1Department of Pharmacology and 2Department of Anesthesia, College of Medicine, University of Iowa, Iowa City 52242; and 3Department of Veterans Affairs Medical Center, Iowa City, Iowa 52246
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ABSTRACT |
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Su, X., R. E. Wachtel, and G. F. Gebhart. Mechanosensitive Potassium Channels in Rat Colon Sensory Neurons. J. Neurophysiol. 84: 836-843, 2000. Single-channel recording techniques were used to characterize mechanosensitive channels in identified (1.1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine methanesulfonate labeled) colon sensory neurons dissociated from adult S1 dorsal root ganglia. Channels were found in 30% (7/23) of patches in a cell-attached configuration and in 43% (48/111) of excised inside-out patches. Channels were highly selective for K+, had a slope conductance of 54 pS in symmetrical solutions, and were blocked by tetraethylammonium, amiloride, and benzamil. Channels were also seen under Ca2+-free conditions. Gadolinium (Gd3+), a known blocker of mechanosensitive ion channels, did not block channel activity. Tetrodotoxin and 4-aminopyridine were also ineffective. The cytoskeletal disrupters colchicine and cytochalasin D reduced the percentage of patches containing mechanosensitive channels. These results indicate that rat colon sensory neurons contain K+-selective mechanosensitive channels that may modulate the membrane excitability induced by colonic distension.
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INTRODUCTION |
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In the rat, experimental
colorectal distension activates pelvic nerve afferent fibers
innervating the colon. Changes in muscle tension within the wall of the
colon cause mechanical deformation of mechanosensitive (MS) afferent
fibers, resulting in action potential generation. Alterations in the
responsiveness of these MS pelvic afferents may be caused by or may
contribute to certain pathological conditions such as inflammation or
pain (Blumberg et al. 1983; Haupt et al.
1983
; Jänig and Koltzenburg 1991
;
Sengupta and Gebhart 1994a
,b
). However, the mechanisms
by which MS fibers are activated by mechanical deformation, and the
factors that regulate their responsiveness, are not well understood.
Presumably mechanical distention is converted into an electrical signal
by MS ion channels in the neuronal cell membrane.
MS ion channels are present in many cell types, including nonsensory
and sensory cells (see reviews by Hamill and McBride 1996; Ohmori 1989
). MS channels have been
classified as stretch-activated (SA) or stretch-inactivated (SI). They
also have been classified, on the basis of their selectivity, as cation
selective, K+ selective (SA K and SI K), or anion
selective. SA cation channels are thought to participate in the process
of mechanotransduction in the action potential generator region of
sensory fibers where pressure is converted into an electrical event. SA
K channels have been reported in Xenopus oocytes
(Hamill and McBride 1992
; McBride and Hamill
1992
; Small and Morris 1994
), renal proximal tubules (Sackin 1987
), Drosophila muscle
(Gorczyca and Wu 1991
; Zagotta et al.
1988
), rat and human skeletal muscle (Burton and Hutter
1990
), the fungus Uromyces (Zhou et al.
1991
), Lymnaea neurons (Morris and Sigurdson
1989
; Small and Morris 1994
), Aplysia neurons (Patel et al. 1998
), glial cells
(Sontheimer 1994
), human macrophages (Martin et
al. 1995
), rabbit corneal epithelia cells (Watanabe et
al. 1997
), rat aortic endothelial cells (Hoyer et al.
1997
), and pig articular chondrocytes (Martina et al.
1997
). Although both SA K and SI K channels have been cloned
(Ji et al. 1998
; Patel et al. 1998
), the
functional role of MS channels remains controversial because of the
diversity of channels identified (McBride and Hamill
1992
; Morris 1992
; Morris and Horn
1991
; Small and Morris 1994
; Wan et al.
1999
).
Little is known about MS channels in colon sensory neurons. MS channels
in afferent fibers in the wall of the colon are not readily accessible,
but cell bodies of MS pelvic nerve afferent fibers in the dorsal root
ganglion (DRG) can be dissociated for study. Experiments have confirmed
that MS channels are also present in the cell bodies of colon sensory
neurons dissociated from the DRG. Channel properties have been
characterized, including cation selectivity, sensitivity to blockers,
and dependence on cytoskeletal elements. A preliminary report of some
of this work appeared in abstract form (Su et al. 1999).
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METHODS |
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The methods were reported previously (Kraske et al.
1998; Su et al. 1998
) and are briefly summarized
here. All protocols were approved by the University of Iowa
Institutional Animal Care and Use Committee.
Labeling of colon sensory neurons
Male adult Sprague-Dawley rats weighing 250-300 g (Harlan, Indianapolis, IN) were anesthetized with sodium pentobarbital (Nembutal). Under aseptic conditions, 70 µl of the dicarbocyanine dye Di-I (1.1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine methanesulfonate, 25 mg in 0.5 ml methanol) was injected at multiple sites into the smooth muscle of the descending colon. The rats were allowed to recover for 1-2 wk to permit Di-I to be transported back to the cell soma, most of which are located in the S1 DRG.
Cell dissociation and culture
Rats were anesthetized with sodium pentobarbital and the S1 DRGs were removed bilaterally, transferred into ice-cold culture media, and minced with micro-scissors. The ganglia tissue was digested in modified L-15 culture media containing collagenase (type Ia, 1 mg/ml), trypsin (type III, 1 mg/ml) and DNase (type IV, 0. 1 mg/ml) at 37°C for 50 min. Chemical digestion was terminated by soybean trypsin inhibitor (2 mg/ml) and bovine serum albumin (1 mg/ml). The tissue fragments were then gently triturated with a siliconized sterile Pasteur pipette and centrifuged at 800 g for 5 min. The neurons were resuspended in modified L-15 media supplemented with 5% rat serum and 2% chick embryo extract and were then plated onto poly-L-lysine-coated glass coverslips. Neurons in the culture media were kept at 37°C in an incubator under a 95%/5% air/CO2 atmosphere saturated with water vapor. Cells were studied within 24 h. Only Di-I labeled DRG cells, identified by their red-orange color under Hoffman contrast optics (×400) in fluorescent light with a Rhodamine filter (excitation wavelength ~546 nm and barrier filter at 580 nm), were selected for study.
Single-channel recordings
Coverslips containing cells were transferred into a 1 ml recording chamber with medium of the following composition (in mM): 140 KCl, 5 NaCl, 0.1 CaCl2, 1 MgCl2, 10 HEPES, and 10 D-glucose. The pH was adjusted to 7.35 with CsOH and the osmolarity was adjusted to 320 mOsmol/l with sucrose. Patch pipettes had resistances of 4-6 Mohm and were coated near the tips with silicone elastomer (Sylgard, Dow Corning, Midland, MI). They were filled with a solution containing (in mM) 5 KCl, 140 NaCl, 0.1 CaCl2, 1 MgCl2, 10 HEPES, and 10 D-glucose. The pH was adjusted to 7.25 with CsOH and the osmolarity was adjusted to 310 mOsmol/l with sucrose. For some experiments, the pipette solution was identical to the bath solution. A reference electrode was connected through an Ag-AgCl pellet to the bath solution via an agar bridge. The offset potential between the pipette and bath solutions was zeroed prior to seal formation.
Channel opening and closing events were recorded from excised
inside-out patches (cytoplasmic side of membrane exposed to bath
solution) obtained from DiI-labeled cells. In some cases, MS channels
were also recorded in the cell-attached configuration to demonstrate
that MS channels were not an artifact arising from patch excision. Seal
resistances averaged 15-20 Gohm. Compensation for whole-cell
capacitance and series resistance was not used. Series resistance was
usually less than 4 Mohm. Steady-state command potentials applied to
the pipette were used to change the voltage across the membrane patch.
In some cases, patch potential was changed in a ramp-like fashion from
+120 to 90 mV over a period of 7.5 s. Currents were recorded
with an Axopatch 1C patch clamp (Axon Instruments), low-pass filtered
at 5 kHz, and recorded digitally on videocassette recorder tape.
Recordings were made at room temperature.
Application of suction
To activate MS channels, negative pressure (suction) was applied through a suction port on the pipette holder. The port was connected via tubing to a glass tuberculin syringe and retraction of the syringe plunger created suction that was transmitted to the patch. A mercury manometer was used to monitor changes in applied suction. In some experiments, a CDX III disposable pressure transducer (Cobe Laboratories Inc., Lakewood, CO) and chart recorder (Gould 2400S) were used to generate permanent records of changes in applied suction. On application of suction, pressure readings reached a stable value within a few hundred milliseconds and readings remained stable in the absence of changes in the applied stimulus. Thus the applied suction was rapidly transmitted to the membrane patch and the magnitude of the suction experienced by the patch remained constant during each application period. Different levels of suction were tested in order of increasing suction to minimize possible cell trauma.
Data analysis
Recordings were digitized at 50 µsec per point and analyzed off-line using pCLAMP software (version 6.0.3, Axon Instruments). The open probability of a single MS ion channel could not be determined directly because the number of channels in each patch was not known. Therefore NPo was used as a measure of open probability where N is the number of channels in each patch and Po is the open probability of a single channel. Each digitized point in the record was binned to create an all-points histogram of point count as a function of current amplitude. Histograms were fit to 1-, 2-, or 3-component Gaussian distributions using the pCLAMP simplex least-squares routine. In the absence of suction, one component was usually adequate to describe the data, which consisted of a single peak arising from baseline noise, and average current amplitude was close to zero. A second component was sometimes needed if the patch showed spontaneous channel activity. Histograms from patches that responded to suction with an increase in channel activity exhibited an additional small peak corresponding to the amplitude level of the open channel. The majority of patches exhibited only a single open channel level in response to suction, and the all-points histograms were well-described by 2-component Gaussian fits. The interval between the two peaks along the abscissa was used to calculate mean single channel amplitude whereas the relative area under the smaller peak yielded channel open probability (NPo). Occasionally the simultaneous opening of two channels could be observed and histograms exhibited a third peak at twice the current level associated with a unitary channel-opening event. To calculate NPo, the area under this peak was doubled and added to the area under the peak corresponding to unitary opening events. When fitting histograms, an additional component was deemed to provide a significant increase in goodness of fit only if its area was greater than 0.1% of the total area.
All data are expressed as means ± SE. Data were compared using Student's t-test or ANOVA for repeated measures using GraphPad Prism (version 2.01). A value of P < 0.05 was considered to be statistically significant.
Drugs
All drugs were purchased from Sigma (St. Louis, MO). In some experiments, tetrodotoxin, tetraethylammonium (TEA), 4-aminopyridine (4-AP), amiloride, benzamil, or Gd3+ was included in bath and/or pipette solutions. In other experiments, both colchicine and cytochalasin D were added in combination to the bath solution.
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RESULTS |
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SI channels were not found in colon sensory neurons
Colon sensory neurons did not exhibit SI channels. Spontaneous
channel activity observed under baseline conditions was not decreased
by suction applied through the pipette holder. In 11 excised patches
held at 30 mV in which NPo averaged
0.21 ± 0.06 without suction, NPo
following application of 40 mmHg suction was unchanged at 0.20 ± 0.06.
Colon sensory neurons contain SA channels
Approximately 43% (48/111) of excised inside-out patches and 30% (7/23) of cell-attached patches contained MS SA channels. SA channels appeared as discrete opening events that were activated by negative pressure (suction) applied through the patch pipette (Fig. 1). MS channels activated rapidly when suction was applied and then quickly turned off when the suction was released. Channel activity did not decline during sustained application of negative pressure over periods of up to 10 s.
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Figure 2 illustrates the effects of
varying levels of suction on channel open probability
(NPo) and single-channel amplitude. Figure 2A is a recording from a representative patch in
which channel openings clearly became more frequent as suction was
increased. Aggregate data in Fig. 2B confirm that channel
open probability increases significantly with suction
(P < 0.05). Although
NPo increased with greater applied
suction, single channel amplitude was unaffected (Fig. 2C),
indicating that suction was not simply producing "holes" in the
membrane that became larger as the level of suction was increased
(Sackin 1987).
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Figure 3 illustrates the effects of
changes in membrane potential on channel properties. Although there was
a slight trend for NPo to increase on
depolarization of the membrane (Fig. 3B), channel open
probability was not significantly voltage-dependent (P = 0.13). The current-voltage relationship of the MS channel was
relatively linear, with a slope conductance of 46 pS between 30 and
+30 mV (Fig. 3C).
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MS channels are selective for K+
The cation selectivity of the MS channel was determined by
measuring its reversal potential in different solutions. The potential across excised patches was ramped from +120 to 90 mV during a 40 mmHg
suction to measure single channel amplitude at a variety of membrane
potentials (Fig. 4). The measured
reversal potential was the voltage at which current amplitude was zero.
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With 140 mM KCl/5 mM NaCl in both pipette and bath, MS channels had a reversal potential of 0 mV, as expected, because the solutions on both sides of the membrane were identical. In asymmetrical solutions with 5 mM KCl/140 mM NaCl in the pipette and 140 mM KCl/5 mM NaCl in the bath, the extrapolated reversal potential for MS channels was +68 mV (extracellular side positive relative to cytoplasmic side of the membrane). According to the Nernst equation, a conductance that is purely selective for K+ should have a reversible potential of +84 mV under these conditions. If this conductance is not purely selective for K+, then the Goldman-Hodgkin-Katz equation can be used to calculate the relative permeabilities of K+ and Na+. Given a reversal potential of +68 mV, the permeability ratio K+:Na+ is 1:0.03. The permeability of Na+ is 3% that of K+, and thus we conclude that this MS channel is highly selective for K+.
MS channels are not Ca2+-activated K+ channels
When Ca2+ was removed from the bath
solution, MS channels were still observed in nine of 13 excised patches
(Fig. 5A).
NPo was not affected by the absence of
Ca2+, indicating that these channels are not
dependent on internal Ca2+ for activation
(Martin et al. 1995).
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MS channels are blocked by amiloride and benzamil but not Gd3+
Gd3+, a known blocker of MS channels
(Kraske et al. 1998; Yang and Sachs
1989
; Zhou et al. 1991
), was added both
to bath and to pipette solutions to determine whether it could prevent
the response to applied suction. MS channels were observed in five of
eight patches exposed to Gd3+
(10
4 M), indicating that
Gd3+ failed to block the MS current (Fig.
5B).
Additional experiments were performed with amiloride and benzamil,
which block MS sodium channels in epithelial cells. No MS channels were
observed in 12 patches with
103 M amiloride or nine
patches with 10
3 M
benzamil in pipette and bath solutions, indicating that both of these
agents blocked MS channel activity. As a control, experiments were also
performed with 10
5 M
tetrodotoxin, a blocker of voltage-gated sodium channels, in the
pipette. Four of 11 patches contained MS channels. In those patches,
NPo was not significantly different
from control (P = 0.1).
MS channels are blocked by TEA but not 4-AP
TEA, a K+ channel blocker, significantly
attenuated MS channel activity. No MS channels were observed in 18 cells exposed to 30 mM TEA in bath and pipette solutions in a
cell-attached configuration, as compared with seven of 23 cells (30%)
in the absence of TEA. In excised patches, zero of five cells exhibited
clear MS channel activity when exposed to
103 M TEA. However, MS
channels were seen in two of five cells at 10
4 M TEA and three of
six cells at 10
5 M TEA.
When MS channels were visible, they were greatly reduced in amplitude
(Fig. 6). These findings are consistent
with the possibility that TEA may be acting as an open channel blocker, producing a rapid flickering of the channel between open and blocked states. Rapid flickerings often present as an apparent reduction in
single-channel amplitude due to bandwidth limitations of the recording
system. MS channels might have been present at the higher concentrations of TEA but would have been difficult to detect because
of their apparently small amplitude.
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Another K+ channel blocker, 4-AP, was ineffective
in blocking the MS K channel. With
102 M 4-AP in both bath
and pipette solutions, MS channels were still observed in seven of 16 patches (44%). In those patches, NPo
was not significantly different from control (P = 0.89). At
30 mV, NPo values were
0.018 ± 0.01 (0 mmHg), 0.09 ± 0.04 (20 mmHg), 0.31 ± 0.11 (40 mmHg), 0.49 ± 13 (60 mmHg), and 0.04 ± 0.02 (release).
MS channels are dependent on the cytoskeleton
The integrity of cytoskeletal elements may be important for
mediating the response to applied suction because gating tension may be
exerted through the lipid bilayer (Martinac et al. 1990; Patel et al. 1998
; see reviews by Hamill and
McBride 1994
; Sachs and Sokabe 1990
) or through
the underlying cytoskeleton (Guharay and Sachs 1984
;
Hamill and McBride 1992
; Marchenko and Sage
1997
; Martin et al. 1995
; Small and
Morris 1994
). In excised inside-out patches, the cytoskeleton
apparently remains attached to the excised patch (Guharay and
Sachs 1984
) and may still function normally. Our finding that
the incidence of MS channels in excised patches (48/111, 43%) was
similar to that observed in the cell-attached configuration (7/23,
30%) supports the conclusion that cytoskeletal elements, if important,
remain attached to the excised patch.
To determine whether cytoskeletal elements might be involved in
mediating the response to applied suction, colchicine (500 µM) and
cytochalasin D (1 µg/ml), which disrupt cytoskeletal elements (Patel et al. 1998; Small and Morris
1994
), were added to the bath solution during study of excised
inside-out patches. Only two of 17 patches (12%) exposed to
cytoskeletal disrupters exhibited MS channel activity. Spontaneous
channel activity was still present even after treatment with colchicine
and cytochalasin D, indicating that the membrane patches were still
viable and that the effect was specific for MS channels.
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DISCUSSION |
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Colon sensory neurons in rat S1 DRG contain MS channels that are selective for K+. These SA K channels have a conductance of 54 pS in symmetrical solutions, are not dependent on external Ca2+, and are not blocked by Gd3+, 4-AP, or tetrodotoxin. They are, however, blocked by TEA, amiloride, and benzamil.
The conductance of 54 pS is similar to that observed for SA K channels
in other preparations where conductances ranged from 26 to 90 pS
(Morris and Sigurdson 1989; Sackin 1987
;
Zagotta et al. 1988
). Other studies, however, identified
a large conductance channel with a reported conductance of 229-601 pS
(Martin et al. 1995
; Zhou et al. 1991
).
Gd3+ was ineffective in blocking these MS
channels at a concentration of 100 µM
(104 M), although it
blocks MS channels in a number of other preparations at concentrations
ranging from 10 to 20 µM (Guilak et al. 1999
; Kraske et al. 1998
; Yang and Sachs 1989
;
Yeh et al. 1997
; Zhou et al. 1991
).
Although sensitivity to Gd3+ is often considered
to be evidence that a channel is MS in nature, Gd3+ is not highly selective for MS channels and
many SA channels are insensitive to block by Gd3+
(Caldwell et al. 1998
; Hamill and McBride
1996
). Gd3+ (100 µM) does not block SA
K channels in Lymnaea neurons (Small and Morris
1995
), MS channels in supraoptic neurons (Oliet and Bourque 1996
), or MS channels in corneal epithelial cells
(Watanabe et al. 1997
).
Unfortunately, no specific antagonist for MS channels is yet available. No ion, ligand, or toxin is known that exhibits a high degree of specificity and binds with a relatively high affinity to MS channels. Furthermore, MS channels represent a broad class of channels, displaying different mechanosensitivities, gating dynamics and modalities, and open channel properties.
MS currents in colon DRG cells, which are highly selective for
K+, were inhibited by TEA, a potassium channel
blocker, but not by 4-AP, another blocker of potassium channels. TEA
has also been reported to block SA K channels in Drosophila
muscle and in Lymnaea neurons at Kd
levels ranging from 10-48 mM (Gorczyca and Wu 1991; Small and Morris 1995
; Zagotta et al.
1988
). TEA did not block SA K channels in the fungus
Uromyces (Zhou et al. 1991
) or in human
macrophages (Martin et al. 1995
). In human macrophages, the SA K channel was blocked by 1 mM 4-AP (Martin et al.
1995
), although in the present study10 mM 4-AP was ineffective
in blocking SA channels in colon sensory neurons.
Channels were also inhibited by 1 mM amiloride or benzamil, which block
MS sodium channels in epithelial cells at concentrations as low as 10 µM (see review by Kleyman and Cragoe 1988). Amiloride has been reported to block SA K channels in Lymnaea neurons
at 2 mM (Small and Morris 1995
) and calcium waves in
articular chondrocytes at 1 mM (Guilak et al. 1999
).
Tetrodotoxin, a blocker of voltage-gated sodium channels, was ineffective.
Further characterization of the SA K channel from pelvic nerve afferents is limited by several factors. 1) Sensory nerve terminals embedded in the smooth muscle of the colon are not directly accessible and therefore recordings were obtained from the soma of pelvic nerve afferent fibers isolated from the DRG. MS channels may not be distributed uniformly within the cell membrane and cell bodies may therefore not be truly representative of the nerve terminals. However, the fact that MS channels are indeed found in the soma suggests that the cell body provides a reasonable model system in which to study channels of nerve terminals. Because the cell body expresses those same channels, it provides a much more convenient preparation for isolating the channel and examining its properties, although the possibility remains that channels found in the soma are not identical to channels in the nerve terminals. 2) Many patches exhibited some spontaneous channel activity even in the absence of suction. In those cells for which NPo was greater than zero prior to the application of suction, the apparent increase in NPo seen with suction could be caused by two factors. Suction could have activated MS channels and also could have increased the open probability of those channels that were already active. Channels that were already open would not normally be considered MS channels because they could obviously be activated by some other gating mechanism. Those channels were nevertheless sensitive to mechanical stimulation and should be considered a legitimate component of the MS response. Therefore their presence in these experiments, although a complicating factor, does not alter the result or the conclusions. 3) MS channels chatter and tend to exhibit very rapid opening and closing kinetics. They may possibly pass through more than one closed state and may occasionally show subconductance levels characteristic of more than a single open state. The complex kinetics of the channel are particularly apparent when channel amplitude is greater at higher applied voltages (see Fig. 3A). However, these experiments were not designed to examine channel flickering or its voltage dependence in detail. The intent of this analysis was not to develop a kinetic model to explain channel gating mechanisms or to account for the number of possible states of the channel but simply to identify those characteristics of the channel that would be most helpful for understanding its physiological role.
The physiological role of MS K channels remains uncertain. Other
studies have reported the existence of additional MS channels in DRG
neurons. Using Fura-2, Raybould et al. (1999) studied
cultured DRG neurons identified as innervating the stomach or colon and found that mechanical stimulation produced an increase in intracellular calcium. The increase was dependent on the presence of external calcium
and was blocked by Gd3+. Gotoh and
Takahashi (1999)
reported a similar increase in intracellular calcium in unidentified neonatal DRG neurons. The response required external calcium and was sensitive to Gd3+ but
did not depend on the concentration of Na+ in the
bath. McCarter et al. (1999)
measured whole-cell
currents in cultured DRG neurons and found that mechanical stimulation activated a nonselective cation conductance that could be blocked by
Gd3+ and benzamil. Thus DRG neurons may contain a
variety of MS channel types.
The MS K channel characterized here may play a unique role in mediating the effects of mechanical stimuli in visceral sensory neurons. MS channels that activate a nonselective increase in conductance to cations or produce an increase in conductance to calcium will normally cause depolarization of the cell, generating an increase in excitability. In contrast, activation of MS K channels should cause membrane hyperpolarization, with a concomitant reduction in excitability. Thus these channels may serve to lessen the sensitization that occurs in response to chronic inflammation.
After experimental inflammation, visceral afferents become sensitized
to mechanical distention of visceral organs (Su and Gebhart
1998; Su et al. 1997a
,b
). A population of
previously mechanical-insensitive afferents will respond to visceral
organ distension after induction of an inflammation
(Häbler et al. 1990
; McMahon and
Koltzenburg 1990
; Sengupta and Gebhart 1994b
).
The hyperalgesia that characterizes functional bowel disorders may
result from a change in the threshold for activation of nociceptive MS
channels. Activation of MS K channels may help moderate the effects of
noxious stimuli and may counteract the deleterious effects of
overstimulation of other SA channels in the cell membrane. SA K
channels could also contribute to intracellular
Ca2+ regulation through control of the membrane
potential (Martin et al. 1995
; Martina et al.
1997
).
The activity of these SA K channels and other MS channels might also be
regulated directly through mechanisms that alter their sensitivity to
stretch. For example, the SA K channels TREK-1 and TRAAK are also
opened by arachidonic acid, and the pressure-activation curve of TREK-1
is altered by changes in pH (Maingret et al. 1999a,b
). Aplysia MS channels are activated by the neuropeptide
FMRFamide (Vandorpe et al. 1994
). Aplysia SA
K channels can also be opened by arachidonic acid but are closed by
serotonin via cAMP-dependent phosphorylation (Patel et al.
1998
). MS K channels are stimulated by cytokines in adherent
but not nonadherent macrophages (Martin et al. 1995
).
After 24 h of stretch, voltage-gated K+
channels in endothelial cells showed a significant depolarizing shift
in the voltage dependence of activation (Fan and Walsh
1999
), suggesting that channel gating may be altered during
chronic mechanical stimulation or prolonged stretch. In aortic
endothelium, the density of SA K channels was higher and the
sensitivity of SA cation channels was greater in spontaneously
hypertensive rats than in normotensive controls, which is consistent
with the hypothesis that MS channels may be regulated by chronic
stretch. Trauma has been shown to enhance MS channel mechanosensitivity
in Lymnaea neurons, possibly due to lowering the nociceptive
threshold (Wan et al. 1999
). MS channel function may
thus be quite adaptable to changes in stimulus intensity and duration.
The cytoskeleton may be an important mechanism for regulating channel
function. Only a small number of patches treated with the cytoskeletal
disrupters colchicine and cytochalasin D retained their sensitivity to
stretch. MS channel activity thus appears to be dependent on the
integrity of the cytoskeleton, which is consistent with previous
reports (Cunningham et al. 1997; Hamill and
McBride 1992
), and cytoskeletal elements may play a role in regulating activation of MS channels (Maingret et al.
1999a
). In contrast, Small and Morris (1994)
reported a link between MS K channels and the cytoskeleton in
Lymnaea neurons whereby the mechanosensitivity of the SA K
channel increased when the cortical cytoplasm was disrupted.
Martin et al. (1995)
found that inhibiting an MS K
channel led to disruption of the cytoskeleton, although activation of
SA channels may also trigger reorganization of the cytoskeleton (see
review by Hamill and McBride 1994
). Cytoskeletal organization and channel activity may be mutually interdependent.
Any relationship between MS channel activity and the cytoskeleton
raises the possibility that MS channel activity may be modulated indirectly by agents that act through biochemical pathways to alter the
cytoskeleton/extracellular matrix (see review by Hamill and
McBride 1996). Guharay and Sachs (1984)
proposed
that cell membrane potential and impulse initiation may be under
metabolic control via cytoskeletal contraction and relaxation. Such
contraction and relaxation could also influence the sensitivity of MS
ion channels and other stretch receptors. Active metabolic regulation of mechanosensitivity is an exciting possibility that remains to be
explored further.
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ACKNOWLEDGMENTS |
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The authors thank M. Burcham for producing the graphics, C. A. Whiteis for assistance with cell culturing, Dr. Mark O. Urban for assistance with GraphPad Prism, and Drs. Klaus Bielefeldt and Mark W. Chapleau for critical comments on this work.
This study was supported by National Institute of Neurological Disorders and Stroke Grant NS-19912.
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FOOTNOTES |
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Address for reprint requests: X. Su, Dept. of Pharmacology, Bowen Science Building, University of Iowa, Iowa City, IA 52242 (E-mail: xin-su{at}uiowa.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 1 July 1999; accepted in final form 25 April 2000.
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REFERENCES |
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