Characterization and regulation of Ca2+-dependent K+ channels in human esophageal smooth muscle

Bernard R. Hurley1, Harold G. Preiksaitis1,2,3, and Stephen M. Sims1

Departments of 1 Physiology and 2 Medicine, University of Western Ontario, and 3 Lawson Research Institute, St. Joseph's Health Centre, London, Ontario, Canada N6A 5C1


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We examined the properties of K+ channels in smooth muscle cells dissociated from human esophagus using patch-clamp recording in the cell-attached configuration. The predominant channel observed had a conductance of 224 ± 4 pS, and current reversal was dependent on K+ concentration. Channel activity was voltage dependent and increased with elevation of intracellular free Ca2+ concentration ([Ca2+]i), consistent with this being the large-conductance Ca2+-dependent K+ (KCa) channel. ACh as well as caffeine caused transient increases in KCa channel activity, and the effects of ACh persisted in Ca2+-free solution, indicating that Ca2+ release from stores contributed to channel activation. Simultaneous patch clamp and fluorescence revealed that KCa channel activity was well correlated with elevation of [Ca2+]i. The functional role of KCa channels in esophagus was studied by measuring ACh-induced contraction of strips of muscle. Tetraethylammonium and iberiotoxin, blockers of KCa channels, increased ACh-induced contraction, consistent with a role for K+ channels in limiting excitation and contraction. These studies are the first to characterize KCa channels and their regulation in human esophageal smooth muscle.

acetylcholine; caffeine; fura 2; patch clamp; contraction


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

LARGE-CONDUCTANCE Ca2+-dependent K+ channels (KCa) have been demonstrated in many types of smooth muscle cells (4, 9, 14, 24, 34). These channels may play an important role in the contraction-relaxation cycle induced by neurotransmitters such as ACh by allowing the efflux of K+ to limit depolarization and contribute to repolarization (9, 34).

Increase of intracellular Ca2+ concentration ([Ca2+]i) can occur due to entry through ligand- or voltage-activated channels or by release from intracellular stores such as the sarcoplasmic reticulum (SR). The second messenger inositol 1,4,5-trisphosphate (IP3) is generated on activation of some receptors and mediates the release of Ca2+ from SR in many cell types, including gastrointestinal smooth muscles (5, 37). A number of Ca2+ influx pathways have been described in smooth muscles. For example, agonist-mediated depolarization activates dihydropyridine-sensitive Ca2+ channels (31), and Ca2+ window currents and nonselective cation channels have also been described (12, 13, 21).

The source of Ca2+-mediating excitation in the esophagus has been the subject of investigation in animal models. Studies of cat esophageal body point to cholinergic excitation being mediated by the M2 muscarinic receptor subtype, with an absolute requirement for Ca2+ influx (7, 35, 36). In contrast, cholinergic excitation in the lower esophageal sphincter utilizes intracellular stores of Ca2+ via a mechanism involving the M3 receptor subtype and generation of IP3 (6, 17, 36). However, recent pharmacological and molecular studies revealed a dominant role for the M3 receptor subtype in excitation of cat esophageal body (29), in support of the general view that contraction of most smooth muscles is mediated by the M3 subtype (11). Consistent with this, studies of human esophagus using the Ca2+-sensitive fluorescent dye fura 2 and contraction of intact tissues indicate that cholinergic excitation involves both Ca2+ influx and release from intracellular stores (32).

Our objectives were to examine K+ channels and their regulation in human esophageal smooth muscle. We describe the large-conductance KCa channel and demonstrate its regulation by ACh and caffeine. Functional studies of muscle contraction reveal that blockade of KCa channels increases the amplitude of ACh-induced contraction, indicating a role for KCa in limiting excitation. These studies are the first to characterize KCa channels in human esophageal smooth muscle and to demonstrate their functional role in limiting excitation. Portions of this work have been presented in abstract form (20).


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Tissue retrieval and isolation of cells. Tissue collection was carried out in accordance with guidelines of the University Review Board for Research Involving Human Subjects and conformed to the Helsinki Declaration. Patient consent was obtained for removal of tissues. Tissues were obtained from 17 patients undergoing esophageal resection for malignancy, as previously described (32). A sample of the entire thickness of the muscularis propria (~1 cm2) was removed from a disease-free region in the distal one-third of the esophagus and placed in ice-cold, oxygenated Krebs bicarbonate solution (see Solutions) for transport to the laboratory.

Muscle cells were dispersed as previously described (32). Segments of esophagus (1 mm wide, 1 cm long) from longitudinal and circular muscle layers were placed in 2.5 ml of dissociation solution consisting of 135 mM K+ solution plus the following: 0.2 mg/ml Sigma blend collagenase type F, 2 mg/ml bovine albumin, 2.5 mg/ml papain, 0.4 mg/ml 1,4-dithio-L-threitol, 10 mM taurine, and 0.5 mM EDTA (pH 7.0). Tissues were generally stored in dissociation solution at 4°C overnight. The following day, tissues were warmed to room temperature for 30-60 min, placed in a gently shaking water bath at 31°C for 60 min, and dispersed by trituration with fire-polished Pasteur pipettes. Cells were studied within 8 h of dispersal.

Solutions. The Krebs bicarbonate solution for retrieval of tissues and contraction studies consisted of (in mM): 116 NaCl, 5 KCl, 2.5 CaCl2, 1.2 MgSO4, 2.2 NaH2PO4, 25 NaHCO3, and 10 D-glucose, equilibrated with 5% CO2-95% O2 (pH 7.4). The Na+ HEPES solution used for channel recording and fluorescence studies contained (in mM): 130 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, 20 HEPES, and 10 D-glucose, adjusted to pH 7.4 with NaOH. To minimize changes in membrane potential in many experiments, we used bathing solution in which NaCl was replaced with KCl, giving 135 mM K+ solution and resulting in the membrane potential being clamped close to 0 mV. The same solutions were used in the recording electrode. In cases in which K+ concentration ([K+]) was altered, we adjusted the concentration of Na+ to maintain the osmolality of the solution. Ca2+-free solutions had the same composition as above, except for the omission of CaCl2 and the addition of 0.5 mM EGTA.

Patch-clamp recording. Dispersed cells were allowed to settle and adhere to the bottom of a 1-ml chamber mounted on the stage of a Nikon inverted microscope and perfused with bathing solution at 1-2 ml/min. Cells selected for study appeared phase bright and contracted in response to ACh or caffeine. Recordings were made in the cell-attached configuration using standard patch-clamp techniques. Single-channel currents were recorded at room temperature (21-25°C) with an Axopatch 200A amplifier (Axon Instruments, Foster City, CA), filtered at 1 kHz, and sampled at 5 kHz using pCLAMP 6.0.2 software (Axon Instruments). Current and voltage were displayed on a chart recorder (Gould RS3200) and stored using a pulse code modulator (PCM-2; Medical Systems, Greenvale, NY). As cells were bathed in solution containing 135 mM K+, the patch potential (Vm) was taken as the negative of the electrode potential. In all traces, upward deflections indicate outward current across the membrane. Test agents were applied by bath perfusion or by glass micropipettes positioned ~50 µm from cells. For all studies, ACh was 100 µM and caffeine was 5 mM in the application pipettes. Control applications of vehicle had no effect on channel activity or [Ca2+]i.

Measurement of [Ca2+]i. Cells were loaded with 0.2 µM fura 2-acetoxymethyl ester (AM) for 20-40 min at 29°C and then allowed to settle to the bottom of a 0.5-ml perfusion chamber mounted on an inverted microscope and perfused. Cells were illuminated by epifluorescence with alternating 340- and 380-nm light from a xenon lamp and a Nikon Fluor ×40 objective. The emission signal was filtered using a 510-nm band-pass filter, detected by a photomultiplier (Photon Technology International), and sampled at 5-20 ratios/s. [Ca2+]i was calculated from the ratio of the fluorescence intensities at 340 and 380 nm after correction for background. System calibration constants were obtained using fura 2 solutions as described previously (32). In combined patch clamp-fluorescence experiments, cells were loaded with fura 2 before seals were established. Current and Ca2+ traces were digitized to ensure proper temporal alignment, and currents were also recorded at high bandwidth on digital videotape and/or using pCLAMP.

Contraction studies. Muscle strips (~2 × 10 mm) from longitudinal and circular muscle layers were mounted in water-jacketed tissue baths containing 10 ml of Krebs-bicarbonate solution bubbled with 5% CO2-95% O2 at 37°C. One end of the strip was attached with a silk thread to a Grass FT03 isometric force transducer coupled to a Grass 79E chart recorder (Grass Instruments, Quincy, MA). Output from the transducer was also digitized (Metrabyte, Taunton, MA) and sampled at 2.5 Hz (Labtech Notebook; Laboratory Technologies, Wilmington, MA).

Drugs and materials. All drugs and chemicals were obtained from Sigma (St. Louis, MO) or BDH Limited (Toronto, Ontario, Canada) unless otherwise indicated. Fura 2-AM and 4-bromo A-23187 were from Molecular Probes (Eugene, OR). Fura 2 was dissolved in dimethylsulfoxide. Iberiotoxin (IbTX) was from Bachem. ACh and caffeine were prepared as stock solutions in water and diluted 1,000-fold into the appropriate bathing solution before application to isolated cells.

Statistics. Values are provided as means ± SE with sample sizes (n) indicating the number of cells studied. Only one patch was obtained per cell, and in total more than 70 cells were studied. Statistical comparisons were made using Student's t-test, where P < 0.05 was considered statistically significant. All traces are representative of at least three experiments on muscle from two or more patients.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Identification of large-conductance KCa channels. The predominant channel recorded in the cell-attached patch configuration had properties consistent with the large-conductance KCa channel (Fig. 1). Isolated smooth muscle cells of both longitudinal and circular muscle layers were spindle shaped and appeared phase bright under phase-contrast microscopy (32). Channel activity was infrequent in resting cells where [Ca2+]i was determined to be close to 100 nM (Fig. 1A). To examine the Ca2+ sensitivity of this channel, we treated cells with Ca2+ ionophore (20 µM 4-bromo A-23187) or mechanically disturbed the cells to elevate [Ca2+]i. Elevation of [Ca2+]i to ~2 µM using A-23187 in the same cell illustrated above greatly enhanced channel activity (Fig. 1A, bottom), demonstrating the Ca2+ sensitivity of this channel.


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Fig. 1.   Identification of large-conductance Ca2+-dependent K+ channels (KCa) in human esophageal muscle. A: in a resting cell with basal intracellular Ca2+ concentration ([Ca2+]i) of ~100 nM, channel activity was brief and infrequent (top trace). Closed channels indicated by C, open channels by O. Channel activity increased when [Ca2+]i was elevated to 2 µM with ionophore (bottom trace). K+ concentration ([K+]) was 135 mM in bath and electrode solution, bath solution contained 1 mM Ca2+, and patch was held at 40 mV. B and C: current-voltage relationship was determined using voltage-ramp commands from -100 to +100 mV over 200 ms. Four overlapping current traces are shown for each condition, and [Ca2+]i was elevated to increase channel activity. In B, extracellular K+ concentration ([K+]o) was 135 mM, slope conductance was 230 pS, and reversal potential was -3 mV. In C, [K+]o was 5 mM, resulting in a slope conductance of 100 pS and reversal potential estimated to be close to -70 mV. D: reversal potential (Vrev), plotted as a function of [K+]o, shifted 44 ± 1 mV per 10-fold change in [K+]o, determined by least-squares regression, indicating selectivity for K+. Error bars are SE, with number of cells studied indicated in parentheses.

Voltage ramp commands were used to determine the voltage dependence of this channel. With 135 mM K+ in the recording electrode, currents were outward at positive potentials and inward at negative potentials, with mean reversal in 10 cells at -3 ± 1 mV (5 longitudinal and 5 circular muscle cells; Fig. 1B). This channel also exhibited voltage dependence, with increased open probability at more positive potentials (Fig. 1, B and C). To determine channel selectivity, we varied extracellular [K+] ([K+]o) in the electrode solution. Reduction of [K+]o to the physiological level of 5 mM shifted the reversal potential negatively (Fig. 1C). Over a range of concentrations, the reversal potentials shifted 44 ± 1 mV per 10-fold change in [K+]o, as determined by least-squares regression (Fig. 1D), indicating the channel is largely selective for K+. Clear reversal of the current was observed at higher levels of activation, but we illustrate traces in which the single-channel events can be resolved. Channel conductance (determined at 0 mV) decreased from 224 ± 4 pS with 135 mM [K+]o to 100 ± 13 pS with 5 mM [K+]o. The large conductance of this channel and its sensitivity to [Ca2+]i, voltage dependence and K+-selectivity documented here are all characteristic of the large-conductance KCa channel. On the basis of the channel conductance and reversal potentials, no differences were evident between KCa channels from cells of circular or longitudinal muscle layers.

ACh elicits transient increases in KCa channel activity. KCa channel activity was infrequent before stimulation, but ACh elicited single-channel currents with short latency (Fig. 2A). Similar responses were observed in 30 of 42 cells stimulated with ACh, from both longitudinal and circular muscle layers. Channel activity returned to baseline levels within seconds after the washout of ACh. Unitary current amplitude was constant before and during the response to ACh, indicating that cell membrane potential did not change during stimulation with ACh, so depolarization cannot account for increased channel opening. The predominant effect of ACh was on the large-conductance KCa channel, because smaller outward currents (e.g., small-conductance KCa channels; see Refs. 23 and 40) or inward currents were not observed in this or other cells illustrated below. Because the channels being recorded were isolated from the ACh by the patch pipette, the increase in channel activity likely indicates the involvement of a cytosolic second messenger.


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Fig. 2.   ACh causes transient increase in channel activity and shifts voltage dependence of channel opening to more negative potentials. A: before application of ACh, channel activity was brief and infrequent (expanded region a at bottom). ACh (100 µM in application pipette) was applied for 10 s, causing contraction of cell (not shown) and increased channel activity (expanded region b at bottom). Channel activity returned to resting level by 10 s after ACh (expanded region c at bottom). Patch potential (Vm) was 30 mV. B: ACh shifted open probability of KCa to more negative potentials, assessed using voltage-ramp commands from -100 to +100 mV over 200 ms. Before ACh (control traces at left), KCa channels opened only at +80 mV. ACh elicited KCa channel opening at approximately -30 mV ([K+]o = 50 mM), with up to 3 channels open simultaneously. C: ACh effect on channel open probability (NPo)-voltage relationship. Current traces at peak of response were averaged and fit by a Boltzmann distribution yielding one-half-activation potential (V1/2) = 33 ± 2 mV and slope factor k = 11 ± 3 mV during stimulation with ACh (n = 4).

To quantify the effect of ACh, channel open probability (NPo) was calculated from leak-subtracted current traces during voltage-ramp commands (average current divided by unitary current amplitude; see Ref. 10). Before the stimulation with ACh, KCa channel opening was only apparent at potentials more positive than ~70 mV (Fig. 2B). ACh shifted the voltage at which KCa currents were observed negatively, with openings now evident near -30 mV (Fig. 2B). Examining currents elicited at the peak of the response to ACh, we found that the relationship between NPo and Vm increased sigmoidally with depolarization (Fig. 2C) and was fit with a Boltzmann distribution of NPo = NPo Max/{1 + exp[(V1/2-Vm)/k]}, where NPo Max is the maximum NPo, V1/2 is the one-half-activation potential, and k is the slope factor. Values of V1/2 and k obtained were 33 ± 2 mV and 11 ± 3 mV, respectively, during the response to ACh (n = 4).

ACh and caffeine act on a common store of Ca2+. The results presented to this point are consistent with ACh activating KCa channels by a rise of [Ca2+]i. In Ca2+-free solution containing 0.5 mM EGTA, ACh continued to cause cell contraction and a typical transient increase in KCa channel activity (Fig. 3, n = 5). These findings indicate that entry of Ca2+ was not necessary for the acute response to ACh and provide evidence for Ca2+ release from stores.


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Fig. 3.   Increase of KCa activity elicited by ACh does not require Ca2+ entry. After establishing seal in solution containing 1 mM Ca2+, we exchanged bathing solution with Ca2+-free solution containing 0.5 mM EGTA. Subsequently, ACh (100 µM in 0 Ca2+ solution) caused a typical transient increase in KCa channel activity. Expanded traces are shown at bottom for times indicated. Vm was 60 mV.

Caffeine releases Ca2+ from intracellular stores in a variety of smooth muscles (4) including human esophageal muscle (32) by acting at ryanodine receptors on the SR. Like ACh, caffeine elicited a prompt and reversible increase of KCa channel activity in esophageal muscle (Fig. 4A). When examined over a range of voltages, caffeine caused KCa channels to open at less-positive potentials (Fig. 4B). A plot of NPo as a function of Vm determined at the peak of the response reveals that caffeine shifted the curve negatively (Fig. 4, B and C), with best fit of the data to the Boltzmann relation yielding values of V1/2 and k of 48 ± 8 and 16 ± 4 mV, respectively (n = 4).


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Fig. 4.   Release of Ca2+ from intracellular stores is sufficient to activate KCa channels. A: caffeine (5 mM) elicited prompt increase of KCa activity, as shown in expanded trace b at bottom. Vm was +30 mV, [K+]o was 135 mM, and there was 1 mM Ca2+ in bathing solution. B: caffeine shifted KCa open probability to more negative potentials. Before application of caffeine (control traces at left), KCa channels opened only at +60 mV. Caffeine elicited KCa channel opening at ~10 mV, with up to 3 channels open simultaneously. C: current traces at peak of response were averaged and fit by a Boltzmann distribution yielding V1/2 = 48 ± 8 mV and k = 16 ± 4 mV during caffeine (n = 4).

The interaction between caffeine and ACh was also studied. In seven cells in which ACh increased KCa channel activity, subsequent application of caffeine caused little or no increase in KCa channel activity (Fig. 5A). After 5-10 min of recovery in Ca2+ bathing solution (to allow for refilling of stores), the order of agonist application was reversed, whereupon caffeine increased KCa activity in every cell. The subsequent application of ACh had no further effect (Fig. 5B). These results indicate that ACh and caffeine release Ca2+ from common intracellular stores, which is sufficient to increase KCa channel activity.


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Fig. 5.   Interaction between caffeine and ACh. A: initial application of ACh caused transient increase in KCa activity. Subsequently, caffeine did not elicit any increase in channel activity. B: after 10-min recovery, cell was rechallenged in reverse order. Application of caffeine now caused typical increase of KCa channel activity, whereas subsequent application of ACh had no effect. Vm = 30 mV, [K+]o = 50 mM, and there was 1 mM Ca2+ in bathing solution. Similar results were observed in 6 other cells.

Elevation of [Ca2+]i accompanies KCa channel activity. To examine whether the increase of KCa channel activity was associated with elevation of [Ca2+]i, we studied fura 2-loaded cells with patch clamp and fluorescence. A rise of [Ca2+]i occurred in all cells in which KCa channel activity was enhanced (6 of 9 cells tested with caffeine and 5 of 8 cells tested with ACh; remaining cells showed negligible changes of [Ca2+]i and channel activity). Typical responses are illustrated in Fig. 6, in which caffeine and ACh caused elevation of [Ca2+]i accompanied by opening of KCa channels. The time course of the rise of [Ca2+]i corresponded well with channel activity, although fura 2 fluorometry reports the global cytosolic Ca2+ levels as opposed to subplasmalemmal Ca2+ levels sensed by ion channels. Our previous studies have confirmed that ACh and caffeine continue to elicit a rise of [Ca2+]i in these cells in Ca2+-free solutions (32), indicating involvement of stores. Thus transient elevation of [Ca2+]i accounts for the acute activation of KCa in esophageal muscle cells.


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Fig. 6.   Elevation of [Ca2+]i accompanies KCa channel activity. Simultaneous Ca2+ fluorescence and patch-clamp recording in fura 2-loaded cells. Caffeine and ACh were applied for times indicated by bars at top of traces, with transient elevation of [Ca2+]i accompanied by increase in KCa channel activity for 2 separate cells. Expanded segments are shown at bottom for regions indicated from response to caffeine. Vm = 50 mV for caffeine response and 80 mV for ACh response. [K+]o = 135 mM. A dashed line indicates brief interruptions in baseline current trace before caffeine.

Ca2+ dependence of KCa channel activity. To quantify the Ca2+ sensitivity of KCa channels, we determined NPo in fura 2-loaded cells with Ca2+ ionophore applied to cause a persistent rise of [Ca2+]i (bath solution containing 1 mM Ca2+). Addition of A-23187 resulted in gradual elevation of [Ca2+]i accompanied by channel activation, monitored using voltage-ramp commands (Fig. 7A). The sensitivity of the channel was estimated from 5 to 10 voltage ramps over 10-s periods of time in which [Ca2+]i changed only 5-10%. Little channel activity was evident at 100 or 500 nM Ca2+, except at very positive voltages, but further elevation of Ca2+ resulted in a clear shift in NPo to more negative values. Boltzmann curves fitted to these data yielded values of V1/2 for various levels of Ca2+ (Fig. 7B). V1/2 was dependent on [Ca2+]i, with the best fit line having a slope of 340 ± 70 mV per 10-fold increase in [Ca2+]i.


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Fig. 7.   Ca2+ sensitivity of KCa channels. A: KCa channel activity was recorded in cell-attached patch configuration while [Ca2+]i was monitored simultaneously. Ca2+ ionophore (4-bromo A-23187) caused rise of [Ca2+]i and activation of KCa channels over the course of several minutes. NPo was determined from average currents elicited during 5-10 voltage ramps at each value of [Ca2+]i. B: Boltzmann curves fit to data in A were used to estimate V1/2, voltage at which NPo was half-maximal. V1/2 was plotted as a function of [Ca2+]i for 3 cells. Electrode solutions contained 5 mM K+.

Functional significance of KCa channels. To assess the role of KCa channels in esophageal muscle, we first examined the effect of channel blockade using tetraethylammonium (TEA), which blocks KCa channels in most tissues, including these cells (G. R. Wade, L. G. Laurier, H. G. Preiksaitis, and S. M. Sims, unpublished observations). Periodic perfusion with ACh caused reproducible increases in tension, and addition of TEA (800 µM) resulted in increased amplitude of ACh-induced contractions (Fig. 8A). This effect was concentration dependent, with 4 mM TEA causing greater augmentation as well as a rise of baseline tension, which in this example was accompanied by oscillatory contractions. The effects of TEA described here were fully reversible, as seen in Fig. 8A, right. Augmentation of the contraction was observed in five of six circular and seven of eight longitudinal muscle preparations from five patients, with peak contraction increased to 1.4 ± 0.1 times control levels for 4 and 8 mM TEA. In addition, increases of baseline tension were observed in 9 of 14 muscle strips. The events underlying the oscillatory contractions have not yet been studied. Because TEA may block other channel types, we examined the effects of IbTX, a selective peptide blocker of large-conductance KCa channels. As illustrated in Fig. 8, B and C, IbTX caused augmentation of the ACh-induced contraction of esophageal muscle, with similar effects in circular and longitudinal muscle layers. IbTX increased the ACh-induced contraction by 1.9 ± 2 times control level (4 muscle strips), supporting the view that KCa channels contribute to recovery after contraction. Muscarinic excitation of human esophageal muscle involves both release of Ca2+ from stores and entry through L-type Ca2+ channels (32). To investigate the involvement of depolarization, we applied nifedipine (10 µM), which reduces the contractile response to ACh, as shown previously (32). In the presence of nifedipine, neither TEA nor IbTX enhanced ACh-induced contraction (20 muscle strips; Fig. 8, B and C, right).


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Fig. 8.   Blockade of KCa channels augments contraction. ACh was applied periodically to strips of muscle at arrowheads (10 µM for 40 s), causing a reproducible increase in muscle tension. A: addition of 800 µM tetraethylammonium (TEA) caused an increase in amplitude of ACh-induced contraction. Further increase of TEA to 4 mM resulted in a rise in baseline and spontaneous oscillatory contractions. Effects of TEA were reversible within 10 min of washout (right). Response shown is from circular muscle. B and C: addition of iberiotoxin (IBTx, 100 nM) to cause selective blockade of KCa channels caused similar enhancement of contraction in both longitudinal and circular muscles, as indicated. Addition of nifedipine caused reduction of ACh-induced contraction (right).


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We have characterized a Ca2+-dependent K+ channel in human esophagus and demonstrated its physiological regulation. Channel opening occurs in response to activation of muscarinic receptors, possibly operating through an IP3-dependent pathway, as well as to caffeine, which stimulates ryanodine receptors, to release Ca2+ from intracellular stores. KCa channels serve to limit excitation and contraction of esophageal muscle.

The KCa channel in human esophagus had a conductance of 224 pS in symmetric K+ and a reversal potential that was dependent on the K+ gradient. These features are shared with large-conductance K+ channels in many muscles (4, 8, 34). The observed dependence of the reversal potential on the K+ gradient (44 mV shift per 10-fold change of [K+]o) was less than predicted by the Nernst equation for a K+-selective channel as well as that reported by others for the KCa channel. This may indicate some limited permeability of human KCa channels to other cations. Several groups have cloned human KCa channels (25, 38). Expression of these channels revealed the characteristic large conductance; however, the K+ selectivity was not examined at varying levels of [K+]o, so it is not possible to say whether permeability to other cations is a common feature. The unitary conductance of the esophageal KCa channel was also dependent on [K+]o, with conductance of 100 pS recorded with the physiological level of 5 mM [K+]o. This is similar to that reported for KCa in several other smooth muscles (8), although the conductance of KCa channels in rabbit vascular muscle was found to be still further reduced due to blockade by internal Mg2+ and Na+ (27). Our recordings of esophageal muscle were carried out in cell-attached configuration with intact cells, including Mg2+ in the bathing and electrode solutions, but we found no evidence for such blockade.

Membrane currents of esophageal muscle have previously been described from recordings at the whole cell level. Voltage-activated Ca2+, transient outward K+, and Ca2+-dependent K+ currents are present in cells isolated from several species, including cat, rabbit, and opossum (2, 3, 33). Human esophageal cells exhibit a similar range of K+ currents, including a delayed rectifier blocked by 4-aminopyridine, and KCa, which we confirm is blocked by TEA, charybdotoxin, and IbTX (G. R. Wade, L. G. Laurier, H. G. Preiksaitis, and S. M. Sims, unpublished observations). The large-conductance KCa channels described here are distinct from the intermediate or small-conductance KCa channels recently shown to be activated by purinergic agonists in murine ileal and colonic muscles (23, 40). Small-conductance KCa channels (5-10 pS) are sensitive to apamin but insensitive to TEA and may coexist with intermediate and large channels in the cell membrane (40). Although the intermediate-conductance channels (~39 pS) are sensitive to TEA (40), we have not detected the presence of such currents in recordings of esophageal muscle.

We studied KCa channels in the cell-attached patch configuration, so the channels under observation were not exposed to applied ACh. The observed activation indicates that ACh mediates its effects indirectly, through the action of a diffusible messenger. The involvement of Ca2+ was supported by the observations that caffeine and Ca2+ ionophore, both of which elevate [Ca2+]i independently of metabotropic receptors, also increased channel opening. Simultaneous fluorescence and channel recording revealed the rise of [Ca2+]i to be closely associated with channel opening, as predicted if Ca2+ were to directly activate channels. Similar findings have been reported for activation of KCa channels in other muscles, including vascular (14), airway (39), and cultured myometrial cells (18). Whole cell fluorimetry studies reveal the time course of global changes of [Ca2+]i. However, imaging is required to determine the magnitude of [Ca2+] changes occurring in compartments adjacent to the membrane or spatially restricted changes that may be sensed by channels, as occurs with Ca2+ sparks (22, 28).

Smooth muscle KCa channels have now been cloned from several sources, including human arterial muscle (25, 38) and canine colonic muscle (41). A common finding is that expression of the pore-forming alpha -subunit alone yields channels with reduced sensitivity to Ca2+. Coexpression of alpha - with regulatory beta -subunits, however, restores the Ca2+ sensitivity of the channels to levels that are found in native cells, supporting the notion that native KCa channels are composed of alpha - plus beta -subunits. Esophageal KCa channels exhibit characteristics most consistent with their being composed of alpha - and beta -subunits, although this remains to be confirmed at the molecular level. Tanaka and co-workers (38) exploited the fact that the plant derivative dehydrosoyasaponin I (DHS-I) activates large-conductance KCa channels only if alpha - and beta -subunits are coexpressed (26). The demonstration that arterial smooth muscle KCa channels are enhanced by DHS-I supports the conclusion that the majority of native channels in coronary smooth muscle consist of alpha - plus beta -subunits. It will be of interest to determine whether DHS-I regulates esophageal KCa channels. The Ca2+ sensitivity of KCa channels can be reported as the shift of the voltage for half- activation for a given change in [Ca2+]i as well as the set point (8). Although the set point for activation of esophageal KCa channels was close to that reported for KCa in other smooth muscles, the V1/2 changed 340 mV per 10-fold change of [Ca2+]i, a somewhat steeper dependence than other smooth muscles (8). However, there may be limitations in measuring local [Ca2+]i in the cell-attached configuration that account for these differences.

We found little evidence for KCa channels being open over the physiological range of resting membrane potentials or [Ca2+]i. However, the observation that TEA increased baseline tension in 9 of 14 preparations studied suggests that KCa channels can contribute to setting the resting membrane potential. Elevation of [Ca2+]i in restricted regions (Ca2+ sparks) underlies spontaneous transient outward K+ currents (STOCs) in vascular (28) as well as esophageal smooth muscle (22). TEA-sensitive STOCs have been observed in cat (33) and human esophageal muscle (G. R. Wade, L. G. Laurier, H. G. Preiksaitis, and S. M. Sims, unpublished observations) and may contribute to setting the resting potential. However, STOCs would not be apparent in cell-attached recordings, possibly explaining how KCa could influence the membrane potential in the absence of steady macroscopic currents.

Our results are relevant to understanding the sources of Ca2+ contribution to excitation and contraction of esophageal muscle. Earlier studies of cat esophagus were interpreted to indicate that store release of Ca2+ was minimal in cells from circular esophageal body, whereas Ca2+ influx was essential for contraction (6, 7, 35). A recent study of receptor subtypes in cat esophagus (29) reveals the presence of functional M3 receptors, consistent with the involvement of Ca2+ stores in muscarinic excitation. When first studied in human esophagus, muscarinic signaling was found to involve both influx and release of Ca2+ from stores (32). The data presented here confirm the existence of Ca2+ stores, as assayed both by fluorescent dye and ion channels as indicators. Moreover, recent pharmacological and molecular studies support the presence of both M2 and M3 receptors in human esophagus (H. G. Preiksaitis, L. G. Laurier, and R. I. Inculet, unpublished observations), although further studies are required to resolve components of the receptor signal transduction pathways. Taken together, our studies emphasize the value of studying human tissues to understand the mechanisms underlying esophageal peristalsis.

TEA and IbTX enhanced ACh-induced contraction, consistent with KCa channels participating in recovery and relaxation of the muscle. It is well established that a rise of Ca2+ initiates contraction of most smooth muscles (37). A rise of Ca2+ would also initiate a negative feedback loop, causing outward K+ current and hastening repolarization of the membrane, thereby limiting Ca2+ entry and contraction. Blockade of KCa leads to depolarization and contraction of several smooth muscles [e.g., canine colon (9) and guinea pig ileum (19)]. Changes of membrane potential have yet to be recorded in human esophagus, but the fact that nifedipine inhibited the effect of channel blockers supports the notion that depolarization causes activation of Ca2+ channels. A role for Ca2+ in mediating relaxation of smooth muscles is emerging. Activation of KCa by localized release of Ca2+ from the SR contributes to relaxation of vascular smooth muscle (28). As well, purinergic agonists, candidates for inhibitory neurotransmitters in the gastrointestinal tract, activate small- and intermediate-conductance KCa channels, apparently due to elevation of [Ca2+]i (23, 40). Further knowledge of the subcellular changes in intracellular Ca2+ will be critical for understanding its role in regulating contraction.

Previous studies have implicated K+ channels in the control of esophageal smooth muscle. Although it is generally accepted that peristalsis is primarily a nerve-mediated phenomenon, TEA can induce waves of contraction that propagate in both directions in an intact esophagus, even with intrinsic nerves blocked (15, 30). Thus K+ channel blockade reveals a myogenic mechanism for peristalsis. Although slower than the oscillatory activity we observed, TEA was shown to evoke phasic contractions in muscle strips from the opossum esophagus (16). Most contractions lasted 2-4 s, but occasional giant contractions lasting up to 150 s were noted, reminiscent of the high-amplitude, prolonged contractions seen in patients with nutcracker esophagus. Moreover, Akbarali et al. (3) suggested that another class of K+ channels, those responsible for transient outward K+ current, is important for timing of the peristaltic contraction. These authors suggest a role for transient K+ channels in the pathogenesis of diffuse esophageal spasm, another motor disorder seen in humans. Because ion channel disorders are recognized as contributing to a number of diseases (1), these observations raise the intriguing possibility that clinically important spastic disorders of esophageal smooth muscle such as nutcracker esophagus or diffuse esophageal spasm may result from abnormal expression or regulation of K+ channels. This possibility awaits further study.


    ACKNOWLEDGEMENTS

We are grateful to Yang Jiao for help in preparation of cells, Dr. Frederik Weidema for assistance with the Ca2+ fluorescence recording, Tom Chrones for advice regarding muscle strips, and Drs. R. I. Inculet, R. A. Malthaner, C. Rajgopal, and S. E. Carroll for providing the esophagectomy specimens.


    FOOTNOTES

This work was supported by grants from the Medical Research Council of Canada and the Physicians' Services Foundation Incorporated. B. R. Hurley was supported by the Heart and Stroke Foundation of Ontario and the Faculty of Medicine Summer Research Training Program Endowment in Foundation Western. H. G. Preiksaitis is the recipient of an Ontario Ministry of Health Career Scientist award, and S. M. Sims was supported by a Medical Research Council Scientist award.

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: S. M. Sims, Dept. of Physiology, The Univ. of Western Ontario, London, Ontario, Canada N6A 5C1 (E-mail: sims{at}physiology.uwo.ca).

Received 11 August 1998; accepted in final form 2 November 1998.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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