Mechanosensitive Potassium Channels in Rat Colon Sensory Neurons

X. Su,1 R. E. Wachtel,2,3 and G. F. Gebhart1

 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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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).


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1. Colon sensory neurons contain mechanosensitive channels. Top: sample recording from an excised inside-out patch. Negative pressure (suction) was applied through the pipette holder and increased in a step-wise fashion at intervals of roughly 7 s during a continuous recording lasting 35 s. The time course of changes in applied suction is illustrated schematically above the current trace. Channel activity increased significantly on application of suction, became more pronounced when the suction was increased, remained stable when suction was held constant, and then returned to baseline levels as the negative pressure was released. Downward deflections are inward currents caused by activation of mechanosensitive (MS) channels. Bottom: a 500-ms segment from the top trace is shown on an expanded time scale to illustrate more clearly the kinetics of individual MS channel-opening events. The short horizontal bars to the left of the trace indicate current levels corresponding to the simultaneous opening of 0 (baseline), 1, or 2 channels. This particular patch contained an exceptionally large number of opening events and it can be seen that multiple channels opened simultaneously. The majority of patches, however, exhibited little channel activity in the absence of suction and revealed only a single open-channel level in response to applied suction.

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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Fig. 2. Effects of applied suction on MS channel properties. A, left: representative single-channel recordings from an excised patch showing graded responses to different levels of negative pressure applied through the recording electrode. Channel openings become more frequent as suction is increased. Downward deflections are inward currents caused by activation of MS channels. The short horizontal bars to the left indicate current levels corresponding to the opening of 0 (baseline) or 1 channel. Traces are not continuous. Holding potential, 0 mV. Right: all-points histograms generated from several seconds of data collected at each level of suction. The control histogram contains only a single large peak because no channel activity is present. The peak is centered around an amplitude of zero and represents baseline noise. Under suction, a second peak appears that becomes larger as suction is increased. Histograms from traces recorded during application of suction are well described as the sum of 2 Gaussian components in which the smaller peak represents channel opening events. The interval between the two peaks along the abscissa is single-channel amplitude whereas the relative area under the smaller peak is channel open probability (NPo). B: averaged data from excised patches showing that NPo becomes larger as suction is increased (two-factor ANOVA, P < 0.05). Patch potential, -30 mV. Each point is the mean ± SE of 12 patches, where each patch was tested successively at 0, 20, 40, and 60 mmHg suction. In some patches, channel activity was present even in the absence of suction. C: averaged data from excised patches showing that single-channel current amplitude is not affected by the magnitude of applied suction. Patch potential, -30 mV. Each point is the mean ± SE of 12 patches, where each patch was tested successively at 0, 20, 40, and 60 mmHg suction.

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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Fig. 3. Effects of membrane voltage on MS channel properties. A, left: representative single-channel recordings from an excised patch at different applied voltages. Downward deflections are inward currents caused by activation of MS channels. The short horizontal bars to the left indicate current levels corresponding to the opening of 0 (baseline) or 1 channel. Right: all-points histograms were fit as 2-component Gaussian distributions. The dotted line shows the change in average current amplitude at different holding potentials. Suction, 60 mmHg. Channel flickering is quite apparent at more negative potentials and the Gaussian component that arises from channel opening events becomes much broader. Although broadening of this peak could be caused by a voltage-dependent change in channel kinetics, it more likely arises from better resolution of channel flickering as channel amplitude increases. B: averaged data showing that NPo is not significantly affected by membrane voltage (two-factor ANOVA, P = 0.13). Suction, 40 mmHg. Each point is the mean ± SE of 9-15 patches. Voltages shown represent applied potentials so that a negative voltage applied to the pipette results in depolarization of an excised patch. NPo was not calculated at +60 mV because channel amplitude was so small. C: averaged data illustrating the relationship between single-channel current amplitude and membrane potential. Current-voltage relationships are similar for different levels of applied suction. Slope conductance between -30 and +30 mV is 46 pS. Each point is the mean ± SE of 8-15 patches.

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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Fig. 4. Mechanosensitive channels are selective for K+. Top: representative current traces from excised patches in response to voltage ramps of 7.5 s duration from +120 to -90 mV during 40 mmHg suction. The applied potential can be determined directly based on the time elapsed since initiation of the ramp. Bottom: summary of voltage ramp data in which current amplitude during channel opening events is plotted as a function of membrane potential. Both vertical and horizontal scales are identical to the raw current trace above. Each curve depicting channel amplitude during the voltage ramp was fit with a regression line (dotted lines). The asterisk indicates the regression line for the cell shown above. The reversal potential (Erev) for the current was then determined from the zero current intercept of the fitted line. The thick line represents the average of the individual regression lines, and the reversal potential for the current was also calculated from the zero current intercept of the averaged line. A: pipette and bathing solutions were identical. The mean reversal potential in symmetrical solutions was 0.49 ± 2.4 mV (n = 5 cells). Erev determined from the averaged line was -0.4 mV. Single-channel conductance was 54 pS. B: the bathing solution contained 140 mM KCl/5 mM NaCl whereas pipettes were filled with 5 mM KCl/140 mM NaCl. The mean reversal potential in asymmetrical solutions was 68.69 ± 2.9 mV (n = 7 cells). Erev determined from the averaged line was 68.2 mV, with the pipette (extracellular surface of patch) positive relative to the bath (cytoplasmic side of patch). Single-channel conductance was 45 pS.

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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Fig. 5. MS channels are not Ca2+-dependent K+ currents and are not blocked by gadolinium (Gd3+). A, left: representative recordings from an excised patch in Ca2+-free bath solution containing 5 mM EGTA. MS channels were observed in 9 of 13 patches tested. Holding potential, -30 mV. Right: aggregate data showing a clear response to suction in the absence of Ca2+ (two-factor ANOVA, P = 0.23 compared with control data in the presence of 0.1 mM CaCl2). Each point is the mean ± SE of 9 patches. B, left: representative recordings from an excised patch with Gd3+ (10-4 M) in both bath and pipette solutions. MS channels were observed in 5 of 8 patches tested. Holding potential, -30 mV. Right: aggregate data showing a clear response to suction in the presence of Gd3+ (two-factor ANOVA, P = 0.94 compared with control data in the absence of Gd3+). Each point is the mean ± SE of 5 patches.

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 10-3 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 10-3 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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Fig. 6. Mechanosensitive channels are blocked by tetraethylammonium (TEA). A: representative control recording showing MS current activity in an excised patch. The short horizontal bars to the left indicate current levels corresponding to the opening of 0 (baseline) or 1 channel. Suction, 40 mmHg. Holding potential, -30 mV. B: representative MS channels observed in an excised patch exposed to TEA (10-4 M) in both bath and pipette solutions. Suction, 40 mmHg. Holding potential, -30 mV. Current amplitude is significantly attenuated and the open channel level appears to be variable. TEA significantly reduced MS current amplitude (P < 0.05) compared with control by Student's unpaired t-test.

Another K+ channel blocker, 4-AP, was ineffective in blocking the MS K channel. With 10-2 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.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 (10-4 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.


    ACKNOWLEDGMENTS

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.


    FOOTNOTES

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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