currents and the slow inactivation
were fitted with the sum of
exponentials. The activation kinetics
were determined using the
approach of the dominant time constant
of activation in which a single
exponential was fitted to the
latter 50% of the activation time
course.
40,41 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 the fit was judged
by the
2 criterion and by
inspection for systematic nonrandom
trends in the difference plot.
Voltage dependence of block was determined normalizing the
leak-corrected current in the presence of drug 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:
 | (3) |
where
z, F, R, and T have their usual meaning in
thermodynamics,
represents the fractional electrical
distance measured from
the inside of the cell membrane, and
Kd* represents the apparent
dissociation
constant at the reference potential (0 mV).
Statistical Analysis
Results are expressed as mean±SEM. 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 at P<.05.
 |
Results
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The segment between transmembrane regions S5 and S6 (P loop)
of
voltage-gated K
+ channels has been implicated as (part of)
the
ion-conducting pore; it controls K
+ selectivity, and
the internal
part of this segment determines internal TEA
binding.
28,29,35,42 Fig 1

illustrates the high degree of conservation in the P loop
and S6
sequences of various cloned mammalian
Shaker-related
channels.
21,43 The functional effects of mutations of
several nonconserved
sites in
Shaker, Kv3.1, and hKv1.5
channels have been interpreted
to indicate that these residues line
part of the ion-conducting
pore.
28,29,44
S(-)-Bupivacaine and R(+)-bupivacaine block WT
hKv1.5 channels in a time-dependent manner after channel opening, with
R(+)-bupivacaine being 7-fold more potent than the
S(-)-enantiomer (Fig 2
); the
Kd values for block were 4.1 and 27.3
µmol/L, respectively.4 Since bupivacaine block of
hKv1.5 channels resembled internal TEA and quaternary ammonium binding,
we tested whether pore mutation T477S affected affinity and/or
stereoselectivity of bupivacaine block (this site is equivalent to the
T441S mutation that reduced internal TEA affinity 10-fold in
Shaker).35 Moreover, since mutations in the
midsection of the S6 sequence of Shaker channels (T469) have
been implicated in the binding of hydrophobic TEA
derivatives,30 the effects of S(-)-bupivacaine
and R(+)-bupivacaine on mutations at T505 (equivalent to
T469 in Shaker) were also studied. T505 was replaced with
isoleucine (T505I) to increase the hydrophobic character of the
side chain. This mutation has been shown to influence alkyl-TEA
derivative block in Shaker channels30 and
quinidine block in hKv1.5.33 We also analyzed the
effects of mutation T505S, which preserves the polar character of
threonine present in the WT channel, and T505V, which retains the
geometry but is nonpolar (valine is the corresponding residue in the
Kv2 and Kv4 subfamilies; Fig 1
). In order to know if the size of the
amino acid at position 505 or only its polar character is a potential
molecular determinant for stereoselective block, we studied the effects
of bupivacaine enantiomers on T505A, which represents a
hydrophobic but smaller substitution at this position. The effects of
S(-)-bupivacaine and R(+)-bupivacaine on L508M
mutant hKv1.5 channels were also studied, since Kv3.1 channels exhibit
a methionine at the equivalent position that has been involved in their
internal TEA binding.29 Finally, since the valine at
position 512 of the hKv1.5 channel is one of the molecular determinants
of quinidine binding,33 the effects of bupivacaine
enantiomers on V512M and V512A were studied.

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Figure 2. Effects of S(-)- and
R(+)-bupivacaine [S(-)- and R(+)-Bupi, respectively]
on WT hKv1.5 channels (WT), T505I, and T505V (left panel) and T505A and
T505S (right panel). Current records were elicited by
250-millisecond depolarization pulses from -80 to +60 mV. For T505V,
the holding potential was -100 mV. Beneath the current records,
the concentration-response curves for both bupivacaine enantiomers are
shown. Tail currents were recorded after repolarization to -40 mV,
with the exception of T505V channels, in which they were recorded
at -60 mV.
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Mutations at Position 505: T505I, T505V, T505A, and T505S
Fig 2
shows currents elicited by the activation of T505I mutant
hKv1.5 channels in the absence and in the presence of 20
µmol/L S(-)- and R(+)-bupivacaine. The
basic hKv1.5 phenotype (fast activating delayed rectifier with
partial slow C-type inactivation) was largely preserved in this
mutation.44 As shown in Table 1
, the voltage dependence for channel
opening was shifted to slightly more negative membrane potentials, and
deactivation kinetics were much slower than in WT channels when
measured at -40 mV (
2 seconds) (Table 1
).19,33,36 The
T505V mutant hKv1.5 channels produced outward K+ currents
with an Eh shifted to more negative potentials than WT
channels and a much faster activation at 0 mV; the latter was due to
the shift in the activation curve. As for T505I, the T505V channels
closed much slower than did WT channels (Table 1
). T505S mutant
channels induced outward K+ currents that exhibited a
voltage dependence and an activation time course that were similar to
those of WT channels but a faster deactivation process (Table 1
).
Activation of T505A mutant channels generated outward K+
currents with an Eh shifted toward more negative
potentials, similar activation kinetics at +60 mV, but a faster
activation time constant at 0 mV. These channels deactivated
slower compared with WT channels but faster compared with T505I and
T505V (Table 1
). Detailed analysis of the gating properties of
these mutants was beyond the scope of the present study, but most
important for the present study was that the drug-channel
interactions (affinity, voltage dependence) were analyzed at
positive potentials (>+10 mV), where intrinsic channel properties were
minimally affected by the mutations.
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Table 1. Characteristics of the Currents Elicited by Eight
hKv1.5 Mutants (T477S, T505I, T505V, T505A, T505S, L508M, V512M, and
V512A) Compared With Those Observed in hKv1.5 WT Channels
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In contrast to the stereoselective block of the WT hKv1.5 channel,
block of T505I mutant channels by bupivacaine enantiomers did not
display stereoselectivity: both enantiomers were equipotent in blocking
these channels (Fig 2
). These data suggested that threonine at position
505 could be a basic requirement for the stereoselective block observed
in WT channels. Fig 2
shows the concentration dependence for block
induced by S(-)- and R(+)-bupivacaine of T505I
mutant channels. Although the concentration dependence for block of WT
hKv1.5 was adequately described by a single binding site
model,4 block induced by either enantiomer in the T505I
mutant was better fit assuming two binding sites, with the fraction of
channels blocked with high affinity being <30%. This lack of
stereoselectivity was observed over the whole concentration range
studied, with Kd values for the predominant
binding site of 20.0±1.4 µmol/L (n=31) and
22.0±6.0 µmol/L (n=26) for S(-)- and
R(+)-bupivacaine, respectively. The nH values
obtained for both enantiomers were close to unity (Table 2
); when the experimental data were fit
to a Hill curve with nH fixed to 1, the
Kd values were similar to those obtained without
any constriction of the Hill coefficient. Similar to block of WT
channels, block of T505I mutant channels by bupivacaine enantiomers was
voltage dependent at all concentrations tested. Using the Woodhull
formalism, the
value was 0.160±0.005 (n=9) and 0.150±0.007 (n=6)
for S(-)-bupivacaine and R(+)-bupivacaine,
respectively (Fig 3
) (Table 3
). This result is consistent
with the proposal that the cationic form of bupivacaine enantiomers
acts as an internal pore blocker.4
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Table 2. Apparent Kd and Association
(k) and Dissociation (l) Rate Constants for
S(-)-Bupivacaine and R(+)-Bupivacaine Block of
WT, T477S, T505I, T505V, T505A, T505S, L508M, V512M, and V512A Channels
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Figure 3. Voltage dependence of block of
S(-)-bupivacaine and R(+)-bupivacaine
[S(-)- and R(+)-Bupi, respectively] of mutant channels at position
505: T505I (A), T505V (B), T505A (C), and T505S (D). For each mutant,
the I-V relation and the relative current
(Idrug/Icontrol)
for each membrane potential are represented. Block steeply
increased in the range of activation of the channel. At membrane
potentials between saturation of activation curve and +60 mV, block
still increased with a shallow voltage dependence, which was fitted to
a Woodhull equation (Equation 5) (continuous line) that yielded the
value referred to the cytoplasmic side. Dashed lines represent
the fits of the activation curves for each mutant in the particular
experiment shown.
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Table 3. Values Obtained for the Block Induced by
S(-)-Bupivacaine and R(+)-Bupivacaine of
hKv1.5 WT Channels, Point Mutations, and Kv2.1 Channels
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To further characterize the molecular requirements of the
stereoselective bupivacaine-induced hKv1.5 block, we studied the
effects of both bupivacaine enantiomers in two additional substitutions
at the T505 position: T505V and T505S. If the stereoselective
bupivacaine block of hKv1.5 requires a polar interaction, we would
expect that block of T505V mutant channels would not be
stereoselective, since in this mutation a -CH3 group
replaces the threonine -OH group. This would not be in the case of
T505S, since serine, like threonine, contains an -OH group. The
mutation T505V decreased the affinity for block by
R(+)-bupivacaine (Kd=13.7±3.5
µmol/L [n=12] compared with 4.1 µmol/L for WT)
but increased the affinity for S(-)-bupivacaine (the
Kd dropped to 18.2±1.8 µmol/L
[n=12] compared with 27.3 µmol/L in WT). The Hill
coefficients were 0.76±0.07 and 0.78±0.18 for
S(-)-bupivacaine and R(+)-bupivacaine,
respectively. Again, when the nH values were fixed to
unity, the Kd values obtained for
S(-)- and R(+)-bupivacaine were 17.2±2.2 and
13.1±3.0 µmol/L, respectively (P>.05 versus
the Kd values obtained without the latter
constriction of the nH). These results suggest that binding
of a single bupivacaine molecule is sufficient to block the T505V
mutant channel. This opposite effect on the affinity for each
enantiomer effectively eliminated the stereoselective block
(P>.05 for Kds for both enantiomers
in T505V). As shown in Fig 2
, the concentration-dependent block of
T505V channels induced by bupivacaine enantiomers was well described by
a single site model, as in the case of WT hKv1.5 channels.4
Fig 2
also illustrates that the T505S mutation preserved the
stereoselectivity for block induced by bupivacaine enantiomers: the
R(+)-enantiomer was 8-fold more potent than was
S(-)-bupivacaine. The low-affinity
Kd values obtained for S(-)- and
R(+)-bupivacaine were 60.1±10.5 µmol/L (n=30)
and 7.4±1.6 µmol/L (n=21), respectively. As in the case
of T505I, the concentration-dependent block of both bupivacaine
enantiomers of T505S channels was best fit by assuming two binding
processes with different affinities, the amplitude of each component
being 50%. The effects of bupivacaine enantiomers on all these mutant
channels were reversible: the amplitude of the current was restored to
90±2% (n=142) after 20 minutes of perfusion of the cells with
drug-free solution.
To determine if the stereoselective block was determined by a
polar amino acid at position 505 or if, on the contrary, it was a
consequence of the presence of a small amino acid at this position, we
studied the effects of S(-)-bupivacaine and
R(+)-bupivacaine on T505A mutant channels. The mutation
T505A decreased 1.6-fold the affinity for block by
R(+)-bupivacaine (Kd= 6.4±0.4
µmol/L [n=12]) but increased 2.6-fold the affinity for
S(-)-bupivacaine (the Kd dropped to
10.3±1.9 µmol/L [n=12]) (P>.05 for
Kds for both enantiomers in T505A). The Hill
coefficients were 0.84±0.13 and 0.85±0.05 for
S(-)-bupivacaine and R(+)-bupivacaine. When the
nH values were fixed to unity, the
Kd values obtained for S(-)- and
R(+)-bupivacaine were 10.5±1.9 µmol/L and
6.6±0.7 µmol/L, respectively (P>.05 versus
the Kd values obtained without the latter
constriction of the nH). These results suggest that binding
of a single bupivacaine molecule is sufficient to block the T505A
mutant channel. This opposite effect on the affinity for each
enantiomer effectively eliminated the stereoselective block. As shown
in Fig 2
, the concentration-dependent block of T505A channels
induced by bupivacaine enantiomers was well described by a single-site
model, as in the case of WT hKv1.5 channels.
Interestingly, a valine residue is found at the T505 equivalent
position in Kv2 and Kv4 families (in Fig 1
). To further test whether a
polar interaction at this position is an important determinant for
stereoselective block of this class of voltage-gated K+
channels, we studied the effects of S(-)-bupivacaine and
R(+)-bupivacaine on Kv2.1 channels. As shown in Fig 4
, bupivacaine-induced block of Kv2.1
channels was not stereoselective, with S(-)- and
R(+)-bupivacaine exhibiting Kd values
of 8.0±0.2 µmol/L (n=15) and 12.4±0.8
µmol/L (n=11) (P>.05), respectively. The Hill
coefficients (nH) were 1.06±0.03 and 1.01±0.06 for
S(-)-bupivacaine and R(+)-bupivacaine,
respectively (Fig 4
). The apparent Kd values
obtained for both bupivacaine enantiomers were similar when the same
data were fitted constraining the Hill coefficients to unity. The
effects of bupivacaine enantiomers on Kv2.1 channels were reversible:
the amplitude of the current was restored to 89±3% (n=26) after 20
minutes of perfusion of the cells with drug-free solution. Other
aspects of block by either enantiomer were qualitatively and
quantitatively similar [
values of 0.172±0.010 and 0.184±0.009
for S(-)-bupivacaine and R(+)-bupivacaine,
respectively; Table 3
]. Taken together, these results suggest that
stereoselective block of WT hKv1.5 channels induced by bupivacaine
involves a specific interaction between the -OH moiety of the residue
at position 505.

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Figure 4. A, Effects of S(-)- and
R(+)-bupivacaine [S(-)- and R(+)-Bupi, respectively]
on Kv2.1 channels. Current records were obtained after applying
depolarizing pulses from -80 to +60 mV for 250 milliseconds. Tail
currents were obtained after repolarization to -40 mV. B,
Concentration-response curve for block induced by
S(-)-Bupi and R(+)-Bupi on Kv2.1
channels.
|
|
Mutations at Positions 508 and 512: L508M, V512M, and V512A
Fig 5A
shows currents for the L508M
substitution in hKv1.5 channels before and after application of 20
µmol/L S(-)- and R(+)-bupivacaine. The
basic hKv1.5 properties were mostly preserved in this mutation (Table 1
).44 The voltage dependence for channel opening
(Eh) was shifted to more positive membrane potentials.
Consequently, the activation kinetics were slower compared with WT
channels, without significant changes in deactivation kinetics measured
at -40 mV (Table 1
). In contrast, V512M mutant channels exhibit an
activation curve shifted to more negative membrane potentials, a
similar activation time constant at +60 mV, and a faster time constant
at 0 mV, presumably due to the negative shift in the activation curve.
Somewhat similar results were obtained for V512A (Table 1
). Both V512M
and V512A channels deactivated slower than WT channels (Table 1
).

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Figure 5. A, Effects of S(-)- and
R(+)-bupivacaine [S(-)Bupi and R(+)Bupi,
respectively] on WT hKv1.5 channels (WT) and indicated L508M and V512M
mutations. Current records were elicited by 250-millisecond
depolarization pulses from 80 to +60 mV. For V512M, the holding
potential was -100 mV. Beneath the current records, the
concentration-response curves for both bupivacaine enantiomers are
shown. Tail currents were recorded at -40 mV for L508M and at -60
mV for V512M. B, Time-dependent block induced by 100 µmol/L
S(-)Bupi and R(+)Bupi on L508M and V512M
hKv1.5 mutant channels. B indicates time constant of the
fast initial drug-induced current decay.
|
|
The methionine substitution of the hydrophobic residues L508 and
V512 abolished the stereoselectivity for block by bupivacaine
enantiomers (Fig 5A
). In both cases, the experimental data could be fit
to a single concentration-response curve. The Kd
values obtained in L508M channels were 35.7±1.5 µmol/L
(n=15) and 24.0±1.4 µmol/L (n=15) for S(-)-
and R(+)-bupivacaine, respectively. The nH
values obtained for both enantiomers were close to unity [1.14±0.06
and 1.14±0.08 for S(-)-bupivacaine and
R(+)-bupivacaine, respectively] (Table 2
), and the
Kd values obtained from fits with the Hill
coefficient constrained to unity were similar. Block induced by
bupivacaine enantiomers of L508M and V512M channels was time dependent,
although this time dependence was only visible at high concentrations
(>100 µmol/L) (Fig 5B
). Similar to block of WT channels,
block of L508M mutant channels induced by bupivacaine enantiomers was
voltage dependent at all concentrations tested, and it was described
with
values of 0.165±0.002 (n=4) and 0.160±0.007 (n=6) for
S(-)-bupivacaine and R(+)-bupivacaine,
respectively (Table 3
). The Kd values obtained
for V512M channels were 29.7±2.6 µmol/L (n=10) and
28.0±5.2 µmol/L (n=11) for S(-)- and
R(+)-bupivacaine, respectively. Again, the nH
values were close to unity (Table 2
). Block induced by bupivacaine
enantiomers of V512M channels was also voltage dependent,
consistent with
values of 0.162±0.005 (n=5) and
0.157±0.008 (n=5) for S(-)-bupivacaine and
R(+)-bupivacaine, respectively (Table 3
). For V512A mutant
hKv1.5 channels, we obtained Kd values for each
enantiomer that averaged 24.4±0.1 (n=2) and 3.2± 0.6
µmol/L (n=2) for S(-)-bupivacaine and
R(+)-bupivacaine, respectively. These results suggest that
stereoselective bupivacaine block of hKv1.5 channels requires at least
two hydrophobic interactions at positions 508 and 512. The decreased
hydrophobia of methionine would act to disrupt this interaction.
Mutation Affecting the Internal TEA Binding Site (T477S)
The threonine at the cytoplasmic turn of the P loop has been
implicated in binding of internally applied TEA.19,35 Since
bupivacaine action resembles internal quaternary ammonium block, we
sought to determine the possible involvement of this site in
bupivacaine binding. Therefore, we analyzed the effect of the
T477S mutations on affinity and stereoselectivity of bupivacaine block.
Outward K+ currents through T477S mutant channels exhibited
a voltage dependence for channel opening similar to that described
previously for WT hKv1.5 channels (Table 1
).19,36,44 The
activation kinetics were also similar, although the deactivation
kinetics at -40 mV were slower (Table 1
).
Fig 6
shows K+ currents from
T477S mutant channels in the absence and in the presence of 20
µmol/L S(-)-bupivacaine and
R(+)-bupivacaine. In these mutant channels,
R(+)-bupivacaine was again more potent than
S(-)-bupivacaine [Kd values of
45.5±4.4 µmol/L (n=35) and 7.8±1.6 µmol/L
(n=46) for S(-)- and R(+)-bupivacaine,
respectively], indicating that the WT stereoselectivity was preserved
in this mutation (Table 2
). Block of T477S mutant channels induced by
either enantiomer was reversible: the amplitude of the current was
restored to 85±1% (n=81) after a 20-minute perfusion of the cells
with drug-free solution. Fig 6
shows the dose-response curve for the
block induced by S(-)-bupivacaine and
R(+)-bupivacaine of T477S mutant channels. Contrary to the
WT channel, the concentration-dependence curve could not be fit using a
single binding site model. A good fit was obtained using a two-site
model, in which each component accounts for
50% of the binding.
Importantly, the stereoselectivity was preserved over the full
concentration range; ie, R(+)-bupivacaine was the more
potent enantiomer for both sites.

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Figure 6. Stereoselective block by
bupivacaine enantiomers of T477S mutant hKv1.5 channels. Top panels
show original records obtained in the absence and in the presence
of S(-)-bupivacaine and R(+)-bupivacaine
[S(-)Bupi and R(+)Bupi, respectively]. Bottom panel shows the
concentration-response curve for both enantiomers.
|
|
Voltage Dependence of Block
To interpret stereoselective effects in a meaningful way, it is
necessary to test whether other aspects of drug binding were preserved.
The voltage dependence of hKv1.5 block by S(-)- and
R(+)-bupivacaine displays a biphasic voltage dependence with
a steep phase coincident with the voltage dependence of channel opening
and a shallow phase at potentials positive to 0 mV.4 Using
the Woodhull formalism,45 the latter can be used to gauge
the equivalent
value, ie, the fraction of the transmembrane
electrical field sensed at the binding site. We routinely determined
this parameter for each mutation by using the fractional
reduction of current during depolarizing steps between 0 and +60 mV.
The values for the apparent
value clustered in a narrow range
(
=0.15 to 0.20) similar to that of WT channels (Table 3
) (Fig 3
).
This was of particular interest in the case of T505I, T505V, T505A and
V512M mutations, which displayed slower deactivation kinetics and
variable negative shifts in the activation curve with respect to WT
channels (Table 1
). Despite these kinetic changes, it is important to
note that we determined the affinity for block at strong
depolarizations (+60 mV), where the differences in gating were minimal.
Time Dependence of Block
Block induced by either bupivacaine enantiomer of T477S, T505V,
T505S, T505A, L508M, and V512M channels was time dependent, as was the
case for WT channels.4 In those cases in which block was
best fit assuming two binding processes with different affinities
(T477S, T505S, and T505I), the rate constants for the lower affinity
process, which accounted for 50% to 70% of block, were
analyzed. As shown in Table 2
, there were no major changes in
the association rate constants for block of T477S by bupivacaine
enantiomers versus WT channels. However, the dissociation rate
constants for these channels were faster than those observed for WT
channels.4 These faster dissociation kinetics suggest that
the lower affinity of S(-)-bupivacaine and
R(+)-bupivacaine reflects a decreased stability of the
drug-channel complex.
The dissociation rate constants of bupivacaine enantiomerinduced
block of T505S channels were increased to the same extent [2.2-fold
and 2.6-fold for S(-)-bupivacaine and
R(+)-bupivacaine, respectively] (Table 2
). Similarly, the
association rate constant was increased 1.05-fold for the
S(-)-enantiomer and 2.2-fold for the
R(+)-enantiomer. This similar effect on both dissociation
and association rate constants explains the decreased affinity observed
for both enantiomers as well as the similar stereoselective block of
these mutant channels versus WT channels by bupivacaine enantiomers.
Compared with that for WT channels, the dissociation rate constant for
the interaction with T505V channels was increased 2-fold for both
enantiomers. On the other hand, the association rate constant was
increased 3-fold for S(-)-bupivacaine, whereas it decreased
1.3-fold for R(+)-bupivacaine. This opposite effect on the
association rate constant for both bupivacaine enantiomers is
sufficient to explain the lack of stereoselective bupivacaine block
observed in the present experiments on these mutant channels (Table 2
).
The dissociation rate constants for the interaction between bupivacaine
enantiomers and T505A channels remained similar to those values
calculated for WT channels (Table 2
). However, the association rate
constant increased 2.5-fold for S(-)-bupivacaine and
remained unaltered for R(+)-bupivacaine (Table 2
). The
modification of these values explains the lack of stereoselective
block of bupivacaine of these channels.
The time dependence of block of T505I channels by bupivacaine
enantiomers could not be resolved experimentally. The following
experimental observations support an open-channel block mechanism:
biphasic voltage dependence with a
value similar to that described
in WT channels and a slower time constant of deactivation versus
control conditions. Therefore, lower limits for the association and
dissociation rate constants were extracted assuming a very fast
transition (compared with WT) from the rising phase of channel opening
into the reduced steady-state level representing a fast
block, ie, block occurring on the time scale of channel opening
(
B<1.5 milliseconds). Under these assumptions, mutation
T505I increased the dissociation rate constant for both enantiomers
10-fold (from 24 to 250 s-1) and the association rate
constant for S(-)-bupivacaine 11-fold. However, it only
increased this parameter 2.65-fold for
R(+)-bupivacaine, which explains the lack of
stereoselective bupivacaine block of these mutated channels.
Compared with that for WT channels, the dissociation rate
constant for the interaction between S(-)-bupivacaine and
L508M channels was not modified, whereas it decreased 2-fold for the
interaction with R(+)-bupivacaine. On the other hand, the
association rate constant between S(-)-bupivacaine and
R(+)-bupivacaine and L508M channels decreased 1.8-fold and
9.6-fold, respectively. As in the case of T505V, the opposite effect on
the association rate constants for both bupivacaine enantiomers is
enough to explain the lack of stereoselective bupivacaine block
observed in these experiments (Table 2
).
Similarly, in V512M mutant channels, the association rate constant for
the interaction between S(-)-bupivacaine and the channel
was similar to that obtained in WT channels, whereas it decreased
3.5-fold for R(+)-bupivacaine. The dissociation rate
constants for both enantiomers were increased 2-fold. Therefore, the
opposite effect on the association rate constants, together with the
similar effects on the dissociation kinetics, can explain the lack of
stereoselective block induced by bupivacaine enantiomers on V512M
channels.
 |
Discussion
|
|---|
Stereoselective block of hKv1.5 channels by bupivacaine strongly
indicates
that it results from binding to a specific binding site,
which
in turn suggests that the three-dimensional configuration of
the
drug can be an important determinant of drug binding to
its receptor
(in addition to other factors, such as molecular
size and hydrophobia).
In the present study, we have localized
putative amino acids in
hKv1.5 responsible for stereoselective
interactions between
enantiomers of the local anesthetic bupivacaine.
Our results suggest
that (1) a polar interaction at position
505 of the channel protein and
(2) hydrophobic interactions
at positions 508 and 512 are required for
stereoselective interactions
between bupivacaine enantiomers and hKv1.5
channels.
Mutation at the Internal TEA Binding Site (T477S)
The fact that the affinity for bupivacaine block was modified in
the T477S mutation suggests a possible overlap of the binding site for
bupivacaine with that for the internal TEA, as has been suggested for
Shaker channels.46 This is in contrast with the
lack of effect of the same mutation regarding quinidine
block.33 The reduced affinity due to an increase in the
apparent dissociation rate constant is compatible with a reduced
stabilization of binding, which may reflect the lack of a
CH3 group in the serine residue. However, the results
obtained with this mutant suggest that this residue is not involved in
determining stereoselective block of hKv1.5 channels by bupivacaine.
Indeed, block induced by both bupivacaine enantiomers was
stereoselective, with R(+)-bupivacaine being 6-fold more
potent than S(-)-bupivacaine, similar to the 7-fold ratio
previously described for WT hKv1.5 channels.4 This could
reflect a more stable interaction of the R(+)-enantiomer
with the receptor. However, difference in potency between both
enantiomers was explained largely by their different association rate
constants, which was faster for R(+)-bupivacaine. Since
these drugs are enantiomers, the difference in association rates
suggests that the S(-)-enantiomer needs to adopt a less
favored conformation than R(+)-bupivacaine, similar to that
observed in WT channels.4
Mutations at Position 505 (T505I, T505V, T505A, and T505S)
The replacement of threonine at position 505 by the hydrophobic
residues, valine, alanine, and isoleucine, completely abolished
the stereoselective block observed in WT hKv1.5 channels. In contrast,
stereoselective block was retained and even enhanced with the
conservative replacement of threonine 505 by serine (T505S), which
preserved the polar hydroxyl side chain. Taken together, these results
suggest that bupivacaine stereoselective block of hKv1.5 channels
is derived from a stereoselective interaction between the drug and the
channel, irrespective of the size of the side chain at this position.
Furthermore, we propose that the hydroxyl group of this threonine is a
basic requirement, since stereoselectivity was only preserved with the
serine substitution but abolished with all hydrophobic substitutions
(T505V, T505A, and T505I). These results could be due to the following:
(1) the association rate constants of block changed in opposite
directions compared with those observed in WT channels (for T505V), (2)
the increase in the association rate constant was more pronounced for
S(-)-bupivacaine than for R(+)-bupivacaine (for
T505I), or (3) the association rate constant remained unaltered for one
enantiomer and increased for the other one (for T505A) (Table 2
). It is
interesting to note that hydrophobic substitutions at 505 enhanced the
affinity for quinidine in this channel33 but had opposing
effects on the bupivacaine affinity. This further indicates
drug-specific interactions at the internal local anesthetic binding
site.
Interestingly, the equivalent 505 residue of Kv2.1 channels (position
389) and Kv4.3 channels is a valine, in contrast to the threonine in
hKv1.5 and Shaker (Fig 1
). Moreover, bupivacaine-induced
block of these two K+ channels is not stereoselective.
These results suggest the existence of a similar bupivacaine receptor
in Kv1, Kv2, and Kv4 channels with the same stereoselective
determinants. In fact, we have observed that block of Kv4.3 channels by
bupivacaine enantiomers is not stereoselective, with a
Kd for both enantiomers of
31
µmol/L (L. Franqueza, J. Eck, C. Valenzuela, M.M. Tamkun, and
D.J. Snyders, unpublished data, 1997). On the basis of these results,
one would expect that block of Kv1.3 (carrying a serine at the T505
equivalent position) would also be stereoselective, but this remains to
be determined. Moreover, the inhibition induced by bupivacaine
enantiomers of the delayed rectifier K+ current
recorded in frog sensory ganglion cells was not stereoselective.
This K+ current is very similar in voltage- and
time-dependent properties to Kv2.1 current; therefore, the results
shown in the present study could explain the molecular basis of the
lack of stereoselectivity observed in native neuronal cells previously
described.18
Mutations at Positions 508 and 512 (L508M, V512M, and V512A)
Replacement of L508 or V512 by a methionine abolished bupivacaine
stereoselective block. However, substitution of V512 by an alanine
preserved it, with Kd values very similar to
those observed in WT channels.4 These results suggest that
stereoselective block induced by bupivacaine involves hydrophobic
interactions at positions 508 and 512, in addition to the polar
interaction with T505. Leucine, valine, and alanine are all hydrophobic
amino acids, whereas methionine is a less hydrophobic residue.
Moreover, the sulfur atom in methionine can act as an electron donor or
acceptor; therefore, it could establish electrostatic interactions
either with the partially positively charged amide or with the
partially negatively charged carbons of the aromatic ring (Fig 7
). The establishment of these
electrostatic interactions would redirect the position of the entire
molecule; thus, it would abolish the stereoselective bupivacaine block
observed in WT channels.4 An alternate explanation would be
that a decrease of the local hydrophobicity or steric hindrance from
the bulkier methionine side chain disrupts the interaction between
bupivacaine and its receptor site. Although these hypotheses could
theoretically explain the experimental results, further
structure-analysis studies at the chemical level are required
in order to discern the exact mechanism by which these mutations
abolish the bupivacaine stereoselective block.
Biphasic Dose-Response Curves Suggest Two Different Open States
With Different Affinities
A surprising finding of the present study is that
dose-response curves obtained for some mutations (T505I, T505S, and
T477S) were better fit assuming two binding processes with different
affinities. This could be explained by the existence of two different
populations of channels with different affinities for bupivacaine
enantiomers. This seems unlikely to occur with a cloned channel that
forms homomultimers. Heteromultimer formation with
endogenous subunits is unlikely, since the
Ltk- cells used do not contain
endogenous voltage-gated ion currents or detectable
K+ channel mRNA.19 The
Ltk- cells do contain the Kvß2.1 subunit,
which resembles the heterologously expressed hKv1.5, but this ß
subunit does not affect antiarrhythmic drug action.47 In
addition, we did not obtain evidence for nonhomogeneous
channel population when examining quinidine pharmacology and channel
gating.33,48 A second possible explanation is that these
mutations are introducing a high-affinity binding site for bupivacaine
in hKv1.5 channels that was not present in WT channels. However, if
there were two separate drug binding sites, then all the current would
be blocked by the high-affinity binding site before the low-affinity
site could be occupied, thus masking low-affinity binding. A third
explanation would be that mutated residues in each subunit of the
homotetramer permits the access of more than one bupivacaine molecule
in the pore. This would then lead to negative cooperative interactions
between them (electrostatic or hindrance interactions) as has recently
been proposed for block of IRK1 channels by polyamines.49
Finally, the hypothesis we favor is that multiple open states exist
with different bupivacaine affinities, even in WT hKv1.5 channels.
Bupivacaine binding to the high-affinity open state could
represent an intermediate transition state in WT channels, as
has been proposed previously to explain the blockade of cardiac
Na+ channels by QX-314.50 Under this framework,
T477S, T505I, and T505S mutant channels would be stabilizing an
ultrafast (higher affinity) interaction between bupivacaine and hKv1.5
channels. In fact, we have preliminary evidence that multiple open
states exist in hKv1.5 channels and that conversion between them can be
influenced by drug concentration.48 Thus, as bupivacaine
concentration increases, an open state with low affinity is favored.
This hypothesis requires a drug-induced shift in gating that is
independent of open-channel block, and indeed, we have observed that
concentrations of racemic bupivacaine below the
Kd for block of hKv1.5 actually modify channel
activity in a manner consistent with the transition from one
open state to another (J. Vicente, M. Longobardo, L. Franqueza, E.
Delpón, D.J. Snyders, and C. Valenzuela, unpublished data, 1996).
Mutations such as T505S could enhance the drug-induced shift to the
low-affinity state and thus unmask bupivacaine-induced transitions from
one open state to the next. The mechanism responsible for the biphasic
dose-response curves requires further investigation.
Limitations of the Present Study
A potential problem of site-directed mutagenesis is that the
observed results may be derived from more generalized perturbations of
the protein. In fact, we observed differences in the deactivation
kinetics of several mutants used in the present study (T505I,
T505V, V512A, V512M, and L508M), which in part resulted from shifts in
the voltage dependence of channel opening (Table 1
). However, we could
not discern a relationship between altered deactivation gating and the
affinity for open-channel block at depolarized potentials; ie, T505V
slowed the deactivation gating, whereas T505A did not, and both
suppressed stereoselective bupivacaine block. Moreover, we
analyzed not only the apparent affinity but also the
association and dissociation kinetics and the
value and checked for
drug-modified tail current kinetics as a qualitative indicator for
open-channel block. The preserved open-channel block mechanism and the
similarity of the
values suggest that no major conformational
changes were introduced to the binding site.
Conclusions
The results shown in the present study suggest a model of
stereoselective interactions between bupivacaine enantiomers and hKv1.5
channels that involves the interaction between bupivacaine and the -OH
group of threonine at position 505, combined with hydrophobic
interactions at positions 508 and 512 of the channel protein. The
enhanced stereoselectivity in the T505S mutant and the lack of
stereoselectivity in T505I, T505A, and T505V mutants as well as in
Kv2.1 and Kv4.3 channels are consistent with this hypothesis.
T505 and V512 are separated by two turns of the S6 helix, so that they
are
11 Å apart. Bupivacaine is
11 Å in length51
with positively charged and hydrophobic moieties at either end.
Therefore, the positively charged end could interact with the T505, and
the hydrophobic aromatic ring could establish a hydrophobic interaction
with the valine residue at position 512, which is perturbed by a
substitution with methionine, whose sulfur atom decreases its
hydrophobicity (Fig 7
). We have previously reported that a decrease in
the length of the N-substituent decreases the potency of the molecule
to block hKv1.5 channels.52 One possible explanation would
be that this alkyl chain interacts with a hydrophobic amino acid (ie,
L508) in the S6 of the channel (Fig 7
). Our results further suggest
that threonine at position 477 is involved in the binding of
bupivacaine, although it does not determine the stereoselective block.
The unusual dose-response curve observed in T477S, T505I, and T505S
mutant channels may reflect a transition-intermediate state between
bupivacaine and the channel.
 |
Selected Abbreviations and Acronyms
|
|---|
 |
= |
fractional electrical distance |
| Eh |
= |
voltage at which 50% of the channels are open |
| I-V |
= |
current-voltage |
| PCR |
= |
polymerase chain reaction |
| TEA |
= |
tetraethylammonium |
| WT |
= |
wild type |
|
 |
Acknowledgments
|
|---|
This study was supported by FIS 95/0318 (Dr Valenzuela), CICYT
SAF960042
(Dr Tamargo), and National Institute of Health grants
HL-47599
(Dr Snyders), HL-46681 (Drs Snyders and Tamkun), and HL-49330
(Dr
Tamkun). The authors want to express their thanks to Dr Ricardo
Caballero
for his constructive criticism of the manuscript. We also
thank
Guadalupe Pablo and Ruben Vara for their excellent technical
assistance.
We thank Chiroscience (Cambridge, UK) for supplying us with
S(-)-bupivacaine
and
R(+)-bupivacaine.
 |
Footnotes
|
|---|
Previously published as preliminary reports in abstract form
(
Biophys J. 1996;70:A400;
Biophys J. 1997;72:A141).
Received February 10, 1997;
accepted September 19, 1997.
 |
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