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The Journal of Neuroscience, January 15, 1999, 19(2):529-538
Capsaicin Binds to the Intracellular Domain of the
Capsaicin-Activated Ion Channel
Jooyoung
Jung1,
Sun Wook
Hwang1,
Jiyeon
Kwak1,
Soon-Youl
Lee1,
Chang-Joong
Kang1,
Won Bae
Kim2,
Donghee
Kim3, and
Uhtaek
Oh1
1 The Sensory Research Group, Creative Research
Initiatives, Seoul National University, College of Pharmacy, Kwanak,
Seoul 151-742, Korea, 2 Research Laboratories of Dong-A
Pharmaceuticals, Yongin, Krunggi 449-900, Korea, and
3 Department of Physiology and Biophysics, Finch University
of Health Sciences, The Chicago Medical School, North Chicago, Illinois
60064-3095
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ABSTRACT |
Capsaicin (CAP) excites small sensory neurons, causing pain,
neurogenic inflammation, and other visceral reflexes. These effects have been proposed to be the result of CAP activation of a nonselective cation current. It is generally assumed that CAP binds to an
extracellular domain of the membrane receptor. However, the exact
binding site is not known because of the lipophilic nature of
CAP. To determine whether the binding domain is extracellular or
intracellular, we tested the effect of a synthetic water-soluble CAP
analog, DA-5018·HCl, on current activation. CAP activated the 45 pS
(at  60 mV) nonselective cation channel from either side of the
membrane. However, DA-5018·HCl, which had a greater potency and
efficacy than CAP, activated the channels only from the cytosolic side of the patch membrane in a capsazepine, a CAP receptor antagonist, reversible manner. When applied extracellularly, DA-5018·HCl did not,
but CAP did, activate whole-cell currents in sensory neurons, as well
as in oocytes expressing vanilloid receptor 1, a recently cloned CAP
receptor. Hydrogen ions, reported as a possible endogenous activator of
cation current, failed to elicit any current when acidic medium (pH
5.0-6.0) was applied intracellularly, indicating that
H+ does not mediate the CAP effect. These results
indicate that CAP and its analog bind to the cytosolic domain of the
CAP receptor and suggest that an endogenous CAP-like substance other
than H+ may be present in the cell.
Key words:
capsaicin receptor; VR1; binding domain; DA-5018; capsazepine; acid; pain
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INTRODUCTION |
Capsaicin (CAP), a pungent chemical
present in hot peppers, produces an immediate pain or hyperalgesia when
applied cutaneously or intradermally (Simone et al., 1987 , 1989;
Geppetti et al., 1988; Park et al., 1995). CAP also causes neurogenic
inflammation mediated by release of neuropeptides, such as substance-P
or calcitonin gene-related peptide, from sensory nerve endings (for
review, see Szolcsanyi, 1996). In addition, CAP paradoxically induces desensitization of sensory neurons to various types of noxious stimuli,
thus producing a long-lasting and naloxone-resistant analgesia (for
review, see Holzer, 1991). Because of this analgesic action, CAP is
often used for alleviating pain caused by diabetic or herpetic
neuropathy or arthritis, and CAP analogs are being explored as
potential analgesics (Watson et al., 1988; Bernstein et al.,
1989; Donofrio et al., 1991; Szallasi and Blumberg, 1996).
The excitation of sensory neurons induced by CAP is believed to
result from large influxes of cations, such as Na+
or Ca2+ (Bevan and Szolcsanyi, 1990). CAP produces
an influx of Ca2+ and other cations in a
dose-dependent manner in cultured sensory neurons (Wood et al., 1988;
Bevan et al., 1992). Studies on cultured sensory neurons further show
the presence of specific ion currents activated by CAP application
(Marsh et al., 1987; Liu and Simon, 1994). Previously, we identified
and characterized a ligand-gated cation channel specifically
activated by CAP and antagonized by capsazepine (CZP), a
functionally defined CAP receptor antagonist in cultured dorsal root
ganglion (DRG) neurons (Bevan et al., 1992; Oh et al., 1996). Recently,
a gene encoding for CAP receptor, vanilloid receptor 1 (VR1), was
cloned from rat DRG neurons (Caterina et al., 1997). The VR1, having
six putative transmembrane domains and two cytosolic domains in both
ends of the protein, exhibits inward currents sensitive to CAP, as well
as noxious heat when expressed in oocytes. The channel property of the
expressed CAP receptor resembles that observed in native channels in
DRG neurons (Oh et al., 1996; Caterina et al., 1997). Thus, activation
of this channel may primarily account for the ionic responses of sensory neurons to CAP.
In previous studies, current responses to CAP were tested by applying
CAP only to the extracellular space, with the assumption that CAP binds
to an extracellular site (Liu and Simon, 1994; Oh et al., 1996;
Caterina et al., 1997; Koplas et al., 1997). Because CAP or its
analogs, such as CZP or resiniferatoxin, are lipid-soluble, it is
possible that they pass through the cell membrane and act on binding
sites present in the intracellular surface of the receptor. Thus, the
location of CAP binding has yet to be determined. In this study, to
understand further the signaling pathways involved in CAP-induced
cellular effects, we used a salt form of a synthetic analog of CAP,
DA-5018·HCl (DA) (see Fig. 1) as an experimental tool to
locate the binding domain of CAP. The results of our study indicate
that the binding sites for CAP are present at the intracellular side of
the cell membrane.
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MATERIALS AND METHODS |
Cell preparation. Cultured DRG neurons were prepared
as described previously (Oh et al., 1996). Briefly, DRGs were dissected from all levels of lower cervical, thoracic, and lumbar spinal cord of
1- or 2-d-old neonatal rats. DRGs were collected in cold culture medium
(4°C) containing DMEM-F-12 mixture (Life Technologies, Grand Island,
NY), fetal bovine serum (10%; Life Technologies), 1 mM sodium pyruvate, 25 ng/ml nerve growth factor (Sigma,
St. Louis, MO), and 100 U/ml of penicillin-streptomycin (Sigma).
Ganglia were washed three times with DMEM-F-12 medium and incubated
for 30 min in the DMEM-F-12 medium containing 1 mg/ml collagenase (Type II; Worthington, Freehold, NJ). The ganglia were then washed three times with Mg2+- and
Ca2+-free HBSS and incubated with gentle
shaking in the warm (37°C) HBSS containing 2.5 mg/ml trypsin (Life
Technologies). The solution was centrifuged at 1,000 rpm for 10 min,
and the pellet was washed two or three times with the culture medium to
inhibit the enzyme. The pellet was suspended in the culture medium and
gently triturated with a Pasteur pipette. The suspension was plated on
round glass coverslips (Fisher, Pittsburgh, PA) placed in small Petri
dishes. The glass coverslips were treated overnight with
poly-L-lysine (Sigma) and dried before use. Cells were
incubated at 37°C in a 95% air-5% CO2 gas mixture.
Cells were used 2-4 d after plating.
Current recording. Borosilicate glass pipettes (Narishige,
Tokyo, Japan) were pulled and coated with Sylgard (Dow Corning, Midland, MI). Tip resistances were ~2 and 5 M for whole-cell and
single-channel current recordings, respectively. After gigaseals were
formed with the glass pipettes, cell-attached and inside-out patch
configurations were used to study single-channel currents as described
by Hamill et al. (1981). A salt bridge (1% agar in 300 mM
KCl) immersed in bath and an Ag/AgCl reference electrode in pipette
solution was used to minimize changes in junctional potentials.
Junctional potentials were canceled before gigaseals were formed.
For whole-cell recording, the cell membrane under a glass pipette was
ruptured by a gentle suction. After forming a whole cell, capacitative
transient was canceled. Single-channel currents were recorded using a
patch-clamp amplifier (Axopatch 200A; Axon Instruments, Foster City,
CA) and filtered at 5 kHz with an eight-pole low-pass Bessel filter.
Data were digitized at 37 kHz with a digital data recorder (VR-10B;
Instrutech, Great Neck, NY) and stored on videotapes for later
analysis. For chart recording, output of amplifier was filtered at 500 Hz (Frequency Device, Havenhill, MA) and fed into a thermal array chart
recorder (TA-240; Gould Instrument System, Valley View, OH). The
digitized data stored on videotapes were imported to a personal
computer (IBM pentium-compatible) for computer analysis of
single-channel currents.
Channel open probability (Po), amplitude, and mean
open time of single-channel currents were obtained using the pCLAMP software (version 6.02; Axon Instruments). Po of
single channels was obtained from the ratio of the areas under the
curves representing open events divided by the sum of the areas under the curves representing open and closed events. The half-amplitude algorithm in the FETCHAN program (Axon Instruments) was used for the threshold amplitude for detecting open events. Channel activity (NPo) was calculated as a product of the number of
channel (N) in the patch and Po.
NPo or Po was collected only from
patches that contained less than five functional CAP-activated channels.
VR1 expression in oocytes and two-electrode recording. Rat
brain mRNAs were isolated using the FastTrack 2.0 mRNA isolation kit
(Invitrogen, San Diego, CA). VR1 cDNA was cloned into a pSDTF vector by
reverse transcriptase-PCR from rat brain mRNA using the
SuperScript kit (Life Technologies). PCR primers, designed based on the
sequence of VR1 (Caterina et al., 1997), are as follows: VR1-HindIII (sense), 5'-CCC AAG CTT GCC GCC ACC ATG GAA CAA
CGG GCT AGC-3'; and VR1-KpnI (antisense), 5'-CCG GTA CCT TAT
TTC TCC CCT GGG AC-3'. cRNA transcripts were synthesized from
XbaI-linearized VR1 cDNA templates using SP6 RNA
polymerase of the MEGAscript kit from Ambion (Austin, TX) as suggested
by the manufacturer. Defolliculated Xenopus laevis oocytes
were injected with 1 ng of VR1 cRNA in 50 nl of water. Three to 5 d after injection, two-electrode voltage-clamp recording was performed
(Ehold = 60 mV) using an oocyte-clamp
amplifier (OC-725C; Warner Instrument, Hamden, CT). The recording
chamber was perfused at a rate of 2 ml/min at room temperature with a
solution containing (in mM): 96 NaCl, 5 HEPES, 2 KCl, 1.8 CaCl2, and 1 MgCl2, pH 7.5 (ND96).
Solutions. Solutions in bath and pipette for single-channel
recordings contained (in mM): 140 Na+, 2 Mg2+, 144 Cl , 5 EGTA, and 10 HEPES, pH 7.2. For whole cell, pipette solution contained (in
mM): 140 K+, 2 Mg2+,
144 Cl, 5 EGTA, 10 HEPES, and 4 ATP, pH 7.2. The
control perfusion solution for whole-cell recording contained (in
mM): 140 Na+, 5 K+, 2 Mg2+, 1 Ca2+, 151 Cl, and 10 HEPES. CAP and CZP (Research
Biochemicals, Natick, MA) were dissolved and stored in 100% ethanol to
make 10 mM stock solutions. A synthetic analog of CAP, DA
(406.9 MW) (Fig. 1), was obtained from
Dong-A Pharmaceutical (US patent 5242944; Seoul, Korea), dissolved in
distilled water, and stored as a stock solution (10 mM).
All other reagents used in cell culture or electrophysiological experiments were purchased from Sigma. All values were expressed as
mean ± SE.
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RESULTS |
Activation of single-channel currents by intracellular CAP
Previously, we showed that CAP activates a nonselective cation
channel when applied to the outer surface of the patch membrane of
cultured DRG neurons (Oh et al., 1996). Because CAP is highly lipid-soluble and thus can cross the membrane easily by diffusion, we
predicted that CAP applied to the inner surface of inside-out patches
would also lead to the channel activation. As shown in Figure
2A, channel activity
was not observed when control solution was applied to the bath
(intracellular side) in an inside-out patch. When 1 µM
CAP was introduced to the bath solution, a rapid activation of
single-channel currents was observed within a few seconds
(n = 21). This activation by intracellular CAP was
completely antagonized by 10 µM CZP, a competitive CAP
receptor antagonist (Fig. 2A). As summarized in
Figure 2B, Po of single-channels induced by intracellular CAP increased from 0.00 to 0.42 ± 0.09 (n = 5). This Po of single-channels,
however, decreased to 0.03 ± 0.01 (n = 5) when 1 µM CAP was applied to the bath together with 10 µM CZP. Intracellular application of resiniferatoxin, a
potent agonist of CAP receptors (Szallasi and Blumberg, 1989), was also
tested for activation of the CAP-activated channel. In inside-out patches, perfusion of resiniferatoxin (1-2 nM)
to the inner side of the membrane also caused rapid activation of
single-channel currents (n = 6; data not shown). The
rapid onset of activation after intracellular application of CAP
suggests that the receptor for CAP may be at the inner membrane
surface. Thus, channel activation by the extracellular application of
CAP may be activating the channel by diffusing into the cell membrane
and acting on binding domains present in the intracellular side of the
patch membrane.
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Figure 2.
Activation of single-channel currents by
intracellular CAP application and block by CZP. A, An
inside-out membrane patch was held at 60 mV in a symmetrical 140 mM NaCl solution. After an inside-out patch was formed, CAP
(1 µM) or CAP plus CZP (1 and 10 µM,
respectively) was applied to bath. Inset shows
single-channel current traces in an expanded time scale.
Straight line in the inset represents the
closed state of the channel. B,
Po of the channel obtained when CAP was applied
to the intracellular surface of five different patch membranes.
C, Blockade by intracellular CZP application of channel
currents activated by extracellular application of CAP. The pipette
contained CAP (1 µM) in the 140 mM NaCl
solution in a cell-attached or inside-out patch. CZP (10 µM) was applied to bath (intracellular side) after an
inside-out patch was formed. D, Activation of
single-channel currents after bath perfusion of 1 µM CAP
in a cell-attached patch.
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To examine whether the channel activated by extracellular CAP can be
blocked by intracellular CZP, we applied CZP to the inner surface of
the patch membrane while CAP was kept in the extracellular surface of
the patch. As shown in Figure 2C, when a cell-attached patch
was formed with a pipette containing 1 µM CAP, activation of single-channel currents was observed (Po = 0.51 ± 0.13; n = 7). After forming an inside-out
patch, the channel activity persisted, although it depressed slightly
(Po = 0.38 ± 0.13; n = 7). In
the same patch, 10 µM CZP applied to the bath (intracellular surface) quickly blocked the CAP-induced single-channel currents (Po = 0.01 ± 0.0; n = 7) (Fig. 2C). The rapid activation of channel by CAP and
rapid inhibition by intracellular CZP also suggest that the binding
site may be at the intracellular side of the membrane. In other
experiments, CAP was applied to the bath after forming cell-attached
patches with pipettes containing only the control 140 mM
NaCl solution. When perfused to the bath, 1 µM CAP
activated single-channel currents 8.4 ± 2.6 sec
(n = 14) after application (Fig. 2D).
It, however, generally required several minutes for CAP to activate the
channels maximally. Activation of the channel in this patch
configuration further suggests that extracellular CAP can diffuse
easily through the membrane to reach the target site.
Activation of single-channel currents by intracellular DA
To determine whether CAP activates the channel by acting at an
intracellular or extracellular binding domain of the membrane, we used
a CAP analog that is charged at physiological pH and thus cannot cross
the cell membrane easily. A salt form of an analog of CAP, DA (Fig. 1),
was synthesized (Park et al., 1993) and used as the water-soluble
ligand for activating the channel. DA at physiological pH was applied
to isolated patches under different configurations. As shown in Figure
3A, 0.5 µM DA
also caused activation of single-channel currents
(Po = 0.73 ± 0.11; n = 5) when
applied to the bath (intracellular side) in an inside-out patch. This
activation by intracellular DA was blocked by 10 µM CZP
(Po = 0.0 ± 0.0; n = 5). To
determine whether extracellular application of DA can activate the
channel, we applied DA to the outer surface of the cell membrane. As
shown in Figure 3B, 1 µM DA in the pipette
solution (extracellular side) failed to activate the
CAP-activated ion channel in either the cell-attached or inside-out patch state (n = 5). In the same patch, however,
application of 0.5 µM DA to the bath (intracellular side)
activated the channel currents (Fig. 3B). The response to
the intracellular application of DA was reversible. After washout,
application of 1 µM CAP to the bath caused openings of
the same channels (n = 5). Because the ionic state of
DA depends on the pH of the solution, we changed the pH of the
extracellular DA. At pH 6.0, the proportion of un-ionized species of DA
is dramatically reduced (0.16% of total DA, see Discussion) compared
with that at physiological pH (2.5% of total DA). At pH 6.0, 1 µM DA in the pipette (extracellular side) did not
activate the channel currents in cell-attached or inside-out patches
(n = 6). However, intracellular application of DA at pH 6.0 readily activated the currents. In contrast, as shown in Figure 3C, at pH 8.6, at which the proportion of uncharged species
is high (37% of total DA), extracellular application of 1 µM DA greatly activated channel currents in all patches
tested (n = 8). The channel currents activated by
extracellular DA at pH 8.6 were abolished by intracellular application
of 10 µM CZP (Fig. 3C). These results clearly
suggest that DA in the predominantly charged state can activate the
channel only from the intracellular side and that binding of CAP and
its analogs takes place at the intracellular side of the membrane.
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Figure 3.
Differential responses of the CAP-activated
channel to an ionized form of the CAP analog, DA, applied to the
intracellular or extracellular surface of patch membranes.
A, Activation and block by CZP of single-channel
currents by 0.5 µM DA applied to bath (intracellular
side) in an inside-out patch. B, Failure of DA in normal
pH (pH 7.2) to activate the CAP-activated channel in a cell-attached or
inside-out patch, with the pipette (extracellular side) containing 1 µM DA. Only when DA or CAP was perfused to the bath was
the rapid activation of the channel currents observed. Time and current
scales are same as shown in C. C, DA at
pH 8.6 in the pipette-activated channel currents in a cell-attached or
inside-out patch. Application of 10 µM CZP to the bath
after forming an inside-out patch blocked the channel currents.
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Single-channel openings by CAP and DA
The channels activated by intracellular application of CAP
exhibited single-channel properties similar to those obtained from the
extracellular application of CAP (Oh et al., 1996). Channel openings
with membrane potentials held at 80 to +80 mV in 40 mV increments in
symmetrical Na+ solution are shown in Figure
4A. Amplitude
histograms were obtained from channel openings at each membrane
potential held at 80 to +80 mV in 20 mV increments, and the mean
amplitude was plotted against membrane potential to obtain a
current-voltage (I-V) relationship. As shown in
Figure 4B, single-channel currents activated by CAP exhibited outward rectification. Slope conductances at 60 and +60 mV
were 44.7 ± 0.9 and 77.5 ± 2.3 pS, respectively
(n = 11). Channel openings produced by intracellular
application of CAP occurred in short and long bursts (Fig.
4A). Single-channel currents activated by DA at each
membrane potential were also obtained, and an I-V
relationship was compared with that obtained from CAP. As shown in
Figure 4B, single-channel currents activated by DA were outwardly rectifying, similar to those activated by CAP. Slope
conductances at 60 and +60 mV were 45.3 ± 0.7 and 75.4 ± 1.1 pS (n = 7), respectively (Fig.
4B). The similarity of single-channel properties also
suggests that DA activates the same channel activated by CAP.
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Figure 4.
Single-channel openings obtained when CAP or DA
was applied to the bath (intracellular surface) in inside-out patches.
A, Traces of single-channel currents activated by 1 µM CAP (right) and 0.3 µM DA
(left) held at different membrane potentials ranging
from 80 to +80 mV. B, I-V
relationships of single-channel currents. Each point was averaged from
11 (CAP) or seven (DA) inside-out
patches. Bars representing SE are so small that they are
inside the triangles or circles.
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Ion selectivity of single-channel currents activated by DA
To characterize further the channel activated by DA, ion
selectivity of the channel current was determined. Because the
CAP-activated ion channel discriminates poorly among cations,
permeability to K+ or Cs+ of the
channel current activated by DA was determined. In inside-out patches
containing multiple CAP-activated channels, a voltage ramp from 80 to
+80 mV in 300 msec duration was applied to get the reversal potentials
in various ionic conditions. Macroscopic current responses to the
voltage ramp were recorded from inside-out patches with the pipette
containing the 140 mM NaCl solution and the bath containing
the symmetrical NaCl or equimolar KCl or CsCl solution. In each patch,
0.5 µM DA was added to the bath solution. As shown in
Figure 5, replacing bath NaCl with KCl
and CsCl did not shift the reversal potential (3.0 ± 0.7 and
5.2 ± 0.6 mV, respectively; n = 6) from 0 mV,
suggesting that the channel activated by DA is permeable to
monovalent cations. The permeability ratios, PK/PNa and
PCs/PNa,
under these bi-ionic conditions calculated from the constant-field
equation (Fatt and Ginsborg, 1958; Hille, 1992) were 1.1 and
1.2, respectively. To determine whether the channel activated by DA was
permeable to Ca2+, the pipette with 140 mM Na+ was replaced with 100 mM Ca2+, and the bath contained 0.5 µM DA in the 140 mM Na+
solution (Fig. 5). The average reversal potential with 100 mM Ca2+ in eight experiments was
26.6 ± 2.2 mV, indicating the high Ca2+
permeability over Na+
(PCa/PNa = 2.9) as
reported previously (Wood et al., 1988; Caterina et al., 1997;
Zeilhofer et al., 1997). These results further suggest that the channel
activated by DA is nonselectively permeable to cations, as observed in
the CAP-activated channel.
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Figure 5.
Ion selectivity of the channel current activated
by 0.5 µM DA applied intracellularly. Macroscopic current
responses to voltage ramps (300 msec) from 80 to +80 mV in a patch
containing multiple CAP-activated channels after the bath solution of
140 mM NaCl was changed to equimolar KCl and CsCl. The
pipette contained 140 mM NaCl, and 0.5 µM DA
was added to each bath solution. To get a macroscopic current in
Ca2+/Na+ bi-ionic condition, the
voltage ramp was applied to a patch containing 100 mM
CaCl2 in the pipette and 140 mM NaCl and 0.5 µM DA in the bath.
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Concentration dependency
We determined the concentration-effect curves for CAP and DA to
compare their efficiencies on channel activation from the intracellular
side. A steady-state level of channel activity was achieved during
intracellular perfusion of agonists at different concentrations. As
shown in Figure 6A, CAP
concentration <0.1 µM rarely activated the channel, but
CAP at  0.3 µM progressively increased the channel
activity, showing a maximal activation at 10 µM. The open
probability (Po) was plotted as a function of
agonist concentration (Fig. 6C). Each data point was fitted by a nonlinear regression to the Hill equation:
In this equation, KD is the half-maximal
concentration of agonists in activating the channel, [Agonist] is the
concentration of CAP or DA, Pomax is the maximal
Po, and n is the Hill coefficient.
KD values for CAP and DA in activating the
channel were 1.0 (n = 11) and 0.32 (n = 7) µM, respectively, indicating that DA has a greater
potency in activating the channel. The intracellular application of DA
also exhibited a greater efficacy as well, because the maximal current
response (Po = 0.84 ± 0.07; n = 7) to DA (3 µM) was ~24% greater than that observed after 10 µM CAP application (Po = 0.70 ± 0.09; n = 11). The Hill coefficients for
CAP and DA were 1.8 and 2.1, respectively. KD
and Hill coefficients obtained by intracellular CAP were not different
from those obtained by extracellular CAP application (KD = 1.1 µM; n = 1.8) (Oh et al., 1996). Furthermore, the slopes of the dose-response
curves suggest that two binding sites may be present for CAP and DA and
that the binding occurs in a cooperative manner.
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Figure 6.
Concentration-dependent activation of the channel
by intracellular CAP or DA. A, CAP, at different
concentrations ranging from 0.03 to 10 µM, was perfused
to the bath in an inside-out membrane patch in a symmetrical 140 mM NaCl condition. B, DA, at different
concentrations ranging from 0.03 to 3 µM, was perfused to
the bath in an inside-out patch in the symmetrical salt solution.
C, A summary of the concentration-response relationship
expressed in Po of the channel versus
concentration of intracellular agonists. Data points were fitted to the
Hill equation, as described in Results.
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Whole-cell and oocyte currents activated by CAP and its analog
As shown in Figure 7, extracellular
application of 1 µM CAP (a half-maximal dose) to a DRG
cell under whole-cell configuration whose membrane potential was held
at 60 mV produced a rapid inward current. After washout of CAP, 0.3 µM DA (a half-maximal dose) was perfused to the bath to
test the effect of the CAP analog on the CAP-induced current. The
perfusion of 0.3 µM DA did not activate the whole-cell
current. After washout, reapplication of 1 µM CAP
produced an inward current again but in a smaller magnitude, showing
apparent desensitization, a property of the CAP-activated channel (Fig.
7A) (Docherty et al., 1996; Liu and Simon, 1996; Oh et al.,
1996; Koplas et al., 1997). However, the application of DA in a
supramaximal concentration (10 µM) exhibited a large,
slowly activating inward current (Fig. 7B), probably because
a substantial amount of an un-ionized form of DA diffused into
the intracellular side. To confirm whether activation of the channel
currents by extracellular DA depends on the amount of un-ionized
fraction of DA, we changed pH of the bath solution containing DA.
Application of 0.3 µM DA at pH 6.0, at which the majority
of DA were ionized (99.8%), did not activate any current at all,
whereas 0.3 µM DA at pH 8.6, at which the proportion of uncharged DA was ~37%, exhibited inward currents with the magnitudes of 63 ± 10% (n = 5) of those of CAP-induced
currents. Inability of DA at a half-maximal concentration in activating
the channel was also tested in oocytes expressing a newly cloned CAP
channel, VR1 (Caterina et al., 1997). As shown in Figure 7C,
the perfusion of the ND96 solution (pH 7.5) containing 1 µM CAP to an oocyte injected with cRNA (1 ng) of VR1
produced a rapid and large inward current. In the same patch, however,
the application of 0.3 µM DA failed to exhibit the
current response. However, as observed in cultured sensory neurons, a
supramaximal dose (10 µM) of DA to the oocyte greatly
activated the current at a much slower rate. Because CAP and its analog
differ only in lipid solubility, the different current responses of
native, as well as oocyte-expressed, channels to extracellular CAP and
DA may imply that the ligand-binding domain of the CAP receptor is
present in the intracellular side of the cell membrane.
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Figure 7.
Current responses of cultured sensory neurons or
oocytes expressing VR1 to the extracellular perfusion of CAP and its
analog. A, Activation of whole-cell currents by bath
application of 1 µM CAP but not by 0.3 µM
DA. Note an apparent desensitization after CAP. B, A
great activation of whole-cell current by bath application of 10 µM DA. Arrow indicates unclamped current
spikes originating in narrow axon trunks. C, Activation
by 1 µM CAP of a CAP receptor expressed in
Xenopus oocytes. A half-maximal dose of DA (0.3 µM) failed to activate the current, but a supramaximal
dose of DA (10 µM) activated the current.
Inset shows a summary of current responses of oocytes
expressing CAP receptors to CAP and DA (n = 6).
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Effect of intracellular acidification
H+ has been suggested as an endogenous
activator of CAP receptors (Bevan and Geppetti, 1994). If this were the
case, an acidic solution (pH <6.0) applied to the inner side of the
patch membrane should activate the CAP-activated ion channel. To test
the action of H+, CAP (1 µM) or acidic
control solution (pH 5.0) was applied to the inner surface of patch
membrane in inside-out patch configuration. When acidic control
solution of pH 5.0 was perfused to the bath, the acidic solution failed
to activate the single-channel currents. However, application of CAP (1 µM) to the same patch elicited a rapid activation of the
channel currents (Fig.
8A). In 30 additional experiments, none of the applications of acidic solution (pH 5.0-6.0) activated the single-channel currents when applied to the cytosolic surface of the patch membrane. Although acidic solution does not activate the channel directly, it is still possible that acid may
activate the channel indirectly via cytosolic signal transduction pathways. To test this, we perfused acidic solution (pH 6.0) to the
bath (extracellular side) in a cell-attached patch containing only
control solution in the pipette. As shown in Figure
8B, application of acidic solution to the bath did
not activate the channel (n = 6). However, 1 µM CAP perfused to the bath easily activated the channel.
In this cell-attached patch, brief spontaneous openings of the channel
were also observed before the application of acid or CAP (Fig.
8B). Because it is known that H+
modulates activity of various ion channels (Hille, 1992), we tested
whether acidic condition affected the CAP-evoked channel currents. As
shown in Figure 8C, the CAP-activated channel activity was
greatly augmented by acidic condition (pH 6.0) in five of eight
patches.
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Figure 8.
A, B, Failure of
acid in activating the CAP-activated channel currents when perfused to
the bath (intracellular surface) of an inside-out
(A) or a cell-attached (B)
patch. Time and current scales in A and B
are same as shown in D and C,
respectively. C, Modulation of the CAP-activated channel
currents by acidic condition. D, Failure of 20 µM amiloride in inhibiting the CAP-activated channel
currents.
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|
Recently, an acid-sensitive channel was cloned in DRG neurons (Waldmann
et al., 1997). Similar to the CAP-activated ion channel, the
acid-sensitive ion channel is permeable to cations. The acid-sensitive channel has two putative transmembrane domains and sequence homology with amiloride-sensitive Na+-channels and is blocked
by amiloride when expressed in Xenopus oocytes (Waldmann et
al., 1997). In DRG neurons, intracellular perfusion of 10 µM amiloride with 1 µM CAP failed to block
the CAP-activated channel current in all six patches tested (Fig. 8D). This result again indicates that the
CAP-activated channel is different from the acid-sensitive channel.
These results suggest that H+ does not activate the
channel directly but possibly modulates the channel.
| |
DISCUSSION |
It is now clear that CAP activates a nonselective cation channel
in cultured DRG neurons. Opening of the channel by CAP would cause
influx of Na+ and Ca2+, resulting
in depolarization of sensory neurons. It is generally assumed that CAP
binds to a receptor site in the extracellular side of the membrane to
produce its hyperalgesic effects. However, it is not clear whether the
CAP binding site is present in the inner or outer surface of the cell
membrane because of the high lipid solubility of CAP. In this study, we
show that intracellular application of CAP can also activate the
channel in a concentration-dependent manner, as expected of a
lipophilic compound. A relatively membrane impermeable analog of CAP,
DA, was able to activate the channel when the analog was applied to the
inner, but not the outer, surface of the patch membrane, indicating
that CAP and its analogs act on the channel from the intracellular
side. The finding that agonists bind to the intracellular side of the
membrane suggests that an endogenous activator of the channel may exist
in the cytosol of cells.
Physicochemical property of DA
The pKa of DA is 8.8. Therefore, the molar ratio of
the ionized form (NH3+) (Fig. 1) of DA
to the un-ionized form (NH2) calculated using the
Henderson-Hasselbalch equation (pKa = pH log[Base/Acid]) is ~40 in the 140 mM NaCl solution at
pH 7.2. This indicates that 97.5% of DA at this pH become ionized.
Thus, at the applied concentration of 1 µM, 0.975 µM DA would be charged. Only a fraction (0.025 µM) of DA remains uncharged and thus can pass through the
cell membrane under this pH condition. Once passing through the cell membrane, the uncharged DA will dissociate and equilibrate again with
charged species. However, the concentration of DA, whether it is
charged or not, would not be high enough to activate the channel,
because DA  0.1 µM rarely activates the channel in
excised membrane patches, as shown in the concentration-response
relationship (Fig. 6). We further showed that application of DA at
physiological pH to the extracellular side of the patch membrane failed
to activate the channel, whereas intracellular application of the DA
readily activated the channel (Fig. 3). In addition, when pH of the
solution became basic (pH 8.6), thus increasing uncharged species of DA (37% of total DA), the extracellular application of DA activated the
channel currents in whole-cell and isolated membrane patches. These
results strongly suggest that the ligand-binding domain of the receptor
is present in the intracellular surface of the cell membrane and that
activation of the channel by extracellular application of CAP or DA is
caused by its diffusion through the cell membrane.
Binding property of the CAP-activated channel
High-affinity binding sites for CAP, as measured by specific
binding of radio-labeled 3H-resiniferatoxin, were
distributed in the DRG, spinal cord, cerebellum, and retina (Szallasi
et al., 1995). Furthermore, Szallasi et al. (1993) showed that
3H-resiniferatoxin bound to the CAP receptor in a
cooperative manner, with a Hill coefficient of 1.7. This is in good
agreement with our results that CAP and DA activate the channel in a
positive cooperative manner (Hill coefficients, 1.8 and 2.1, respectively). This positive cooperativity with the Hill coefficient of
~2 was also seen in VR1 when expressed in oocytes (Caterina et al.,
1997). Judging from the data obtained from both binding assays and
current recordings from native or heterologously expressed channels,
the channel complex has at least two binding sites for CAP. Thus, the
binding of a molecule of CAP to one subunit of the channel may
accelerate binding of CAP to another subunit, exhibiting a positive
cooperativity. Hydrophobicity analysis of VR1 predicts two relatively
long cytosolic loops at both ends of the receptor protein and four
short extracellular segments between putative transmembrane domains
(Caterina et al., 1997). As suggested in part by the present study,
these cytosolic domains may interact with CAP to cause an opening of
the channel.
Endogenous activator of the CAP-activated channel
Analogous to what has been described for opiate receptors,
distribution of high-affinity binding sites for CAP (Szallasi et al.,
1995) and the presence of the CAP-activated ion channel in sensory
neurons (Liu and Simon, 1994; Oh et al., 1996) suggest the existence of
an endogenous ligand. Such an endogenous substance, however, has not
yet been identified. Previous studies have suggested that proton
(H+) is a possible candidate for the endogenous
substance, because acidic solution and CAP exhibit similar effects on
various tissues, such as sensory neurons, muscle, and bladder (for
review, see Bevan and Geppetti, 1994). Application of acidic solution
has been reported to cause inward whole-cell currents that are similar to the CAP-induced currents (Bevan and Yeats, 1991; Geppetti et al.,
1991). CAP and H+ even show similar responses to NGF
deprivation in primary cultures of DRG neurons (Bevan and Winter,
1995). Although these findings suggest that proton may be an endogenous
ligand for the CAP receptor, there is no direct evidence indicating
that proton is the activator of the CAP receptor. There are reports
against the assumption that H+ activates the
CAP-activated channel (Bevan et al., 1992; Steen et al., 1992). For
example, Rb+ efflux induced by low pH from cultured
DRG neurons was not blocked by CZP (Bevan et al., 1992). In the
previous study, we failed to observe the activation of a CAP-activated
channel by the extracellular application of acidic solution (pH 6.0)
(Oh et al., 1996). Recently, both acid-sensitive and CAP-sensitive
channels were cloned in DRG neurons (Caterina et al., 1997; Waldmann et
al., 1997). In addition to the difference in their primary structures,
there are clear lines of physiological evidence suggesting that the two channels are not identical. First, the acid-sensitive
domain of the channel is present at the extracellular side of the
membrane. Second, the acid-sensitive channel shows high permeability of Na+ over K+
(PNa/PKa = 13).
Last, the acid-sensitive channel is blocked by amiloride, but the
CAP-activated channel is not (Fig. 8). Thus, together with other
reports, the results of the present study further indicate that proton
may not be the endogenous ligand that binds to the CAP receptor.
However, we could not exclude the possibility that protons would
modulate the channel activity allosterically, partly as shown by
Caterina et al. (1997). We also observed the modulatory effect of
protons on the channel currents, because CAP applied in acidic solution
(pH 6.0) exhibited a greater channel activity than CAP in normal pH
(Fig. 8).
Clinical implication of DA
DA was developed as an analgesic, similar to other CAP
analogs (Dray et al., 1990; Dray, 1992; Szallasi and Blumberg, 1996). These analogs have agonistic activities for CAP receptors and induce
desensitization of sensory neurons to noxious stimuli. The
desensitization of sensory neurons is believed to be responsible for
the analgesic effect of the CAP analogs. Cellular mechanisms underlying
the desensitization of sensory neurons by the CAP analogs are not known
clearly. However, swelling of sensory neurons or cellular damage
induced by Ca2+-dependent cytosolic enzymes caused
by a large influx of Na+ and Ca2+
through CAP receptor is suggested (for review, see Bevan and Szolcsanyi, 1990; Szolcsanyi, 1993). Because DA is soluble in physiological solution, it can hardly pass through the cell membrane to
activate the channel. Thus, our finding that the ligand binding site is
present in the intracellular side of the CAP receptor suggests that DA
is not suitable for clinical use. This appears true especially when the
concentration of DA is low, but as the concentration of DA increases,
the concentration of the un-ionized form of DA gets greater so that it
is able to affect the CAP-activated channel from outside the cell
membrane. For example, we observed in the present study that DA at
half-maximal dose (0.3 µM) did not activate, but at 10 µM, it greatly activated whole-cell current, even when
applied to the bath (extracellular side) (Fig. 7). Under this
experimental condition, the un-ionized form of DA reached ~0.25
µM, which is close to a half-maximal dose in activating the channel. Therefore, these results indicate that depending on its
dose present in extracellular matrix systemic application of DA can
have pharmacological actions on sensory neurons and, thus, can be
applicable clinically.
In summary, we report that CAP activates a nonselective cation
channel by acting on binding sites present in the intracellular side of
the membrane receptor. We assume that the binding of CAP is directly on
the channel, because a cloned gene, VR1, alone confers the sensitivity
to CAP when expressed in oocytes. The present results suggest the
possible presence of an endogenous CAP-like substance in the cytosol of
sensory neurons. Because the CAP-activated current accounts for the
cellular responses of sensory neurons to CAP, the present finding will
help to further elucidate the mechanisms underlying sensory
transduction induced by CAP.
| |
FOOTNOTES |
Received July 14, 1998; revised Oct. 15, 1998; accepted Oct. 23, 1998.
This work was supported by Creative Research Initiatives of the Korea
Ministry of Science and Technology.
Correspondence should be addressed to Uhtaek Oh, The Sensory Research
Group, Creative Research Initiatives, Seoul National University,
College of Pharmacy, Kwanak, Shinlim San 56-1, Seoul 151-742, Korea.
| |
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Abstract
Introduction
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