© 1999 American Heart Association, Inc.
Original Contribution |
Local Anesthetic Anchoring to Cardiac Sodium Channels
Implications Into Tissue-Selective Drug Targeting
From the Departments of Physiology and Medicine (R.A.L., R.G.T., P.H.B.), University of Toronto; Centre for Cardiovascular Research (R.A.L., R.G.T., P.H.B.), The Toronto Hospital, Toronto; and Toronto Research Chemicals, Inc. (K.H., D.S.D), North York, Ontario, Canada.
Correspondence to Peter H. Backx, The Toronto Hospital, General Division, CCRW 3-802, 101 College St, Toronto, Ontario, Canada M5G 2C4. E-mail p.backx{at}utoronto.ca
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Abstract |
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Abstract—Local anesthetics inhibit Na+ channels in a variety of tissues, leading to potentially serious side effects when used clinically. We have created a series of novel local anesthetics by connecting benzocaine (BZ) to the sulfhydryl-reactive group methanethiosulfonate (MTS) via variable-length polyethylether linkers (L) (MTS-LX-BZ [X represents 0, 3, 6, or 9]). The application of MTS-LX-BZ agents modified native rat cardiac as well as heterologously expressed human heart (hH1) and rat skeletal muscle (rSkM1) Na+ channels in a manner resembling that of free BZ. Like BZ, the effects of MTS-LX-BZ on rSkM1 channels were completely reversible. In contrast, MTS-LX-BZ modification of heart and mutant rSkM1 channels, containing a pore cysteine at the equivalent location as cardiac Na+ channels (ie, Y401C), persisted after drug washout unless treated with DTT, which suggests anchoring to the pore via a disulfide bond. Anchored MTS-LX-BZ competitively reduced the affinity of cardiac Na+ channels for lidocaine but had minimal effects on mutant channels with disrupted local anesthetic modification properties. These results establish that anchored MTS-LX-BZ compounds interact with the local anesthetic binding site (LABS). Variation in the linker length altered the potency of channel modification by the anchored drugs, thus providing information on the spatial relationship between the anchoring site and the LABS. Our observations demonstrate that local anesthetics can be anchored to the extracellular pore cysteine in cardiac Na+ channels and dynamically interact with the intracellular LABS. These results suggest that nonselective agents, such as local anesthetics, might be made more selective by linking these agents to target-specific anchors.
Key Words: local anesthetic • cardiac • Na+ channel • methanethiosulfonate • drug targeting
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Introduction |
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Local anesthetics and related Na+ channel modifiers preferentially bind to the open or inactivated conformations of Na+ channels,1 2 thus blocking Na+ current and reducing cellular excitability. As a result, these agents are used clinically to treat a variety of conditions. For example, the elimination of pain and the management of seizures are achieved by blocking nerve Na+ channels with local anesthetics.2 On the other hand, these agents are used in controlling the symptoms associated with skeletal muscle diseases, such as hyperkalemic periodic paralysis and other related muscle myotonias.3 In heart, Na+ channel blockers are used to treat patients with ischemia-related arrhythmias4 and the congenital long-QT syndrome, LQT3.5 There is also a recent report suggesting that local anesthetic might be useful in the treatment of torsade de pointes and related arrhythmias.6 The ability of local anesthetics to target different tissue-specific Na+ channel homologues in various clinical applications highlights the nonselective nature of these agents. Indeed, indiscriminate blockade of Na+ channels regardless of tissue location is responsible for many of the side effects associated with their use. Despite these observations, local anesthetics do block Na+ channels with slightly higher potency in heart compared with nerve or skeletal muscle channels possibly because of longer cardiac action potentials, which promotes high-affinity binding to inactivated channels.1 7 8 Alternatively, this modest preferential block of cardiac Na+ channels might result from an intrinsically greater affinity of drug binding9 10 and/or differences in steady-state inactivation gating.11
Despite these modest differences in drug binding between
tissue-specific Na+ channel homologues, local
anesthetics bind to all voltage-gated Na+ channel
pores and inhibit ionic current by either directly occluding ion flow
through open channels or by promoting channel
inactivation.1 2 Moreover, the site for local anesthetic
binding has been localized to a pair of aromatic residues within the
sixth transmembrane segment of domain IV,12 which are
conserved between various tissue-specific Na+
channel homologues13 14 15 16 and therefore cannot be
responsible for the observed differences in binding. Molecular models
of Na+ channels place these aromatic residues
deep within the channel pore on its intracellular face.17
This location is supported by biophysical studies establishing that
internally perfused, permanently charged local anesthetics traverse
70% of the transmembrane electric field to reach its binding
site.18 On the other hand, externally applied
Cd2+ senses 21% of the transmembrane electric
field when binding to a pore-lining cysteine residue located in the
ascending limb of the P (pore) loop in domain I of cardiac
Na+ channels (Figure 1A
).19 20 These observations
suggest that the external Cd2+ binding site in
domain I is located in close proximity to the local anesthetic binding
site (LABS) in domain IV.
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The relationship of these 2 pore regions was assessed in this study by
linking the hydrophobic local anesthetic benzocaine (BZ) to the
sulfhydryl reactive methanethiosulfonate (MTS) group via a variable
length linker (L) (Figure 1B
). We took advantage of previous
studies establishing that the unique cysteine residue in the external
pore of cardiac Na+ channels19 20 21
(Figure 1A) reacts readily with externally applied
sulfhydryl-modifying agents. We hypothesized that these drugs would
anchor to the extracellular pore cysteine and interact with the
intracellular LABS provided the linker was sufficiently long (Figure 1C).
The hydrophobic local anesthetic benzocaine was chosen for
these studies, because this agent does not rely on hydrophilic pathways
when binding to the LABS,7 which might otherwise hamper
drug access. Our experiments demonstrate a very close physical
relationship between the unique cysteine in the external pore of the
cardiac Na+ channel and the LABS. Our results
suggest a novel paradigm for creating target-specific drugs wherein a
nonselective drug, such as a local anesthetic or
Ca2+ channel blocker, is linked to an anchor that
is designed to interact with a chosen, distinct molecular site on the
desired channel protein.
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Materials and Methods |
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Site-Directed Mutagenesis
Site-directed mutagenesis was performed using uracil-enriched single-stranded DNA according to the methods of Kunkel22 and confirmed by dideoxy nucleotide sequencing.23 A 1.9-kb BamHI-SphI or a 2.5-kb SphI-KpnI fragment of the rSkM115 Na+ channel pore mutant was subcloned into pGEM-11f+ or pGEM-7f+ (Promega), respectively. The mutant cassette was subcloned into the full-length rSkM1 Na+ channel construct in pGW1-CMV (British Biotechnology).
Heterologous Channel Expression and Recording
Oocytes were removed from female Xenopus laevis frogs
anesthetized by immersion in a solution containing 0.2%
3-aminobenzoic acid ethyl ester (tricaine; Sigma). Oocytes were
digested for 60 to 90 minutes with 1.5 mg/mL collagenase
(Type 1A, Sigma) dissolved in a solution containing (in mmol/L)
NaCl 82.5, KCl 2, MgCl2 1, and HEPES 5 (pH 7.6
with NaOH). Nuclear injections were performed on healthy stage IV
through VI oocytes with either the hH124 or rSkM1
-subunit cDNA (5 ng per 50 nL) subcloned into pcDNA3 (Invitrogen) or
pGW1H (British Biotechnology), respectively. The rSkM1 was coexpressed
with the rat brain ß1 subunit25
(1:5 wt/wt ratio) to reconstitute native channel
behavior.26 Oocytes were incubated in a solution (ND96)
containing (in mmol/L) NaCl 96, KCl 2,
BaCl2 1, MgCl2 1, and HEPES
5 (pH 7.6) and supplemented with 5 pyruvate, 0.5 theophylline, and 50
µg/mL gentamicin.
Two-electrode voltage-clamp recordings were performed at room
temperature using a Warner OC-725C amplifier on oocytes 1 to 5 days
after injection. Only oocytes with peak Na+
currents <6 µA were used in our studies to minimize voltage-clamping
errors. Agarose-plugged electrodes (TW120F-6; World Precision
Instruments) were pulled using a Sutter P-87 horizontal puller and were
filled with 3 mol/L KCl, having a final resistance of 1 to 3 M
.
Recording solution was ND96.
Rat Ventricular Myocyte Isolation and Patch-Clamp
Recording
Rat ventricular cardiac myocytes were isolated as
described previously.27 Briefly, male Sprague-Dawley rats
(250 to 300 g) were heparinized (3000 U/kg) and
anesthetized with 150 mg/kg pentobarbital. Hearts were excised
and retrogradely perfused for 5 minutes with a Krebs-Hensleit buffer
consisting of (in mmol/L) NaCl 123, KCl 5.4,
CaCl2 1, MgSO4 1.2,
NaH2PO4 1.2,
NaHCO3 20, and glucose 5.6 and
equilibrated with a 95% O2-5%
CO2 mixture (pH 7.4). Next, the hearts were
perfused for 5 minutes with a calcium-free Krebs-Hensleit solution
followed by perfusion for 8 minutes with the same solution containing
collagenase (type II, 0.6 mg/mL, Boehringer
Mannheim) and protease (type XIV, 0.05 mg/mL, Sigma). At the end of the
digestion period, hearts were perfused for 5 minutes with an
enzyme-free high-K+ solution (KB) consisting of
(in mmol/L) KCl 120, MgCl2 1,
K2-EGTA 0.5, glucose 10, and HEPES 20 (pH to 7.4
with KOH). The hearts were dismounted, and the atria and aorta were
removed. Ventricular tissue was cut into small pieces, and
cells were mechanically isolated by trituration with a Pasteur pipette
and filtering through nylon mesh. Cells were stored in KB solution
until required. Only calcium-tolerant, rod-shaped, quiescent myocytes
with clear cross-striations were used for
electrophysiological
recordings.
Na+ currents were recorded using the
whole-cell patch-clamp configuration28 with an Axopatch
200-A amplifier (Axon Instruments). Rat cardiac ventricular
myocytes were placed in a 1-mL bath and perfused with extracellular
solution with the following composition (in mmol/L):
tetramethylammonium chloride 135, NaCl 5, CaCl2
1, MgCl2 1, glucose 10, and HEPES 10 (pH to 7.4
with tetramethylammonium hydroxide). Recording electrodes were
fabricated from borosilicate glass (TW150F-4, World Precision
Instruments), pulled on a Sutter puller, and heat polished to a final
resistance of <1 M when filled with intracellular solution. The
intracellular solution consisted of (in mmol/L) CsF 150, EGTA 10,
and HEPES 10 (pH to 7.2 with CsOH). Series resistance compensation was
60% to 80%.
Experimental Protocols and Data Analysis
Whole-cell Na+ current-voltage
relationships, steady-state inactivation, and recovery from
inactivation were recorded from oocytes or rat
ventricular myocytes. Cells were then exposed to the
methanethiosulfonate benzocaine (MTS-LX-BZ [X represents 0, 3,
6, or 9]) compounds or benzocaine (500 µmol/L) for at
least 10 minutes to allow for complete channel modification. Washout of
the drugs was performed for 5 to 10 minutes with recording
solution. MTS-LX-BZ, benzocaine, and benzyl methanethiosulfonate
(MTSBN) were dissolved in DMSO and diluted to the desired
concentration. Lidocaine and 2-hydroxyethyl methanethiosulfonate
(MTSHE) were dissolved in ND96. The final concentration of DMSO
(0.01%) had no significant effect on any parameters
measured. MTS-LX-BZ compounds were synthesized at Toronto
Research Chemicals, Inc. MTSBN and MTSHE were purchased from
Toronto Research Chemicals, Inc. Benzocaine and lidocaine were
purchased from Sigma.
Whole-cell currents were elicited by 50-ms step depolarizations from
–80 to +60 mV from a holding potential of –100 mV (oocytes) or –120
mV (cardiac myocytes). Leak and capacitance subtraction were performed
as described previously.31 Steady-state inactivation was
determined using a standard 2-pulse protocol. Data were normalized to
the current amplitude recorded at –140 mV. Normalized steady-state
inactivation relationships were fit with a single Boltzmann
distribution,
A1/(1+exp[(V–V1/2)/k])+A2,
where V1/2 is the voltage of half inactivation,
k is the slope factor, A1 is the
amplitude of the relationship, and A2 is
the amplitude of the noninactivating
component. Recovery from inactivation was assessed with a 2-pulse
protocol with identical 50-ms step depolarizations to –10 mV from a
holding potential of –100 mV separated by a variable recovery
period. The data for the recovery from inactivation were fit with the
biexponential function,
1–{Afast/[1+exp(–x/
fast)]+Aslow/[1+exp(–x/slow)]},
where Afast and
Aslow are the amplitudes and
fast and slow
are the time constants of the fast and slow components, respectively.
Data acquisition and analysis were performed using
custom-written software.
Curve fitting was performed using a nonlinear least-squares iterative method that uses Marquardt-Levenberg algorithms. Statistical analysis was performed using a 1-way ANOVA followed by a Student-Newman-Keuls test.29 A P value <0.05 was used to denote statistical difference between groups. Data are expressed as mean±SEM.
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Results |
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Figure 2A
shows that exposure of
human heart (hH1) Na+ channels expressed in
Xenopus oocytes to 500 µmol/L benzocaine resulted in
a 43±3% reduction in peak current measured at –10 mV (n=5). The
effects of benzocaine on peak currents were completely reversed after
10 minutes of drug washout. The application of MTS-L0-BZ at a
concentration of 500 µmol/L reduced peak hH1
Na+ currents to 62±8% (n=7) of control (Figure 2A).
This reduction was not reversed after drug washout (ie,
reduced to 49±8% of control), which indicates that most channels were
irreversibly modified by MTS-L0-BZ (also see below). In contrast,
application of MTS-L0-BZ (500 µmol/L) to rat skeletal muscle
Na+ channels (rSkM1) lacking a pore cysteine
resulted in a 35±5% (n=5) current reduction, which was
completely reversed on drug washout (Figure 2A).
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To establish whether MTS-L0-BZ interacted specifically with the
cysteine pore residue in hH1 channels (Figure 1), an equivalent
cysteine residue was introduced into the rSkM1 channel pore
(Y401C).19 Y401C channels resemble cardiac
Na+ channels in relation to block by tetrodotoxin
and Cd2+ and to single-channel
conductance19 but are otherwise similar to wild-type rSkM1
channels. A 10-minute exposure of Y401C channels to 500 µmol/L
MTS-L0-BZ followed by a 5-minute washout resulted in an irreversible
reduction in current amplitude (38±4%, n=5), as observed in hH1
channels (Figure 2A). Longer washout periods did not restore
peak current amplitude to predrug values. These data are
consistent with a specific interaction of MTS-L0-BZ with a
cysteine residue in the extracellular face of the pore.
To further verify that MTS-L0-BZ reacted specifically with the cysteine
pore residue in both hH1 and Y401C, the effects of this drug on the
Cd2+ sensitivity of the channels were examined.
Modification of hH1 and Y401C channels with external application of
methanethiosulfonate compounds is known to reduce sensitivity to block
by Cd2+ and Zn2+ by
cross-linking with extracellular reduced free sulfhydryl
groups.20 21 30 Accordingly, application of reducing
agents such as DTT is expected to reverse the effects of
methanethiosulfonate modification on Na+ channels
after drug washout. Modification with MTS-L0-BZ (500 µmol/L)
reduced the sensitivity of hH1 channels to Cd2+
block (IC50) from 50±8 to 1552±245
µmol/L (n=3; P<0.05) and for Y401C channels from 16±3 to
1081±322 µmol/L (n=3; P<0.01) (Figure 3C). The
Cd2+ sensitivity of the MTS-L0-BZ–modified Y401C
channels is similar to that observed for the wild-type rSkM1
channel.30 Figures 3A and 3B show that the
application of MTS-L0-BZ reduced peak Y401C Na+
currents and the rate of recovery from inactivation after washout.
Subsequent application of the sulfhydryl-reducing agent DTT (10
mmol/L) completely restored peak current amplitude (Figure 3A),
the rate of recovery from inactivation (Figure 3B), and
Cd2+ sensitivity of Y401C channels previously
modified with MTS-L0-BZ (Figure 3C). These data suggest that
MTS-L0-BZ reacts with the pore cysteine residue and that the effects
are not the result of nonspecific modification of the channel
protein.
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Effects of Methanethiosulfonate-Benzocaine (MTS-L0-BZ) on
Na+ Channel Inactivation
Local anesthetic agents alter the inactivation properties of
Na+ channels, leading to leftward shifts in the
voltage dependence of steady-state inactivation and a slowing in the
rate of recovery from inactivation.7 8 9 10 11 These effects
stem from high-affinity binding of these compounds to the
inactivated state of Na+ channels,
which results in a stabilization of the inactivated
state.7 8 Therefore, we investigated whether anchoring
benzocaine to an external pore residue on Na+
channel truly allows access to the LABS, thus leading to the changes in
Na+ channel inactivation as observed with other
local anesthetics. The effects of MTS-L0-BZ (500 µmol/L) on
steady-state inactivation of hH1 channels were studied using a standard
2-pulse protocol; channels were depolarized to various conditioning
potentials for 50 ms from –140 to –20 mV from a holding potential of
–100 mV followed by a 50-ms test pulse to –10 mV. Only data from
–120 to –20 mV are shown in Figure 2. The 50-ms conditioning
pulse was sufficient to allow for equilibrium binding of benzocaine
because of the fast kinetics of benzocaine binding to the
channels.7 31 32 MTS-L0-BZ shifted the steady-state
inactivation curve of hH1 channels from –60.2±1.7 to –71.7±2.8 mV
(n=7; P<0.01), which was not reversed on washout
(–75.7±3.0 mV, n=7; P<0.01) (Figure 2B, Table 1). A similar shift of the
steady-state inactivation curve was observed with 500 µmol/L
benzocaine (–60.9±1.9 mV control versus –68.8±2.7 mV benzocaine;
P<0.01); however, this was reversible on drug washout
(–63.7±2.5 mV; n=4) (Figure 2B). These results establish that
the MTS-L0-BZ can stabilize the inactivated state of hH1
Na+ channels in a manner similar to that observed
with benzocaine.
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MTS-L0-BZ (500 µmol/L) also produced leftward shifts of the
steady-state inactivation curves in rSkM1 channels from –54.7±0.7 to
–61.2±2.0 mV (n=6; P<0.05), which could be largely
restored to predrug values on washout of MTS-L0-BZ (–58.4±1.0 mV)
(Figure 2B). However, in Y401C mutant channels, the shift in the
steady-state inactivation curve was irreversible on washout of
MTS-L0-BZ (–55.8±1.2 mV control; –64.7±3.8 mV MTS-L0-BZ;
–67.3±2.6 mV wash, n=6).
To further examine the effects of MTS-L0-BZ on channel inactivation,
the recovery from inactivation was measured; this gives another measure
of drug binding to Na+ channels by providing an
index of the rate of exit from the inactivated state. The
recovery from inactivation for hH1 channels was best fit with a
biexponential function with fast (fast) and
slow (slow) time constants of 13.3±0.7 and
255±70 ms (n=4), respectively (Figure 2C). Benzocaine (500
µmol/L) significantly prolonged the fast (28.2±2.9 ms, n=4;
P<0.01) and slow time constants (672±157 ms) without
significantly (P>0.4) affecting the fraction of channels
recovering at the faster rate (94±3% [n=4] in control versus
91±2% [n=4] with benzocaine). The rate of recovery returned to
control values on drug washout (fast,
11.8±1.2 ms; slow, 555±264 [n=4]). By
contrast, 500 µmol/L MTS-L0-BZ significantly slowed only the
fast rate of recovery from inactivation (fast,
67.4±6.3 ms [n=7]; P<0.01) but did not significantly
change slow (302±43 ms [n=7]) after
modification and washout (Figure 2C, Table 1).
Furthermore, MTS-L0-BZ (500 µmol/L) produced a significantly
greater slowing in the rate of recovery from inactivation than 500
µmol/L benzocaine (P<0.01), potentially because of an
enhanced effective concentration or slower drug dissociation as a
result of anchoring the agent. These data are consistent with a
stabilization of the inactivated state by anchored
MTS-L0-BZ.
MTS-L0-BZ (500 µmol/L) slowed the fast rate of recovery from
inactivation of rSkM1 channels from 5.3±1.0 to 18.3±3.9 ms (n=4;
P<0.05), which was reversible on washout of the agent
(8.2±1.7 ms) (Figure 2C). However, in Y401C channels, MTS-L0-BZ
slowed the fast recovery rate from 4.3±0.9 to 26.5±3.3 ms (n=4;
P<0.01), which remained significantly prolonged (22.1±3.5
ms, P<0.01) after washout (Figure 2C). These results
demonstrate that, in contrast to wild-type rSkM1 channels, treatment
with MTS-L0-BZ of hH1 or Y401C mutant rSkM1 Na+
channels is irreversible, which suggests covalent anchoring to the pore
cysteine. However, the effects of MTS-L0-BZ on steady-state
inactivation and recovery from inactivation on Y401C channels were less
pronounced than those observed with hH1, which supports previous
studies demonstrating that the cardiac isoform is more sensitive to
local anesthetic agents than are skeletal muscle
Na+ channels.9 10
Effect of MTSBN and MTSHE on hH1 Na+ Channels
Sulfhydryl modification of hH1 Na+ channels
with the methanethiosulfonate derivatives reduces peak current
amplitude.20 21 However, the effects of sulfhydryl
modification on inactivation properties of the channel have not
previously been studied. To determine whether the effects of MTS-L0-BZ
on Na+ channel inactivation result simply from
nonspecific consequences of the pore cysteine modification, effects of
MTSBN and MTSHE on hH1 channels were examined. MTSBN is similar to
MTS-L0-BZ in that an aromatic group is linked to the
methanethiosulfonate moiety, whereas MTSHE mimics MTS-L0-BZ
without the aminobenzyl group. Modification of hH1 channels with
500 µmol/L MTSBN resulted in a 62±3% (n=4) reduction in peak
current after modification and washout of the drug (Figure 4A, Table 1). This was similar to
the decrease in peak current observed with MTS-L0-BZ modification
(49±8%). As with MTS-L0-BZ, MTSBN caused irreversible reductions in
peak current and caused similar shifts and changes in slope factors of
the steady-state inactivation relationship (Table 1). However,
the degree of slowing of the fast recovery rate by MTSBN was
significantly less than that with MTS-L0-BZ (P<0.001).
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In contrast, MTSHE had no effect on any of the inactivation
parameters measured and reduced peak currents to a lesser
extent than either MTS-L0-BZ or MTSBN (Table 1). These data
suggest that simple sulfhydryl modification of the channel pore does
not result in alterations in channel inactivation. The effects of
MTS-L0-BZ are dependent on the presence of the aromatic group, given
that MTSBN elicited similar effects on both steady-state inactivation
and recovery from inactivation as compared with MTS-L0-BZ.
Effect of MTS-L0-BZ on Native Cardiac Na+
Channels
To ensure that the isoform specificity of MTS-L0-BZ was applicable
to native cardiac Na+ channels, the effects of
MTS-L0-BZ on native rat cardiac Na+ currents were
examined. Using the whole-cell patch-clamp recordings,
MTS-L0-BZ (500 µmol/L) reduced peak rat cardiac
Na+ current (Figure 5A) and shifted the voltage midpoint of
the steady-state inactivation relationship leftward from –68.5±1.4 to
–92.8±1.7 mV (n=6; P< 0.01) (Figure 5B; Table 1).
As in expressed hH1 channels, this hyperpolarizing shift was
maintained despite extensive washout of the drug
(V1/2,–90.4±5.0 mV; Figure 5B, Table 1),
which is consistent with irreversible anchoring of
MTS-L0-BZ to native cardiac Na+ channels. This
notion is further supported by the irreversible slowing in the rate of
recovery from inactivation at –120 mV (Figure 5C). MTS-L0-BZ
prolonged the rate of recovery from 8.3±1.5 to 46.1±3.0 ms (n=6;
P<0.01) after drug modification and washout. Therefore,
MTS-L0-BZ elicits similar effects on both native and heterologously
expressed cardiac Na+ channels.
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Effect of MTS-L0-BZ on the LABS
A method for further establishing if anchored MTS-L0-BZ interacts
with the LABS involves examining whether MTS-L0-BZ can compete with the
binding of other local anesthetics.33 Exposure of hH1
channels to lidocaine elicited 2 distinct time components for the
recovery from inactivation (data not shown). The fast component
(<20 ms) reflects normal gating channels, whereas the slow
component (>300 ms) is indicative of lidocaine binding to the
inactivated state of the channel.7 8
Increasing lidocaine concentrations enhances the fraction of slowly
recovering channels without altering the time constant.7 8
Plotting the fraction of the hH1 channels recovering at the slow time
constant as a function of the lidocaine concentration allows estimation
of the IC50 for lidocaine inhibition of
Na+ current8 (Figure 6). An IC50 of
22±3 µmol/L (n=5) was calculated, which is very similar
to those reported previously in native Na+
channels (9.7 µmol/L)8 and in hH1 channels
expressed in Xenopus oocytes (16
µmol/L)9 or human embryonic kidney cells (21
µmol/L).10 As expected, the local anesthetic benzocaine
(500 µmol/L) shifted the affinity of lidocaine for the
inactivated state to 123±13 µmol/L (n=4;
P<0.01) (Figure 6A). Irreversible modification of
hH1 channels with MTS-L0-BZ (500 µmol/L) resulted in a 1.5-fold
larger reduction in lidocaine sensitivity of the channel than
benzocaine (187±29 µmol/L, n=4; P<0.01) (Figure 6B).
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) and presence
(
) of 500 µmol/L benzocaine (A) or after irreversible
modification by 500 µmol/L MTS-L0-BZ (
; B). Data were fit
with a Hill equation as described in Figure 3
Recent mutagenesis studies have identified critical residues that
influence local anesthetic binding to the rat brain
Na+ channel.12 34 These residues are
located in the sixth transmembrane-spanning region of domain IV of the
Na+ channel subunit. One residue in
particular, F1764, when mutated to alanine resulted in near-complete
abolition of use-dependent and voltage-dependent block by local
anesthetics.12 34 Similarly, alanine substitution of the
equivalent residue in rSkM1 (F1579A) has been shown to reduce local
anesthetic sensitivity.35 Therefore, the effects of
MTS-L0-BZ on rSkM1 Na+ channels containing the
F1579A mutation were examined, as well as the Y401C pore mutation, to
allow covalent anchoring of the drug. By comparison with Y401C channels
(Figure 2B), Y401C/F1579A mutant Na+
channels had a rightward-shifted voltage midpoint of the steady-state
inactivation relationship from –55.8±1.2 mV (n=5) to –44.8±0.7 mV
(n=5) (P<0.01) (Figure 7A)
and an accelerated rate of recovery from inactivation from 4.30±0.90
ms (n=4) to 1.00±0.04 ms (n=5) (P<0.01) (Figure 7A). Modification of Y401C/F1579A mutant
Na+ channels by 500 µmol/L MTS-L0-BZ had
no effect on peak current amplitude (101±4%, n=5) (data not shown).
Furthermore, there was no significant effect on the voltage dependence
of steady-state inactivation (Figure 7A). The voltage midpoints
for the steady-state inactivation relationship were –43.3±1.1 and
–44.4±1.4 mV (n=5) during exposure and washout of MTS-L0-BZ,
respectively, which were not significantly different from control
values (–44.8±0.7 mV). There was also a modest but significant
slowing in the fast rate of recovery from inactivation from 1.00±0.04
to 1.23±0.06 ms (n=5; P<0.05) with 500 µmol/L
MTS-L0-BZ present (Figure 7B) and 1.30±0.06 ms
(P<0.05) after drug washout. These data further suggest
that MTS-L0-BZ modifies Na+ channel inactivation
by binding to the local anesthetic receptor after anchoring to the
external pore site.
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Dependence of Channel Modification on Linker Length
The results above establish that tethering benzocaine to the
sulfhydryl-reactive methanethiosulfonate group via an ethyl alkyl
linker allowed irreversible modification of hH1
Na+ channels. On the basis of the structure of
the local anesthetic MTS-L0-BZ, it is conceivable that varying the
linker properties could influence drug action. Indeed, we hypothesized
that excessively short linker lengths may prevent or reduce the
interaction of the anchored drug with the LABS, thus decreasing drug
efficacy. Alternatively, very long linkers could increase diffusion
times or produce steric effects that would also reduce drug efficacy.
Moreover, the precise chemical properties of the linker may also have
effects on drug modification and/or delivery. Therefore, altering the
linker properties may affect the ability of the MTS-L0-BZ to modify
Na+ channels. Initially, the linker length was
increased with 3, 6, or 9 alkyl groups. However, increasing the alkyl
linker length reduced the drug solubility and reduced the rate of
anchoring to hH1 channels at low concentrations. For that reason,
linkers were constructed using polyethylether units,
(–OCH2CH2–)n
(n=1, 2, or 3), which markedly enhanced their aqueous solubility in
comparison with the long alkyl linker derivatives. Extending the single
sulfide linker (–S–; 0.3 nm) in MTS-L0-BZ with an ethylether
sulfide unit
(–O–CH2–CH2)–S–
increased the linker by 3 bond lengths (extended length, 0.65 nm),
which is referred to as MTS-L3-BZ. Incorporation of 2 ethylether units
(–O–CH2–CH2–O–CH2–CH2)–S–
produced a linker 6 bond lengths longer (extended length, 1 nm), which
is referred to as MTS-L6-BZ, and so forth. The application of
polyethylether-based drugs (500 µmol/L) with 3, 6, or 9 bond
lengths did not enhance the reduction in peak hH1
Na+ current (Table 2) or produce a further leftward-shift
steady-state inactivation curve (Figure 8A, Table 2) when compared with
MTS-L0-BZ (Figure 8A). Indeed, shifts in the steady-state
inactivation curves with the extended linker compounds followed the
sequence MTS-L0-BZ>MTS-L6-BZ=benzocaine>MTS-L3-BZ>MTS-L9-BZ (Figure 8A, Table 2).
|
|
A very different rank potency emerged when the effects of MTS-L0-BZ and
the various linker lengths on recovery from inactivation were examined.
MTS-L6-BZ prolonged the fast rate constant of recovery from
inactivation more than MTS-L0-BZ or MTS-L3-BZ (Figure 8B, Table 2).
Further lengthening of the linker to 9 bond lengths
(extended length, 1.35 nm) did not elicit any further modification. In
fact, MTS-L9-BZ was less able to slow the rate of inactivation compared
with MTS-L6-BZ (32.8±3.5 versus 92.7±11.2 ms, P<0.05).
The rank potency for slowing the rate of recovery from inactivation was
MTS-L6-BZ>MTS-L0-BZ=MTS-L3-BZ>MTS-L9-BZ=benzocaine, which suggests
that a 6-atom linker is more efficacious at modifying the recovery from
inactivation properties of hH1 channels.
In addition to the marked slowing in the recovery from inactivation,
MTS-LX-BZ compounds also produced use-dependent reductions in peak
current. When hH1 channels were depolarized to –10 mV for 20 ms
(holding potential, –100 mV) at a stimulation frequency of 5 Hz, there
was a 9±1% reduction in peak current amplitude measured at the 30th
pulse (n=18, Figure 8C). Application of 500 µmol/L
benzocaine produced a modest increase in use dependence as compared
with control (13±1%, n=4, P< 0.05) (Figure 8C).
All MTS-LX-BZ analogues tested at 500 µmol/L resulted in a
significant use-dependent reduction in peak current amplitude compared
with control (measured at the 30th pulse), as shown in Figure 8C, but only MTS-L0-BZ (31±5%, n=3) and MTS-L6-BZ (42±5%,
n=5) resulted in a significantly greater use dependence than benzocaine
(P<0.01). This rank potency was similar to that seen with
slowing of the recovery from inactivation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The application of MTS-LX-BZ compounds irreversibly modified native and expressed cardiac as well as mutant Y401C rSkM1 Na+ channels, unless treated with the reducing agent DTT. In contrast, the drug effects on wild-type rSkM1 Na+ channels were reversed on washout. The effects of MTS-LX-BZ compounds were more prominent in hH1 Na+ channels when compared with rSkM1 or Y401C channels, which supports previous findings that the cardiac isoform is more sensitive to local anesthetics.9 10 These observations establish that MTS-LX-BZ agents were anchored onto the channel via an extracellular pore cysteine uniquely found in cardiac Na+ channels. Anchoring these compounds to the channel pore could affect Na+ channels in any number of ways. For example, tethering of the drug could reduce current by simply physically occluding the pore. However, the effects of anchored MTS-LX-BZ are largely indistinguishable from those observed with benzocaine alone (although see below), which suggests that interactions between the anchored drugs and LABS are also involved. This conclusion is supported by a number of experimental observations. Drug anchoring was able to reduce the potency of lidocaine binding to the channels, which is expected if the 2 drugs were competing for the same LABS, as established previously.7 33 Additionally, mutation of the phenylalanine residue (ie, F1579A) located in S6 of domain IV abolished the effects of anchored benzocaine. This residue has been shown to reduce both tonic and phasic block by local anesthetics in Na+ channels,12 34 35 as expected if this residue forms an important part of the local anesthetic receptor. Finally, anchored compounds such as MTSHE (Figure 4
)
or MTS-LX (ie, linkers alone attached to MTS), which lack the
aromatic portion of MTS-LX-BZ (data not shown), did not have the
effects on Na+ current characteristically
associated with the aromatic structures of local
anesthetics.36 37 On the other hand, the aromatic compound
MTSBN did produce local anesthetic-like modulation of channel function
(Figure 4), which suggests that the chemical interaction of the
aromatic group is required for interaction with the LABS,
consistent with previous studies involving
benzene36 and phenol.37 Despite evidence for
the involvement of interactions between anchored MTS-LX-BZ agents and
the LABS, probably via the aromatic structure, some of the reductions
in peak current (Table 1) can be attributed to the partial
occlusion of the pore. Indeed, MTSHE and MTS-LX modification caused a
25% reduction in peak current but, again, without significantly
affecting other channel properties. Local anesthetics modify Na+ channels by dynamically binding and unbinding to their binding site in a time- and voltage-dependent manner. This behavior readily explains the leftward shifts in steady-state inactivation, use-dependent blockade, and slowed rates of recovery from inactivation. Because modification of Na+ channels by anchored MTS-LX-BZ closely resembles the changes by benzocaine, our results demonstrate that the interaction of the anchored drug with the LABS involves dynamic, voltage-dependent binding and unbinding, presumably of the aromatic portion of the drug. This dynamic binding of the anchored MTS-LX-BZ compounds could arise from voltage-dependent changes in the intrinsic affinity of the drug for the LABS or possibly from voltage-dependent changes in the spatial arrangement of the anchored drug to the LABS or both. In essence, the anchoring of MTS-LX-BZ agents should increase the apparent local anesthetic concentration at the extracellular face of the channel to extremely high levels. This would be expected to produce a very potent block, and indeed MTS-L0-BZ causes larger shifts in the steady-state inactivation curves than 500 µmol/L benzocaine. However, the extent of channel modification by these anchored compounds also depends on energetic, steric, and entropic factors that are probably very different from unanchored local anesthetics. For example, if interactions with the LABS require protein distortion, the apparent affinity of drug binding will be reduced. Alternatively, the anchored drug might bind to other sites remote from LABS or cause local changes of the channel protein structure in the anchored region, thereby reducing the apparent binding affinity for the LABS.
Application of MTS-L0-BZ to cardiac and Y401C channels results in the
tethering of benzocaine to an external pore residue via a linker with
an approximate length of 0.3 nm when fully extended (ie, –S–). The
extent of channel modification by anchored MTS-L0-BZ was more potent
than that observed after the application of 500 µmol/L free
benzocaine. Although initially surprising, this result is not entirely
unexpected. For example, P-loop residues involved in forming the
putative selectivity filter of Na+ channels can
influence local anesthetic access and binding of the drug with its
receptor.38 39 In addition, as outlined above, previous
studies have demonstrated that the pore-lining residues and the LABS
are in close proximity within the channel pore. The fractional
electrical distance (
) of the pore-lining cardiac-specific cysteine
in domain I was estimated to be 0.2114 from the outside,
whereas is estimated to be 0.7 for the local anesthetic blocking
site18 from the cytoplasmic side. Assuming that the
voltage drop within the pore occurs over a span of 3 nm, these data
suggest that the axial distance between the anchoring site and the LABS
is <0.3 nm. However, this distance could be grossly overestimated if
the Na+ channel structure in the P-loop region
resembles that of K+ channels. In Shaker
K+ channels, 80% of the voltage drop occurs
between residues Y449 and T441.40 Assuming that the
recently published crystal structure of the
K+ channel derived from Streptomyces
lividans (KcsA) is representative of Shaker
K+ channel pores, the bulk of the voltage drop occurs over
a distance of only 1.2 nm.41 In any event, our results
suggest that the pore cysteine in cardiac Na+
channels is located a very short distance from the LABS.
The potency of the different anchored MTS-LX-BZ compounds depended on channel property studied and did not, at first glance, follow an obvious pattern with respect to linker length. Because steady-state inactivation conveys information on the properties of Na+ channel inactivation at equilibrium, it should give a thermodynamic measure of the energetics of binding to the channel. The degree of shift in steady-state inactivation decreased with increasing chain length, which suggests that anchored MTS-L0-BZ and MTSBN, with a linker length of only 0.6 nm (–S–CH2), bind more strongly to the LABS than do the longer compounds. However, we cannot rule out the possibility that kinetic factors might also play a role, because the pulses used in these studies were limited to 50 ms to prevent slow inactivation. Alternatively, the interaction of the linker with the channel may also contribute to the differences observed between the different linker length and contribute to the energetics of drug binding. Structure-activity studies on local anesthetics have revealed that small changes in drug structure can influence both state-dependent binding and potency.32 42 43 44
In contrast to the steady-state inactivation curves, rates of recovery from inactivation and use-dependent measurements are strongly influenced by the kinetics of drug interaction. Our results show that MTS-L6-BZ slowed recovery from inactivation to a greater extent and elicited greater use-dependent reductions in peak current than the other compounds tested, whereas MTS-L0-BZ had a more potent effect on these parameters than either MTS-L3-BZ or MTS-L9-BZ. Interestingly, MTSBN was less effective than the MTS-LX-BZ compounds at prolonging the recovery from inactivation. The more potent effects of MTS-L6-BZ compared with compounds with a shorter linker might simply reflect easier access of the benzocaine moiety to the LABS possibly because of reduced mechanical strain of the channel. However, this explanation would not readily explain the greater efficacy of MTS-L0-BZ compared with MTS-L3-BZ. Longer linkers, on the other hand, may result in slower diffusion times for drug interaction with the local anesthetic receptor. As already mentioned, the physiochemical properties of the linker could have a strong influence on the affinity and kinetics of binding to the LABS. One potential explanation for the pattern observed is that, as the linker length is increased, drug binding to the LABS is energetically destabilized, but the kinetics of drug binding and unbinding are slowed. The rates of recovery from inactivation and use dependence would therefore depend on a balance between changes in energetics and kinetics with length. Similar results were obtained previously using various analogues of benzocaine, establishing that the affinities and kinetics of binding to Na+ channels depend critically on drug structure.32 Regardless, the pattern of channel modification with linker length demonstrates that factors other than the physical length of the linker are important determinants of anchored drug action. Further studies using linkers with different chemical structures may provide further insights into these experimental observations, and we are currently investigating these possibilities.
In summary, we created novel compounds comprised of the following 3 components: an anchoring domain (MTS), a variable-length linker, and the generic nonselective local anesthetic (benzocaine). These anchor-linker-drug compounds were found to selectively modify the cardiac Na+ channel homologue by anchoring the local anesthetic, benzocaine, to the unique external free sulfhydryl and subsequently dynamically binding and unbinding to the LABS. Despite their selectivity, these agents might prove to have limited therapeutic value as tissue-specific local anesthetics because of possible reactions with other free sulfhydryl groups or because covalent modification, via disulfide links, may produce long-lived complexes that may have undesirable consequences. However, selective delivery of local anesthetics to Na+ channels may not necessitate covalent modification of target proteins but, rather, could involve reversible binding of an "anchor" to a unique epitope, thereby effectively increasing the apparent local concentration of the nonselective drug. Indeed, we are currently designing anchor-linker-drug compounds using this approach to develop tissue-specific Na+ and Ca2+ channel modifiers. We believe that this approach may prove to be of general utility in targeting inherently nonselective drugs to a single member of a homologous protein family via a selective anchor.
|
Acknowledgments |
|---|
This research was supported by a Medical Research Canada (MRC) grant to P.H.B. and by the Tiffin Trust. P.H.B. is an MRC Scholar. R.A.L. is a recipient of an Ontario Graduate Scholarship. We thank Tin Nguyen for her excellent technical support.
|
Footnotes |
|---|
1 Both authors contributed equally to this study.
Received August 20, 1998; accepted April 23, 1999.
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