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Department of Neuroscience, Albert Einstein College of Medicine, Bronx, New York
Received December 23, 2002; accepted March 3, 2003
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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3.8) was increased at low
external pH, suggesting that the uncharged form of the drug is important for
blockade. The effect of FFA did not seem to be mediated by direct binding of
the drug to the pore of the gap junction channel. Internal application of high
concentrations of FFA by addition to patch pipettes did not cause inhibition
of channel currents. The magnitude of inhibition was neither voltage-dependent
nor influenced by the nature of permeant ion. Single-channel recordings
indicated that FFA reduced the channel-open probability without modifying the
current amplitude and induced slow transitions between open and closed states.
We propose that FFA inhibits gap junctions by inducing a conformational change
in the protein upon binding to a site that is presumably located within the
membrane.
). Gap
junction channels formed by various connexins can be closed by a number of
factors including pH and phosphorylation by tyrosine kinases and by
transjunctional voltage (for review, see
Harris, 2001). In addition, a
large number of drugs are known to block gap junctional communication
(Spray et al., 2002). These
include long-chain alcohols (e.g., heptanol and octanol), volatile anesthetics
(halothane and ethrane), glycyrrhetinic acid derivatives, oleamide,
aminosulfonates (e.g., taurine), tetraalkylammonium ions, and the antimalarial
drug quinine (Johnston et al.,
1980; Davidson et al.,
1986; Burt and Spray,
1989; Bevans and Harris,
1999; Musa et al.,
2001; Srinivas et al.,
2001). Recently, arylaminobenzoates were shown to reduce the
spread of Lucifer yellow through gap junctions composed of Cx43
(Harks et al., 2001) and to
inhibit putative hemichannel currents in Xenopus laevis oocytes
(Zhang et al., 1998;
Eskandari et al., 2002).
The effects of arylaminobenzoates on several ion channels have been well
documented. Arylaminobenzoates, diphenylcarboxylate and its derivates, are
highly lipophilic molecules that were first systematically studied as blockers
of chloride currents (DeStefano et al.,
1985). Subsequently, diphenylcarboxylate and its derivatives such
as flufenamic acid and niflumic acid were shown to inhibit currents through a
wide variety of channel types, including nonselective cation channels,
voltage-dependent potassium channels, L-type calcium channels, and chloride
channels such as the cystic fibrosis transmembrane conductance regulator
(Gögelein et al., 1990;
White and Aylwin, 1990;
Alton et al., 1991; Richard and
Dawson, 1993; Ackerman et al.,
1994; Wang et al.,
1997; Schultz et al.,
1999). In addition, these drugs potentiate the large-conductance
Ca2+-activated K+ channel current
(Farrugia et al., 1993;
Ottolia and Toro, 1994;
Greenwood and Large, 1995).
Effects of fenamates on ligand-gated ion channels have also been reported
(Woodward et al., 1994).
The mechanism of blockade of these drugs varies for different channel
subtypes. Arylaminobenzoates inhibit chloride channels by binding in the
permeation pathway in a manner consistent with open channel block
(McCarty et al., 1993;
McDonough et al., 1994). The
mechanism of inhibition of some other channels by arylaminobenzoates is less
clear, but it might involve the alteration of channel gating
(Lee and Wang, 1999). In this
study, we characterized the effect of arylaminobenzoates on gap junction
channels formed by various connexins at the macroscopic and the single channel
level by directly measuring channel currents using the dual whole-cell
patch-clamp technique. Our results indicate that block by flufenamic acid is
reversible, with both inhibitory potency depending on the concentration and pH
of the external solutions. In addition, we determined that these drugs do not
cause open-channel block of gap junction channels, but they seem to close
junctional channels by affecting gating.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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DNA Construction and Transfection. All electrophysiological
experiments described here were performed on N2A cells that were either stably
transfected with connexins or transiently cotransfected with connexin and
enhanced green fluorescent protein cDNAs in separate vectors as described
previously (Srinivas et al.,
2001). The connexins used in the study were rCx26, hCx32, rCx43,
rCx46, and mCx50 (where r, h, and m refer to rat, human, and mouse cDNAs,
respectively). Transiently transfected cells were dissociated at 8 to 12 h
after transfection, plated at low density on 1-cm round glass coverslips, and
used within 48 h thereafter.
Electrophysiology. Junctional conductance was measured between cell
pairs using the dual whole-cell voltage-clamp technique with Axopatch 1C or 1D
patch-clamp amplifiers (Axon Instruments, Union City, CA) at room temperature.
The solution bathing the cells contained 140 mM NaCl, 5 mM KCl, 2 mM CsCl, 2
mM CaCl2, 1 mM MgCl2, 5 mM HEPES, 5 mM dextrose, 2 mM
pyruvate, and 1 mM BaCl2, pH 7.4. Patch electrodes had resistances
of 3 to 5 M
when filled with internal solution containing 130 mM CsCl,
10 mM EGTA, 0.5 mM CaCl2, 3 mM MgATP, 2 mM Na2ATP, and
10 mM HEPES, pH 7.2. pH values of external and internal solutions were
measured and adjusted before each experiment. Macroscopic and single-channel
recordings were filtered at 0.2 to 0.5 kHz and sampled at 1 to 2 kHz. Data
were acquired using pClamp software (Axon Instruments) and plotted using
Origin 6.0 software (OriginLab Corp, Northampton, MA).
Each cell of a pair was initially held at a common holding potential of 0 mV. To evaluate junctional coupling, 200-ms hyperpolarizing pulses from the holding potential of 0 to –10 mV were applied to one cell to establish a transjunctional voltage gradient (Vj), and junctional current was measured in the second cell (held at 0 mV). To determine the voltage dependence of drug action, 7- to 10-s hyperpolarizing or depolarizing pulses were applied every 20 s to test potentials between –30 and 30 mV. Single-channel currents were investigated in weakly coupled cell pairs (1 to 2 channels) without the use of uncoupling agents by applying –20 mV pulses to one cell of a pair. Gating events were recognized as simultaneously occurring events of equal amplitude and opposite polarity in current traces for both cells in the pair. The channel-open probability was determined from recordings of cell pairs that contained a single active channel and was defined as the absolute fraction of time that was spent in the open state.
Drugs were applied with a gravity-fed perfusion system. Solution exchanges were complete within 30 s. Concentration-response curves for drug-induced uncoupling were determined by exposure of each cell pair to two to three concentrations of each drug. In all experiments, reversibility was assessed by washout of the drug. The magnitude of inhibition caused by drugs is expressed as the fraction of the conductance (gj) in the absence and presence of the drug, "gj, % control". Concentrations of drugs ([D]) that caused a half-maximal inhibition (EC50), an index of the potency of a drug, and the Hill coefficients (nH) of concentration-response relationships were estimated by fitting the data to the equation: gj, % control = 1/[1 + ([D]/EC50)nH]. Data are presented as means ± S.E.M.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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60% within 3 min, whereas at the higher concentration,
the drug caused near-maximal decrease in Ij within less than 1 min. For both
concentrations, washout of drug resulted in complete recovery of the currents
within 2 min, i.e., the block was quickly reversible
(Fig. 1A, top trace).
Figure 1B shows the
concentration dependence of FFA-induced inhibition of Cx50 junctional currents
determined by the exposure of 4 to 10 cell pairs to each drug concentration.
Nonlinear least-squares fit of the individual data points to the Hill equation
(see Materials and Methods) yielded an EC50 value of 47
µM for the FFA-induced inhibition of Cx50 gap junction channels. This
EC50 value for the effect of FFA is comparable with those found for
block of other channels as well as for putative hemichannels formed by Cx50
(Schultz et al., 1999;
Eskandari et al., 2002). The
Hill coefficient for inhibition of gap junctions was 3, suggesting that
binding is not caused by a simple 1:1 interaction between FFA and individual
gap junction channels. A Hill coefficient greater than unity was also observed
for the inhibition of putative Cx50 hemichannels expressed in oocytes
(Eskandari et al., 2002).
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We recently demonstrated that the potency of blockade of gap junctions by
quinine was much greater for certain connexin subtypes than for others
(Srinivas et al., 2001). To
determine whether FFA selectively inhibited gap junctions formed by specific
connexin subtypes, we tested the effects of the drug on gap junction channels
formed by several other connexins, including Cx26, Cx32, Cx40, Cx43, and Cx46.
Figure 2A shows the effect of
FFA (30 and 100 µM) on Cx26 gap junction channel currents. FFA at
concentrations of 30 and 100 µM reversibly decreased Cx26 junctional
currents by 46 and 100%, respectively, with onset and reversal even more
rapid than those seen with Cx50. Although these data suggest that the drug is
marginally more potent at inhibiting these channels compared with its effect
on Cx50 channels, the effect of FFA on other connexins, however, was similar
to that observed for Cx50 channels (Fig.
2B). At the high concentration of 100 µM, FFA blocked Cx32,
Cx43, Cx46, and Cx40 currents by 85 to 95% (means ± S.E.M. are 98
± 2%, n = 6, for Cx26; 89 ± 3%, n = 5, for
Cx32; 88 ± 4%, n = 3, for Cx40; 96 ± 2%, n =
7, for Cx43; and 86 ± 4%, n = 4, for Cx46). These results
demonstrate that inhibition of gap junction channels by FFA is only marginally
selective among these several different connexins.
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We also determined the effects of several other commonly used chloride-channel blockers on gap junction channels. These included derivatives of FFA such as NPPB, MFA, and NFA as well as unrelated molecules such as clofibric acid and the stilbene derivative DIDS. In addition, the importance of the benzoate moiety and the bulky hydrophobic chain were also investigated by determining the effect of 2-amino-4-phenylbutyric acid and 2-aminobenzoic acid, respectively (compare structures with NPPB in Fig. 3A). The effects of the various derivatives of flufenamic acid on Cx50 junctional currents are summarized in Fig. 3. Application of 30 and 100 µM NPPB caused a decline of 15 ± 3% (n = 4) and 96 ± 4% (n = 4) of the junctional current, respectively, suggesting that the potency of NPPB at causing blockade of gap junctions is not greatly different from that of FFA. Similar results were obtained with MFA (means ± S.E.M.: 7 ± 4, n = 4, at 30 µM and 90 ± 2, n = 4, at 100 µM) (Fig. 3). In the case of NFA, higher concentrations were required to reduce Cx50 junctional currents, with 300 µM and 1 mM inhibiting channels currents by 54 ± 5% (n = 4) and 97 ± 4% (n = 4), respectively. In contrast, the application of high concentration of the unrelated derivative DIDS led to little closure of Cx50 channels. The magnitude of inhibition caused by 100 and 300 µM was 2 ± 1 and 4 ± 2%, respectively. Similarly, clofibric acid caused little reduction of Cx50 junctional currents (n = 4). More importantly, o-aminobenzoic acid and 2-amino-4-phenylbutyric acid caused little effect on Cx50 channels, strongly indicating that both the phenyl side chain and the benzoate ring are important for the inhibition of gap junction channel currents.
To determine how externally applied FFA accesses its binding site,
strategies that have been used to investigate the site of block by this drug
in chloride channels were applied (Walsh
and Wang, 1998; Zhang et al.,
2000). Most arylaminobenzoates, including FFA, are weak acids with
pKa values in range of 3 to 5 and therefore can exist in
both the membrane-permeable uncharged form and the anionic-charged form,
depending on the pH of the external solution. At pH 7.4, the vast majority of
the drug is charged, but a small fraction is uncharged and is free to cross
cell membranes. To determine whether the uncharged form of the drug is
important for inhibition, the effect of FFA was investigated at different
external pH values (Fig. 4). Fig.4A shows the effect of 60
to 90 s of exposure to FFA (40 µM) on Cx50 junctional currents at
pHe values of 6.7, 7.4, and 8.0 (where uncharged FFA proportions
are calculated as 0.13, 0.03, and 0.007% by using the Henderson-Hasselbach
equation). Junctional currents were measured initially and during FFA washout
in external solution at pH 7.4. Varying pHe values for 60 s in the
absence of FFA caused no significant change in the junctional conductance
(Fig. 4B, 
). As
illustrated in Fig. 4A, the
magnitude of reduction of Cx50 junctional currents by external FFA (40 µM)
was maximal at low external pH and minimal at high external pH. The magnitude
of block induced by 40 µM FFA was 4 ± 3% at pH 8.0 (n = 7),
41 ± 8% at pH 7.4 (n = 4), and 95 ± 3% at pH 6.7
(n = 4) (Fig. 4B).
These results demonstrate that arylaminobenzoate inhibition of the gap
junction channel is enhanced by lowering the pH of the external solution.
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In the case of chloride channels, arylaminobenzoates access the binding
site in the uncharged form but bind to their receptor in the charged form
after protonation in the cytoplasmic milieu
(McCarty et al., 1993;
McDonough et al., 1994;
Walsh and Wang, 1998).
Evidence that the charged form binds to the receptor and causes open-channel
blockade was provided in those studies by 1) potent blockade by drug applied
via patch pipettes, 2) voltage-dependence of block, 3) reduction of magnitude
of blockade by substitution of chloride by a larger permeant ion, and 4) rapid
flicker in the single-channel records in the presence of the drug. We applied
each of these criteria to determine whether FFA caused open-channel block of
gap junction channels.
Initially, FFA was added at a high concentration to patch pipettes and the
magnitude of inhibition was measured (Fig.
5). Application of high concentrations of FFA (1 mM) caused little
or no reduction of Cx50 channel currents even after 10 min of dialysis of drug
into cells (mean ± S.E.M. of inhibition is 2 ± 0.1%, n
= 5). However, extracellular application of a 10-fold lower concentration of
the drug to the same cell pair caused a marked decrease in the current (mean
± S.E.M.: 88 ± 3%, n = 5) that was fully reversible
upon removal of external FFA. Application of the drug at a higher internal pH
of 8.0, which would favor higher concentrations of the charged form of the
drug in the patch pipette, also caused little reduction of the junctional
current (mean decrease after 10 min 5 ± 3%, n = 7),
whereas external application of 100 µM FFA again decreased the current
significantly by 82 ± 5%, suggesting that the binding site for FFA (at
least for the charged form) is not intracellular. Not surprisingly, the
inhibition of gap junction channels by FFA was not voltage-dependent. Voltage
dependence of block was assessed by measuring the magnitude of steady-state
block induced by external FFA at voltages between –30 and +30 mV, in
which the inherent closure of Cx50 channels induced by transjunctional voltage
is minimal (Srinivas et al.,
1999). The normalized values of junctional conductance
(Gj) in the absence () and presence (
) of externally
applied 50 µM FFA are plotted at various transjunctional voltage gradients
(Fig. 6C). The steady-state
inhibition induced by FFA (50 µM) was similar at all voltages. The
magnitude of inhibition at Vj of 10 and 30 mV were 44 ± 3%
(n = 3) and 42 ± 4% (n = 3), respectively.
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Permeant ions are known to modulate the potency of open-channel blockers by
competing with the drug at its binding site. Block of chloride channels was
shown to be significantly reduced when chloride concentrations in the pore
were lowered and when larger permeant ions were used as charge carriers
(McDonough et al., 1994). To
determine whether permeant ions affected the reduction of gap junction channel
currents by FFA, chloride was replaced with the larger glutamate ion. As
illustrated in Fig. 6, the
magnitude of inhibition of Cx50 channels by FFA with glutamate as the primary
anion was similar to that observed in the previous experiments with
Cl– internal solution. The magnitudes of inhibition caused by
30 and 100 µM were 12 ± 3% (n = 3) and 93 ± 6%
(n = 4), respectively, values that are close to those observed when
chloride was used as the primary anion, indicating that block by FFA is not
influenced by the nature of the permeant ion.
Block at the Single-Channel Level. Single-channel studies were
undertaken to investigate further the mechanism of channel closure of gap
junction channels by FFA. The effect of FFA (50 and 100 µM) on a Cx50
single channel at a Vj of 30 mV is shown in
Fig. 7. At this voltage, Cx50
channels are open 80 to 90% of the time with occasional transitions from an
open state of 210 pS to a subconductance state of 40 pS, as has been reported
previously (Fig. 6) (control:
Srinivas et al., 1999). These
open-substrate transitions are typically observed in response to
transjunctional voltage gradients and are usually "fast" with
increase and decay times of 1 to 2 ms
(Bukauskas and Peracchia, 1997;
Verselis et al., 2000;
Harris, 2001)
(Fig. 8). External application
of FFA (50 and 100 µM) caused a reduction in open-channel time. At 50 µM
FFA, channels exhibited transitions between the open and closed state in the
entire duration of the recording, whereas at 100 µM FFA, the channels were
rarely open. The open probability of the channel was reduced from 0.85 in
control solution to 0.5 at 50 µM FFA and to 0.1 at 100 µM FFA, values
that are very close to the magnitudes of inhibition of gj by these
FFA concentrations obtained in macroscopic measurements
(Fig. 1B). Similar results were
obtained in two other experiments.
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Even though the channel activity was reduced for prolonged intervals, the
single-channel conductance was not appreciably modified. Amplitude histograms
of the recordings in the absence and presence of FFA are illustrated to the
right of each recording. In control recordings, histograms had three peaks at
0, 1.2, and 6.3 pA corresponding to the closed state, subconductance state (40
pS), and open state (210 pS), respectively
(Srinivas et al., 1999). In
the presence of 50 µM FFA, histograms had one peak at 6.3 pA corresponding
to the open state (210 pS) and a second peak corresponding to the closed state
(0 pA). Similarly, at 100 µm FFA, two peaks at 0 and 6.2 pA corresponding
to open and closed states were observed. Similar results were obtained from
three such experiments (mean ± S.E.M. values: 207 ± 12, 199
± 10, and 203 ± 8 pS for control, 50 µM, and 100 µM FFA,
respectively, verifying the lack of significant reduction in unitary
conductance during channel blockade by FFA).
Closer inspection of recordings in the presence of FFA revealed that the
drug induced "slow" transitions between the open and closed states
and not flickery transitions that are characteristic of open-channel block
(Fig. 8). The recordings in
Fig. 8 were obtained by
applying short (16-s) pulses in the absence and presence of FFA (50 µM).
The junctional current (Ij) and the current in the pulsed cell
(I2) are both illustrated in this figure to demonstrate that slow
events occur simultaneously in both cells' current recordings and are thus
junctional in origin. In the absence of FFA, channels exhibited fast
transitions between the fully open and subconductance states. Slow transitions
to the fully closed state were only rarely observed (arrowhead, control).
Application of FFA led to an increase in slow transitions between the open and
closed states, although fast transitions were also occasionally observed
(arrowhead, FFA). Increase and/or decay times of these slow transitions varied
between 8 and 100 ms (Fig. 9). Such slow closures to the fully closed state
detected here have been observed in response to certain drugs and uncoupling
agents (e.g., low pH, heptanol, halothane, and quinine) and have been
attributed to a gating mechanism (the so-called chemical gate)
(Bukauskas and Peracchia, 1997;
Verselis et al., 2000;
Harris, 2001). The presence of
slow transitions induced by FFA reflects an action of the drug on this gating
mechanism.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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) and also inhibited putative hemichannel currents expressed
in oocytes (Zhang et al.,
1998; Eskandari et al.,
2002). We extended these observations by directly measuring the
effects of FFA and related analogs on junctional currents using the dual
whole-cell patch-clamp technique. Closure of junctional channels by these
drugs was rapid and quickly reversible, concentration-dependent, and exhibited
only marginal selectivity with regard to connexin subtype. The efficacy of
inhibition was markedly enhanced at low external pH values and decreased at
high external pH values, indicating that the uncharged form of the drug is
important for the effect of FFA. The results of other experiments, illustrated
in Figs. 6,
7,
8, suggested that the effect of
FFA is not caused by the binding of the molecule within the pore, but rather
by the binding of the drug to a modulatory site, presumably located within the
membrane, that induces channel closure. A Hill coefficient of 3.0
suggests that binding of more than one molecule of the drug is required to
effect channel closure.
Arylaminobenzoates affect a number of processes within the cell. Thus, it
is possible that inhibition of gap junction channel currents is caused by an
indirect action of the drug. However, the rapid onset and reversibility
suggests that the effects of these drugs are most likely mediated by a direct
action of the drug on the channel and are not caused by indirect effects such
as lowering the intracellular pH and/or increasing intracellular calcium,
factors which have also been shown to reduce coupling between cells. Internal
solutions contained 10 mM HEPES and 10 mM EGTA, which should be sufficient to
buffer small internal pH changes expected to occur when exposing cells to
submillimolar concentrations of weak acids. In addition, increasing the
concentration of HEPES or substituting EGTA with BAPTA had little effect on
the potency of inhibition (data not shown). These results are consistent with
those obtained by Harks et al.
(2001), who demonstrated that
the effects of the drug are not caused by changes in pH, calcium, protein
kinase C, or cyclooxygenase activity.
Preliminary structure-activity studies also support the hypothesis that gap junction channels contain a distinct receptor for arylaminobenzoates. Analogs of flufenamic acid, such as NPPB and meclofenamic acid, inhibited gap junction channels with potencies similar to those observed for FFA. Niflumic acid was less potent at inhibiting gap junction channels, but complete inhibition was achieved at concentrations of greater than 300 µM. In contrast, unrelated analogs such as clofibric acid and the stilbene derivative DIDS had little or no effect on gap junction channels. The potency of arylaminobenzoates at inhibiting gap junctions corresponds with their octanol/water partition coefficients (P), with the least potent blockers having low logP values.
Structure-activity studies of chloride-channel blockers defined four
elements critical for arylaminobenzoate blockade: 1) the anionic carboxylate
group, 2) the negative partial charge in meta (or para)
position to the carboxylate group, 3) the amino "bridge" carrying
a positive partial charge, and 4) the bulky hydrophobic side chain
(Walsh and Wang, 1998;
Walsh et al., 1999). Compounds
that lacked the benzoate ring and/or those that decreased the acidity of this
ring caused a marked attenuation in the potency. Although we did not conduct
elaborate structure-activity studies, our results similarly indicate that a
benzoate group and a bulky hydrophobic side chain are important determinants
of inhibition. Compounds that lack the benzoate ring (APB) and/or the bulky
phenyl ring (e.g., o-aminobenzoic acid) did not cause significant
inhibition of gap junction channels at submillimolar concentrations.
Similarly, compounds such as clofibric acid that contain substantial
modification in this part of the molecule did not block gap junction channels
at 1 mM or lower, whereas niflumic acid, which contains a nicotinic acid
moiety instead of a benzoate ring, was less potent compared with FFA, MFA, and
NPPB. However, additional studies are clearly essential to determine the
importance of other regions of the molecule.
The effects of FFA were dependent on the external pH. External pH is known
to markedly affect ion channel block by tertiary amine drugs such as local
anesthetics and antimalarials as well as weak acids such as arylaminobenzoates
by altering the ratio of charged and the membrane-permeable uncharged forms
(Hille, 2001). We showed that
the inhibitory effect of FFA, which has a pKa between 3
and 4, is increased at low external pH and markedly decreased at low external
pH. These results indicate that extracellularly applied FFA reaches its
blocking site by permeating across the membrane in its uncharged form and
strongly indicate that the uncharged form of the drug is the active
species.
Inhibition of gap junction channels by flufenamic acid does not seem to be
mediated by binding of the anionic form of the drug in the pore of the gap
junction channel, as has been reported to be the mode of action on the cystic
fibrosis transmembrane conductance regulator channel
(McCarty et al., 1993).
Several lines of converging evidence suggest that an alternative mechanism is
involved. Internal application of the drug via patch pipettes did not cause
inhibition of gap junction channels even with 10-fold higher concentration and
after sufficient time was allowed for the diffusion of the drug to
intracellular compartment (Srinivas et
al., 2001, show experiments using other uncouplers). Although it
is possible that the drug diffuses out of the cell before it reaches the
binding site, the use of high concentrations would be expected to cause a
moderate amount of blockade (DeCoursey,
1995). In addition, application of the drug at high internal pH,
where the majority of the drug is charged and therefore would be expected to
diffuse out of the cell more slowly than at pH 7.2, did not cause any
detectable reduction by internally applied drug. Moreover, the recovery of
gj in response to extracellularly applied FFA while internal FFA
concentration remained high provides evidence that the binding site is not
accessible from the cytoplasm. The inhibitory action of FFA was not affected
by the nature of the permeant ion. If the effect were mediated by binding
within the pore, it should be influenced by the concentration and the nature
of the permeant ion. In the case of chloride channels, the potency of these
drugs was markedly reduced when the concentration of chloride ions in the pore
was increased or when chloride was substituted with thiocyanate ions
(McDonough et al., 1994).
Although we did not systematically vary the concentration of chloride in our
internal solutions, we conducted experiments wherein the magnitude of
inhibition was assessed when chloride was replaced by glutamate and determined
no significant change in the degree of reduction of channel currents caused by
30 and 100 µM FFA.
Finally, single-channel currents measured in the presence of FFA provided
additional evidence that the effect of the drug is not caused by open channel
block. A hallmark of open channel block is the rapid flickering of
single-channel current between open and closed states as a result of binding
and unbinding of the drug to a site in the pore of the channel
(Hille, 2001). If the binding
and unbinding rates of the blocker are fast, the single-channel current
amplitude will seem to be reduced (Hille,
2001). In our experiments, however, single-channel currents in the
presence of FFA did not exhibit rapid flickering between open and closed
states, and single-channel current amplitudes were not altered by the presence
of the drug.
The predominant effect of FFA on single channels was a reduction in the
open probability of the channel and an increase the incidence of slow gating
transitions between open and closed states. In these respects, FFA is similar
to almost all other gap junction channel blockers and inhibitors studied,
including low pH, halothane, n-alkanols, and quinine
(Harris, 2001). Most of these
uncoupling agents elicit closure by binding to regions outside the pore. For
example, low pH affects coupling by protonation of histidine residues in the
cytoplasmic loop (Stergiopoulos et al.,
1999; Duffy et al.,
2002), whereas n-alkanols have been reported to exert
their effects by altering the fluidity of cholesterol-rich domains surrounding
the connexin protein (Bastiaanse et al.,
1993). Slow transitions elicited by these uncoupling agents have
been attributed to a distinct form of closure that presumably involves
conformational rearrangements of the channel
(Trexler et al., 1996).
Whether FFA and its analogs similarly induce channel closure by this same
mechanism is difficult to determine experimentally. Our results nevertheless
indicate that FFA and its analogs seem to function as allosteric modifiers of
channel gating, presumably by binding directly to the channel. Because these
drugs are highly lipophilic molecules, it seems most likely that they cause
closure by partitioning into the plasma membrane in their uncharged forms and
inducing a conformational change in the channel protein. An alternate
possibility is that these drugs act by altering bulk membrane fluidity
surrounding the channel protein, as has been proposed for other gap junction
channel inhibitors. Additional studies are required to address this issue.
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Footnotes |
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ABBREVIATIONS: FFA, flufenamic acid; MFA, meclofenamic acid; NFA, niflumic acid; NPPB, 5-nitro-2-(3-phenylpropylamino)-benzoic acid; DIDS, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid; CFA, clofibric acid; APB, 2-amino-4-phenylbutyric acid; Vj, voltage gradient; gj, conductance; Ij, junctional current; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; pHe, external pH.
Address correspondence to: Dr. Miduturu Srinivas, Department of Neuroscience, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY, 10461. E-mail: msriniva{at}aecom.yu.edu
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References |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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) at external pH values of 6.7, 7.4, and 8.0
are shown. A 90-s exposure to solutions at external pH values of 7.0 and 8.2
in the absence of FFA did not cause a significant change in gj
(percentage of control at pH 7.4; 
