Ion channel diversity in the feline smooth muscle esophagus
Anne Marie F.
Salapatek1,
Junzhi
Ji2, and
Nicholas E.
Diamant2
1 Department of Physiology, University of Toronto, Toronto,
Ontario M5S 1A8; and 2 University Health Network and University
of Toronto, Toronto, Ontario M5T 2S8, Canada
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ABSTRACT |
We have characterized ion-channel identity
and density differences along the feline smooth muscle esophagus using
patch-clamp recording. Current clamp recording revealed that the
resting membrane potential (RMP) of esophageal smooth muscle cells
(SMC) from the circular layer at 4 cm above the lower esophageal
sphincter (EBC4; LES) were more depolarized than at 2 cm above LES.
Higher distal Na+ permeability (but not Cl
permeability) contributes to this RMP difference. K+
channels but not large-conductance Ca2+-activated
K+ (BKCa) channels contribute to RMP at both
levels, because nonspecific K+-channel blockers depolarize
all SMC. Depolarization of SMC under voltage clamp revealed that
the density of voltage-dependent K+ channels
(KV) was greatest at EBC4 due to increased
BKCa. Delayed rectifier K+ channels
(KDR), compatible with subtype KV1.2, were
present at both levels. Differences in
KCa-to-KDR channel ratios were also manifest by
predictable shifts in voltage-dependent inactivation at EBC4 when
BKCa channels were blocked. We provide the first evidence
for regional electrophysiological differences along the esophageal body
resulting from SMC ion channel diversity, which could allow for
differential muscular responses to innervation and varied muscular
contribution to peristaltic contractions along the esophagus.
patch clamp; delayed rectifier K+ channels; large-conductance calcium-activated K+ channels; esophageal
motility
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INTRODUCTION |
IN THE SMOOTH MUSCLE
ESOPHAGUS, swallow-induced peristaltic contractions are usually
considered to be the result of the local muscle responding passively to
neural signals that direct inhibition and excitation along the
esophagus; and consequently, neural control has been considered to be
sufficient for regulation of peristalsis (9, 12, 18, 20, 21, 47,
63). As a result, most studies to date have generally ignored
any significant role for the muscle. However, when adequately
stimulated (6-8, 12, 17, 25, 26, 29, 37, 40, 42-44, 54,
57, 63, 65, 68) and with excitation in the face of nerve
blockade (with TTX), a single or repetitive contraction can traverse
the smooth muscle esophagus at velocities similar to swallow-induced
peristalsis (25, 42, 54). That is, the muscle itself
provides another level of control for peristalsis. Therefore, muscle
properties, especially if regional differences exist, hold the
potential to impact on the nature of peristaltic contractions and the
responses of the muscle to its innervation.
Regional differences in neural influence have been described along the
esophageal body, the excitatory (cholinergic) influence most prominent
proximally (2, 12, 13, 19, 21, 40, 57, 67) and the
inhibitory (nitrergic-nitric oxide) influence most active distally
(2, 10, 35, 67). However, there are no obvious objective
differences in neural elements to explain the differing neural
influences (34, 38, 39, 46, 56, 57). Therefore, regional
differences in muscle properties may be present to provide an
alternative basis for the gradients in excitatory and inhibitory neural
influences. Although in both the cat (37, 68) and opossum
(16, 18), resting membrane potential (RMP) of the lower
esophageal sphincter (LES;
40 to
50 mV) is consistently reported as
less negative than that seen in the esophageal body (
50 to
60 mV)
(13, 14, 17, 29), information about the RMP along the
esophageal body (EB) is inconsistent. Decktor and Ryan
(17) demonstrated a progressive decrease of the RMP
along the opossum circular muscle esophagus (
52.8 to
43.5 mV),
whereas others comparing opossum circular muscle between 8 and 2 cm
above the LES have found no difference in the RMP (13, 14). To date, there are no studies of regional differences in ion channels along the smooth muscle EB.
In light of these inconsistencies and the potential importance of
muscular properties to coordinated esophageal contractile activity, the
present experiments were performed to determine whether regional
differences exist in the EB smooth muscle, with attention to
1) the RMP and elucidation of some of the critical ions that
contribute to its generation and 2) voltage-activated currents and characterization of K+-channel subtypes
[voltage-dependent K+-channels (KV)] that
underlie these responses. Studies were performed in isolated SMCs (SMC)
dissociated from circular and longitudinal muscle layers along the EB.
Portions of this work have been published elsewhere in abstract form
(49, 52).
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MATERIALS AND METHODS |
Cell isolation.
Fasted mongrel cats of either sex were euthanized with an overdose of
pentobarbital sodium (100 mg/kg) following a protocol approved by
University Health Network Animal Care Committee and following the
guidelines of the Canadian Council on Animal Care. The esophagus and
upper gastric region were excised and placed in cold (4°C)
Krebs-Ringer solution equilibrated with 5% CO2 and 95%
O2 and having the following composition (in mM): 115.0 NaCl, 4.6 KCl, 1.2 NaH2PO4, 1.2 MgSO4, 22.0 NaHCO3, 2.5 CaCl2, and
11.0 glucose. The esophagus was opened on the greater gastric curvature side, and the mucosa was removed by blunt dissection. Two muscular equivalents of the LES were revealed within a thickened ring of muscle
composed of clasp fibers with oblique gastric sling fibers on either
side (44). For study of esophageal body muscle, circular and longitudinal muscle strips (2 mm wide) were cut from 4 and 2 cm
above the LES clasp region.
These smooth muscle strips were cut into cubes ~2 mm3,
and SMC were isolated enzymatically as previously described
(51). In brief, after tissues were incubated with enzymes,
they were washed with enzyme-free dissociation solution and then
mechanically agitated with siliconized Pasteur pipettes to disperse the
tissue and isolate single SMC. All cells used were studied at room
temperature (22-24°C) within 8 h of isolation.
Electrophysiological recording and data analysis.
Isolated esophageal SMC were allowed to settle and adhere for 30 min to
the bottom of a recording chamber, which was mounted on an inverted
microscope (Olympus CK20, Olympus America, NY). The cells were then
washed by perfusion with Ca2+-containing external solution
(in mM): 140.0 NaCl, 4.5 KCl, 2.5 CaCl2, 1.0 MgCl2, 10.0 HEPES, and 5.5 glucose, pH adjusted to 7.35 with NaOH. Patch electrodes were made using borosilicate glass
capillary tubes (Sutter Instruments, CA) using a micropipette puller
(model PP-83, Narishige, Japan). Pipettes were fire polished using a
microforge (MF-83, Narishige) to resistances of 3-6 M
. In all
experiments, unless otherwise stated, patch pipettes were filled with a
standard high K+ pipette solution containing the following
(in mM): 140.0 KCl, 0.5 CaCl2, 1.0 MgCl2, 10.0 HEPES, 4.0 Na2ATP, and 0.3 EGTA, pH adjusted to 7.2 with
KOH. The calculated calcium concentration, 113 nM, was estimated using
MaxChelator software (version 6.72) (4). In some
experiments, all K+ flux was blocked by substituting KCl
with 122 mM CsCl and 22 mM TEA in the standard pipette solution. The
current flow between the pipette and the bath solution was compensated
to achieve a zero baseline before seal formation and after
breakthrough. Standard tight-seal recording techniques for whole cell
and cell-attached single-channel recording were employed
(24). The resting potential was measured immediately after
achieving the whole cell configuration, and at least 5 min were allowed
for equilibration between the pipette solution and the intracellular
solution and to permit membrane conductances to stabilize.
Whole cell membrane potentials and currents, and cell-attached patch
single-channel recordings were made with an Axopatch-1D patch-clamp
amplifier (Axon Instruments, CA) and recorded online by a computer (IBM
AT) using pClamp software (Axon Instruments, version 6.0). Whole cell
currents were sampled at 2 kHz and filtered at 1 kHz. Single-channel
currents were sampled at 2 kHz and filtered at 500 Hz. Whole cell
capacitances and access resistances were routinely and transiently
recorded throughout the experiment using standard methods. Leak current
was assessed by observing the presence of increasing current flow at
the holding potential, where initial compensation had set current flow
to zero. Cells with leak currents >10 pA were considered leaky and
were not further studied. For whole cell studies, membrane currents
were obtained under voltage-clamp conditions in response to 20 mV
depolarizations from a holding potential of
50 mV, stepping from
70
to +70 mV and held for 250 ms. Current-voltage curves were then
constructed for steady-state currents (at the end of each current
trace) at each voltage step. The peak outward current at +70 mV was
then measured and compared after each pharmacological manipulation.
Whole cell data were analyzed using Clampfit software (pClamp 6.0 suite, Axon Instruments). SMC could be patch clamped for up to 2 h
in normal external solution with no significant change in cell currents
or membrane potential (data not shown). In experiments performed in the
cell-attached patch configuration, SMC were kept in high K+
solutions and studied with pipettes containing normal extracellular solution. Test pulses of 330 ms at varying holding potentials were
applied every 3 s. Single-channel data were analyzed using Fetchan
and pSTAT software (pClamp 6.0 suite).
To study whole cell current kinetics, steady-state activation and
inactivation curves were obtained using a two-pulse protocol. To
measure current activation, whole cell K+-channel currents
were elicited by a series of test pulses between
60 and +90 mV in
10-mV steps from a holding potential of
50 mV and returned to a
subsequent potential of
30 mV for 150 ms to evoke tail currents. The
voltage-dependent activation was derived from a measure of the peak
level of the tail current. Each peak tail current was then normalized
to the maximal tail-current amplitude and plotted against the voltage
of the test pulses. Smooth lines were obtained from best fits of data
to the Boltzmann equation: I/Imax
{1 + exp[(V1/2
Vm)/k]}
1, where
I/Imax is the relative current,
Vm is the membrane potential (the voltage applied),
V1/2 is the half-maximal voltage of activation, and k is
the slope factor. V1/2 and k were determined for
each current obtained from individual cells.
To study whole current inactivation, cells were held at
50 mV and
then a 4.5-s depolarizing conditioning pulse to different voltages was
followed by a 300-ms test pulse to +30 mV. Conditioning and test pulses
were separated by 5 ms before returning to the holding potential (
50
mV). Steady-state inactivation curves were normalized by dividing the
current amplitude (I) during the test pulse by the maximal amplitude
obtained in the absence of a conditioning pulse (Imax) and
the data are fitted with the Boltzmann function: I/Imax = {1 + exp[(Vm-V1/2)/k]}
1. The
V1/2 measured here is interpreted as the half-maximal voltage of inactivation.
Data are expressed as arithmetic mean ± SE. Because variation
between cells in any region was approximately the same as interanimal variation (not >3 mV), multiple observations for the same manipulation (pharmacological and/or electrophysiological) made on cells in one
region from one animal were averaged, and the letter n
represents the number of animals with each n representing at
least six cells. The data sets involving the effects of
K+-channel blockers on the peak steady-state current or at
peak voltage applied were analyzed by one-way ANOVA. The statistical significance of differences in the means was determined by
Tukey's-Kramer multiple-comparisons test, in which P values
<0.05 were considered significant and denoted in the following manner:
*P < 0.05, **P < 0.01, ***P < 0.001.
Solutions and drugs.
Test solutions were applied by perfusion (2-3 ml/min) or added to
the bath to obtain the final concentration as indicated. In one series
of experiments, NaCl was replaced isosmotically by arginine
chloride (both L- and D-isoforms were tested
and yielded similar results). Changes in extracellular K+
concentration were made by isosmotic substitution of NaCl for KCl,
whereas changes in Cl
concentration were made by
isosmotic substitution of NaCl for Na+ aspartate. Drug
concentrations applied were chosen based on other studies done in our
laboratory that showed them to be maximally effective. Stock solutions
of 4-aminopyridine (4-AP) and nifedipine were made up in DMSO and
diluted further (100- to 1,000-fold) with extracellular solution.
Tetraethylammonium chloride (TEA) was dissolved in ddH2O.
Iberiotoxin (IbTX; RBI, Natick, MA) or
-dendrotoxin (Alomone Labs,
Jerusalem, Israel) was dissolved in ddH2O and stored at
22°C, with each aliquot being defrosted once and used over a 6-h
study period. All drugs were added to the chamber in microliter
volumes. Routine controls with the vehicles used for dissolving each
reagent were done to exclude nonspecific effects of the diluent. All
chemicals, excluding those noted exceptions, were purchased from Sigma
Chemical (St. Louis, MO).
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RESULTS |
Two hundred eighty-eight SMCs were patch clamped. The
whole cell capacitance was routinely recorded with no significant
difference in mean values for circular muscle 4 cm above the LES (EBC4)
and 2 cm above the LES (EBC2), these being 62.7 ± 4.6 and
65.3 ± 4.2 pF, respectively. Longitudinal muscle cells were
visually longer and had significantly larger cell capacitances (an
indicator of larger cell surface area) than circular muscles with mean
capacitance measures of 74.6 ± 4.7 and 71.2 ± 2.7 pF for
longitudinal muscle at 4 (EBL4) and 2 cm above LES (EBL2). These
observed differences in size and capacitance among circular and
longitudinal muscle cells are important for studies in which whole cell
current densities are determined (see KV
density). Muscle cell contractility to 10 mM KCl and
subsequent relaxation with the removal of KCl from the extracellular
media were used as indexes of cell viability.
Because little difference in RMP existed along the longitudinal muscle,
subsequent studies were directed primarily toward examination of the
circular muscle layer of the esophageal body with comparison of the
EBC4 and EBC2 regions. Limited characterization studies were also
performed on the longitudinal muscle layer, and these data are
presented where available.
RMP.
The mean RMP of each region is shown in Fig.
1A and Table
1. For these whole cell measurements,
the cells were studied with a standard high-K+ pipette
solution under current clamp conditions. The values recorded in
esophageal body SMC from the circular layers EBC4 and EBC2 were
significantly different from each other,
51.7 ± 2.4 vs.
45.9 ± 2.6 mV, respectively (n = 15).
Therefore, the RMP of cells from the circular muscle was less negative
distally. This gradient in RMP was not seen in the longitudinal muscle.
RMP for SMC from the longitudinal layer of the esophageal body from
EBL4 and EBL2 were not significantly different from each other,
53.3 ± 2.3 and
50.9 ± 2.1 mV, respectively
(n = 12). The RMP at EBL2 was significantly more
negative than that of adjacent circular muscle at EBC2 (Fig.
1A).

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Fig. 1.
A: resting membrane potential (RMP) of
isolated smooth muscle cells (SMC) from the feline esophagus 4 (EB4)
and 2 cm (EB2) above the lower esophageal sphincter (LES). Longitudinal
muscle corresponds to the filled bars and circular muscle to the open
bars. The brackets designate statistically significant differences
between recorded parameters. The error bars represent standard
deviation. The RMP of circular muscle cells was less negative distally.
The RMP of longitudinal muscle cells from the 2 sites was not
different. At 2 cm above the LES, the RMP of circular muscle cells was
significantly less than that of longitudinal muscle cells.
B: effect of exchanging extracellular K+
concentration ([K]) on RMP of circular muscle cells. With increasing
extracellular [K], the RMP followed the Nernst equation for
EK. At [K] 30 mM, the RMP deviated significantly from
EK at both levels, the deviation most marked in EBC2
cells.
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RMP and extracellular K+
concentration.
The relationship between extracellular potassium concentration
([K+]), and RMP was examined in cells from the circular
muscle layer at EBC4 and EBC2. Figure 1B plots the mean
resting potential as a function of the extracellular
[K+]. The smooth curves were obtained by fitting the data
with the Goldman-Hodgkin-Katz constant-field equation (22,
27). SMC from EBC4 more closely followed the straight line drawn
according to the Nernst equation for a K+ channel-mediated
current that has a slope of 58 mV/10-fold change in extracellular
[K+]. However, at extracellular K+ <30 mM,
the curves for SMC from both levels studied were less negative and
deviated from those estimated to result solely through activity of
K+ channels. Therefore, at rest, permeability to other ions
was also contributing to the RMP observed.
Na+ and Cl
are two critical ions that could
potentially contribute to the RMP, and the relative role of these two
ions was assessed by two approaches: calculation of the relative
permeabilities to Na+ (PNa/PK) and
Cl
(PCl/Pk) obtained from the
curve fitting (22, 27) and observing shifts in resting
potentials with application of Na+- and
Cl
-free extracellular solution to the SMCs (Table 1). The
calculated relative permeabilities are 0.13 for Na+ and
<0.01 for Cl
in muscle cells from EBC4 compared with
0.16 and <0.01 in muscle cells from EBC2, respectively. These
calculated values are supported by the observed shifts in resting
potentials seen with the application of Na+- and
Cl
-free extracellular solution to cells from these two
areas (see Table 1). Also shown in Table 1, Cl
replacement had no significant effect on the RMP at either layer. On
the other hand, isotonic replacement of extracellular NaCl with
arginine chloride (D- or L-isoform)
significantly hyperpolarized all muscle cells, the greatest effect in
EBC2 cells. Therefore, at rest, there is a significant Na+
but not Cl
permeability in cells at all four locations.
Furthermore, the contribution of Na+ entry to the RMP is
greater at EBC2 than at EBC4 and likely accounts, in large part, for
the decreasing negativity in RMP distally.
Effect of K+ channel blockers on RMP.
As shown in Table 1, contribution of a K+ conductance to
the RMP was further assessed through the effects of
K+-channel blockers. Application of the nonspecific
K+-channel blocker TEA (10 mM) significantly depolarized
all muscle cells by >16% (with maximal effects as great as 36%).
Application of 1 mM 4-AP similarly depolarized all muscle cells significantly.
To assess whether the large-conductance Ca2+-activated
K+ (BKCa) channel contributed to the RMP, the
effects of low-dose TEA (1 mM) or IbTX (200 nM), a selective large
conductance BKCa blocker, were determined (see Table 1).
With the use of either KCa blocker, there was a lack of
significant effect on resting potentials at either level or at either
layer of the esophageal body, suggesting that BKCa channels
do not contribute to resting potentials in either the circular or
longitudinal musculature of the body. However, the effects of 4-AP,
with the largest percentage depolarization occurring in EBC4 cells
(34%), indicate that another K+ channel sensitive to 4-AP,
perhaps a delayed rectifier (KDR, see below), is present in
all SMC and contributing to their RMP.
KV density.
Outward currents were elicited in freshly isolated SMC derived from
each esophageal region, with a standard high-K+ pipette
solution and a standard depolarizing voltage protocol applied (see
MATERIALS AND METHODS). Typical families of current traces
and the mean peak steady-state outward currents obtained using the
above-described voltage protocol are shown in Fig.
2. When KCl in the pipette solution was
substituted by CsCl (122 mM) or a nonspecific K+-channel
blocker (22 mM TEA) was present in the bath, outward currents were
abolished (data not shown). These results indicate that the outward
currents were carried primarily by K ions passing through
K+ channels. To adjust K+-channel current
magnitudes for cell size, peak outward currents were divided by the
measured cell membrane capacitance and expressed in pA/pF (Fig.
3), a measure of K+ channel
density. Regional differences were seen.

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Fig. 2.
Voltage-activated outward K+ currents (KV)
evoked by the voltage protocol pictured at A, top
and B, top. A: circular muscle at 4 (EBC4) and 2 (EBC2) cm above the LES. B: longitudinal muscle
at 4 (EBL4) and 2 (EBL2) cm above the LES. Outward currents were
largest in EBC4. 4-aminopyridine (4-AP; 1 mM) significantly reduced
outward currents evoked by voltages >30 mV in all regions studied as
shown by the current-voltage curves plotted.
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Fig. 3.
Pharmacological characterization of KV
density. K+-channel density recorded from EBC4 is
significantly higher than that recorded from EBC2 (n = 12). Iberiotoxin (IbTX) reduced outward current at EBC4 only, whereas
subsequent addition of 4-AP (1 mM) reduced outward currents at all
regions to similar low levels.
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In the circular muscle of the esophageal body, the currents were
significantly larger and noisier in cells from more proximal EBC4 cells
(3,150 ± 22.3 pA, n = 12) than in EBC2 cells
(1,495 ± 32.4 pA, n = 12). Current density was
also different between EBC4 (49.8 ± 2.1 pA/pF, n = 12) and EBC2 (23.4 ± 1.4 pA/pF, n = 12). The
outward currents (EBL4: 1,575 ± 24.3 pA, n = 12, and EBL2: 1,661.4 ± 43.3 pA, n = 12) and current
densities (EBL4: 20.6 ± 3.1 pA/pF, n = 12, and
EBL2: 23.4 ± 1.4 pA/pF, n = 12) of longitudinal
muscle cells were not different from each other. Their values were
significantly lower than values of circular muscle at EB4 but similar
to those at EB2.
Voltage-activated KCa and KDR channels.
The channels contributing to the K+-channel currents evoked
by depolarization were assessed pharmacologically. KCa
channels are more sensitive to TEA and are blocked efficiently by low
doses (1 mM) (11). IbTX is a specific large-conductance
KCa-channel blocker. IbTX reduced the current density in
EBC4 circular muscle but not in EBC2 muscle (Fig. 3). Therefore, the
increased K+-current density in EBC4 appears to be due to
the activity of BKCa channels, activity of which is little
present at EBC2. Muscle cells in the longitudinal muscle layer at
either EBL2 or EBL4 were unaffected by low-dose TEA (data not shown) or
by IbTX (Fig. 3).
Several lines of evidence point to an important role for
KDR currents. K+ currents of the
KDR type are sensitive to 4-AP. As shown in the current-voltage relationship pictured in Fig. 2, 1 mM 4-AP
significantly reduced peak outward current (at +70 mV) in SMC at all
levels including EBC4, EBC2, EBL4, EBL2; the reduction was 60 ± 5%, 88 ± 11%, 89 ± 9%, 86 ± 13%
(n = 6), respectively. As shown in Fig. 3, additional
significant reduction in outward current magnitude was observed when
4-AP (1 mM) was applied with IbTX to cells from all esophageal regions.
The sensitivity of the remaining current to 4-AP as well as to high
doses of TEA (10 mM) suggests that the residual current is carried
predominantly by KDR channels. This hypothesis is supported
by the fact that the transient outward type of K+-channel
current (KTO), which is also sensitive to 4-AP, is seen only as a small remnant current at either circular muscle location (see
Fig. 4). The KTO is seen to
be more quickly inactivating than that seen for the putative
KDR. Therefore, in addition to the contribution of
BKCa at EBC4, a sizeable portion of the remaining current
in both EBC4 and EBC2 is likely attributable to a 4-AP-sensitive KDR activity. Of note, at EBC2, a 4-AP-resistant
KDR current remains in Fig. 4B.

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Fig. 4.
Transient outward K+ channel (KTO)
currents. Transiently-activating currents were elicited by depolarizing
pulses from a more hyperpolarized holding potential of 90 mV as shown
in the inset. Activity of the large-conductance
calcium-activated K+ current was inhibited with
tetraethylammonium choride (TEA) or low calcium (8 nM
intrapipette Ca2+). A: EBC4. After addition of
TEA (1 mM), a more rapidly inactivating current is obtained.
B: EBC2. When a pipette containing low Ca2+ (8 nM; high EGTA, 11 mM) is applied, a similar more rapidly inactivating
current is obtained, as in A. This current is blocked by
addition of 4-AP (1 mM) and is compatible with a KTO
current. A small 4-AP-insensitive current component remains.
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Finally, Fig. 5 demonstrates that
KDR single channels could routinely be recorded in
both EBC4 and EBC2 cells in the cell-attached configuration. The
extracellular solution was high in K+ with
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid (BAPTA)-AM (100 µM), a cell-permeant Ca2+ chelator
also present in the bath, and the pipette solution contained high
Na+. The mean slope conductance of the KDR
channels in these tissues is 18.8 ± 1.2 pS (n = 5), and 4-AP (1 mM) greatly reduced KDR activity (not
shown), providing further evidence of their identity. In addition,
dendrotoxin (DTX; maximal dose used, 200 nM) reduced EBC4 peak currents
by 62 ± 11% (n = 5) and all currents at voltages greater than or equal to
30 mV (Fig. 6)
to levels not unlike that seen with 4-AP (Fig. 2). Furthermore, DTX
also reduced EBC2 peak currents to a greater extent, by 80 ± 9%
(n = 4). This finding provides further evidence that
KDR currents are a major contributor to voltage-activated
currents at both esophageal levels.

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Fig. 5.
Delayed rectifier K+ (KDR)
single-channel characterization. Single-channel currents were recorded
in the cell-attached patch configuration from EBC2 and EBC4 in the
presence of the Ca2+ chelator
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid-AM (100 µM). A: unitary K+-channel
current amplitude increased with increased voltages applied. These
currents were reduced by 1 mM 4-AP (intrapipette; not shown).
B: plotting the unitary current against the voltage applied
yielded a slope conductance of 18.8 pS at either location (data shown
here pooled, 26 cells from 4 animals).
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Fig. 6.
Effect of specific KDR blocker dendrotoxin (DTX) on
K+ currents. K+ currents shown were
recorded using a 1-step protocol from 50 to +70 mV
(inset). A: basal K+
currents ( ) were significantly reduced by addition of
DTX (200 nM, ) at all voltages greater than 30 mV.
B: current-voltage curves demonstrate that DTX blocked
>60% and 80% of the K+ current at EBC4 and EBC2,
respectively.
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Voltage-dependent activation and inactivation of KDR
and KCa currents.
On the basis of the pharmacological analyses described above, both
KDR and KCa channels contribute to the observed
outward current with varying involvement along the smooth muscle
esophagus. For example, within esophageal body circular muscle, both
KDR and KCa channels are involved at EBC4,
whereas at EBC2, only the KDR channel plays a significant
role. The varying densities and ratios of KDR to
KCa channels would also be expected to affect the kinetics
of activation and inactivation of the whole cell K+ current.
Figure 7 shows that the voltage-dependent
activation curve obtained from muscle cells of the circular muscle
layer at EBC2 is shifted slightly but not significantly to the right
relative to muscle cells from EBC4, with V1/2 being +11.3
and +12.6 mV, respectively, and the slope factor k equal to
13 ± 2 mV compared with 10 ± 1 mV.

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Fig. 7.
Total outward current voltage-dependent activation along
the esophageal body. With the use of a standard double-pulse protocol
pictured at the top, K+ currents were evoked.
A: typical activation tail current traces obtained from EBC2
and EBC4. B: currents obtained were plotted against the
maximal current obtained. There was no significant difference in
voltage-dependent activation in cells from EBC4 and EBC2.
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To assess the inactivation of K+ channels in esophageal
muscle cells from different regions, whole cell K+-channels
currents were evoked by a double-pulse protocol (pictured at the top of
the Fig. 8). The voltage-dependent
inactivation curve of circular muscle cells from EBC4 is significantly
shifted rightward relative to the inactivation curve for circular
muscle cells from EBC2, with V1/2 being 0 mV compared with
19 mV, and k being 11.2 and 15, respectively. That is,
more K+ channels are available to repolarize esophageal
muscle at EBC4 than EBC2. Therefore, because differences in
K+-channel density and subtype expression exist between
EBC4 and EBC2, it is likely that the presence of BKCa
channels at EBC4 contributes to the shifted inactivation properties.
This hypothesis was tested by application of IbTX, a BKCa
blocker, to EBC4 cells, and assessment of the whole cell current
inactivation was repeated. With IbTX present, there was a leftward
shift in EBC4 currents to values not significantly different from EBC2
alone (V1/2 to
21 mV, k to 13). Therefore, the
regional difference in inactivation is likely due to a difference in
KCa-channel activity between EBC4 and EBC2.

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|
Fig. 8.
Total outward current voltage-dependent inactivation
along the esophageal body. With the use of a standard double-pulse
protocol pictured at the top, K+ currents were
evoked. A: typical inactivation traces obtained during the
second voltage pulse (+30 mV) obtained from circular SMCs from EBC2 and
EBC4. B: currents obtained were plotted against the maximal
current obtained. The inactivation curve for EBC4 was significantly
shifted rightward compared with that of EBC2 cells. Application of the
specific BKCa-channel blocker IbTX (10 7 M)
resulted in a leftward shift in the inactivation curve for EBC4, which
then was not significantly different from that of EBC2.
|
|
 |
DISCUSSION |
Our studies demonstrate conclusively, for the first time, that
there are significant regional differences in circular smooth muscle
properties along the esophageal body and between the circular and
longitudinal muscle layers. This regional diversity includes differences in the RMP, differences in the identity of the ion channels
setting the RMP, and differences in ion channel density on voltage
activation. It is reasonable to expect that these
electrophysiological differences present in esophageal smooth
muscle would have important functional implications. For example, in
stomach, small bowel, and colon, there are regional differences in
muscle characteristics such as RMP (16) and frequency of
electrical slow-wave activity, which play important roles in regulating
coordinated motor activity and contractile responses when the muscle is
adequately excited (15). As well, such regional
electrophysiological diversity is well described and is of functional
importance (53) in other muscle tissues such as heart
(3), vascular smooth muscle (33), and neurons
(62) and has been effectively used to establish new
considerations of etiology and pathogenesis and to design new therapies.
Gradients in RMP in the esophagus.
A gradient in resting potential exists in circular muscle cells
isolated from regions along the esophageal body (Fig. 1A, Table 1), with the RMP of muscle cells from the upper EBC4 level significantly more negative than muscle cells from the EBC2 level. Unlike the gradient along the circular smooth muscle esophagus, no
gradient in resting potential was observed along the longitudinal muscle layer, the RMP being similar to the more negative value at EBC4.
The magnitude of the RMP difference in circular muscle is like that
recorded in the intact opossum esophagus with intracellular microelectrode (17) by one group but not confirmed by
others (13, 14) who found the RMP to be uniformly more
negative (approximately
50 mV). Similarly, Kannan et al.
(29) found the RMP at 2 cm above the LES to be
49 mV.
Therefore, in intact muscle, the consensus to date indicates that there
is little, if any, gradient in RMP along the circular smooth muscle
esophagus. If so, absence of a consistent difference in RMP along the
intact circular muscle layer in the face of differences in isolated
single cells raises important implications. For example: is the RMP of
cells at one or the other level changed by electrical coupling through
structures such as gap junctions to cells that tend to have a more
negative RMP, such as muscle cells in the longitudinal layer (Table 1) or to other cells such as interstitial cells of Cajal
(31)? If the ion channels and currents responsible for the
RMP and those activated by voltage changes show regional differences,
resetting the RMP would have effects on factors such as the activation
and inactivation of voltage- and receptor-activated currents, the resulting membrane electrical events, and, presumably,
excitation-contraction coupling. This aspect would be most relevant distally.
The RMP is established primarily by a resting K+
conductance, because the membrane potential shifted in response to
changes in extracellular [K+] in all muscle cells studied
(Fig. 1B). The K+ content of combined circular
and longitudinal esophageal smooth muscle in the opossum is greater
distally, which would be compatible with a more positive RMP distally
(55). Our studies fit with a difference in the
K+ content in the circular muscle. However, the
relationship between the extracellular [K+] and RMP
deviated from that predicted by the Nernst equation for a purely
K+-selective current, indicating that the membrane
potential is not determined solely by a resting K+
permeability. In circular muscle cells from level EB4 and EB2, the
membrane permeability to Na+, with a
PNa/PK of 0.12 and 0.16, respectively, also
contributes to the RMP and probably accounts for the majority of the
difference from the calculated equilibration potential for
K+ (Ek =
85 mV). The observed
hyperpolarization of all SMCs on application of Na+-free
extracellular solution is consistent with a resting Na+
permeability. The lack of evidence of a voltage-activated
Na+ channel indicates that another voltage-independent
channel, such as a nonselective cation channel, may be involved.
Nonselective cation channels are readily demonstrated in other gut
smooth muscles (58) and have been implicated in setting
the RMP as well as voltage and ACh-stimulated Ca2+
influxes. In addition, Na+ exchangers could also be
involved because blockade of Na+ exchangers can
hyperpolarize esophageal muscle (48).
The identity of some of the K+ channels involved in setting
RMP in the muscle cells of the esophagus was assessed pharmacologically by the ability of various K+-channel blockers to shift the
resting potential (Table 1). The ability of high doses of TEA (10 mM)
or 4-AP to significantly shift RMP in all esophageal body muscle
suggests that K+ channels sensitive to these blockers, such
as KDR channels, are the primary determinant. Furthermore,
our KV current-voltage curves (Fig. 2) indicate that
KCa are not active until voltages greater than
30 mV are
applied, whereas KDR channels are activated at lower
voltages (
50 mV). Similar qualitative differences in the voltages
required to activate KDR and KCa channels have
been described in human esophageal muscle (64). Therefore,
KDR channels are the major K+ channels
contributing to RMP at both esophageal levels.
Recent studies demonstrate that very small KDR channel
density differences can greatly influence the level at which the
resting potential is set as well as influence its stability
(32). In this context, it may be that a small increase in
KDR density at EBC4 compared with EBC2 would contribute to
a more negative RMP at EBC4. Two observations support this suggestion:
1) although the level to which the remaining
IbTX-insensitive RMP was reduced by 4-AP was similar, 4-AP reduced the
remaining RMP by 34% at EBC4, whereas at EBC2, the reduction was only
20% (shown in Table 1); and 2) the KDR density
(as indicated by the IbTX-resistant voltage-activated current in Fig.
3) was marginally smaller (although not statistically different) at
EBC2 than at EBC4. However, these assays are not sensitive enough to
prove this conclusively.
BKCa do not contribute to RMP differences in the esophageal
body because IbTX did not shift the RMP significantly. This is unlike previous studies in the canine sphincter muscle, in which KCa channels are involved in setting resting
potentials as evidenced by the ability of IbTX or nifedipine to
depolarize these cells (51). This difference between
esophageal body and sphincter regions likely relates to their
differences in function in which esophageal body tissues transiently
contract and sphincter tissues are tonically contracted. Our recent
study supports this hypothesis, because unique
Ca2+-handling mechanisms of LES clasp cells were postulated
to be responsible for KCa activation in this muscle
(50). In opossum, the BKCa channel is
activated at potentials more positive than
30 mV
(36). In the human, single-channel KCa
currents were seen only at voltages more positive than +70 mV
unless ACh was added to shift the opening potential to near
30 mV
(28), and with whole cell recording, the BKCa
is only activated at positive membrane potentials (64).
Our cat studies reveal activation of the BKCa at greater
than
30 mV, as in the opossum, but no evidence for a role in
maintaining the RMP (Fig. 2). Furthermore, if spontaneous transient
outward currents are due to transient activation of a
KCa current (60, 64) by release of
Ca2+ from intracellular stores to aid in setting the RMP,
it is unlikely that the BKCa plays a significant role in
the cat and even less likely in the human.
KV channels in the esophagus.
Voltage-activated K+ currents were demonstrated in all
muscle cells studied. It was found that varying K+ channel
density accounted for the significant differences in the magnitude of
the depolarization-elicited outward current recorded in different
muscle cells (Figs. 2 and 3). Muscle cells from the proximal esophagus
(EBC4) had the highest K+ channel density. The
KDR current accounted for >60% of this total outward
K+ current as assessed by the effects of 4-AP (Fig. 2 and
3) in all esophageal body muscle cells studied. BKCa
contribution was assessed by the ability of IbTX or low-dose TEA (data
not shown) to block the outward K+ current. Whereas the
BKCa current accounted for ~30% of the outward current
in muscle cells from the circular layer at EBC4, it did not play a
significant part in cells at any level in longitudinal layer or in
circular layer cells at EB2. In human esophageal muscle, a sustained
outward current is seen in both the circular and longitudinal muscle
cells. This current is partially inhibited by selective BKCa blockade, but it is unclear whether this was seen in
both circular and longitudinal muscle cells (64). Our
findings suggested that not only did differences in channel density
exist, but there were also differences in the ratio of KDR
to KCa channels along the esophageal body, which may
account for some of the functional differences observed along the
esophagus and between muscle layers with voltage activation. Species
differences may also exist.
Voltage activation of KDR currents has been reported in
opossum esophagus in cells from the circular muscle layer (1, 36, 64). Depolarizing from a holding potential of
70 mV, Akbarali et al. (1) noted a KDR as remaining after TEA,
4-AP, and nifedipine and to activate at potentials over
50 mV.
However, in the cat, when depolarizing from a holding potential of
50
mV in the presence of IbTX, we see a more typical KDR that
is 4-AP sensitive. Similar KDR activity is seen in human
esophageal muscle (64). Although the effect of 4-AP to
depolarize the RMP was more prominent at EBC4, the 4-AP-sensitive
K+ current density was not different between EBC4 and EBC2.
Furthermore, single-channel studies in the cell-attached configuration
showed that the KDR channels at both EBC4 and EBC2 had a
similar unitary conductance of 18.8 pS (Fig. 5). Recent genetic
identification of a large number of voltage-activated
K+-channel genes reveals six families of KV,
KV1-KV6, having a range of electrophysiological
activity and biophysical properties (11, 23). On the basis
of these findings, the KDR we see in esophageal circular
muscle cells is most similar to the KV1.2 isoform, which is
reported to have a relatively large single-channel conductance of 18 pS
and is sensitive to TEA and 4-AP blockade in doses similar to those we
found effective (11). This KDR isoform is
sensitive to blockade by the specific toxin DTX such that 100 nM
abolishes its current. In our studies (Fig. 6), 200 nM DTX reduces the
outward current >60%, which is similar to the percentage of
4-AP-sensitive outward current remaining after KCa currents
are blocked with IbTX in our voltage-activation studies (Fig. 3).
Therefore, a significant portion of the KV current is
likely due to KDR currents with KV1.2
pharmacological and biophysical properties. The presence of
KV1.2 and KV1.5 isoforms has been identified in
human esophageal circular and longitudinal smooth muscle
(64).
In addition to pharmacological analyses, the differential distribution
and densities of BKCa and KDR in circular
muscle of the esophageal body were also reflected in the whole cell
K+-current kinetic studies of voltage-dependent activation
and inactivation properties in these regions. There was no significant
difference in voltage-dependent activation between EBC4 and EBC2, and
the slope of activation was relatively low (Fig. 7). Therefore,
differences in voltage-dependent activation are likely not responsible
for any differences in activation responses in these tissues, the activation reflecting primarily the characteristics of the
KDR channel. On the other hand, the voltage-dependent
inactivation was significantly different, with that of EBC4 shifted to
the right relative to that recorded for EBC2 (Fig. 8). These findings indicate that the cumulative population of KV channels,
that is both KCa and KDR, is available over a
wider voltage range at EBC4. As a result, at EBC4, fewer K+
channels would inactivate early, and repolarization of this tissue would be expected to be more rapid, providing a potential basis for the
shorter duration of contraction seen in the proximal esophagus (45). That is, the relative preponderance of these two
K+ currents will affect the rate of repolarization, it
tending to be more rapid with more BKCa activity. Of
interest, as in insulinoma cells, chromaffin cells, neurons, crayfish
muscle, and uterine myocytes, these data suggest that KCa
at EBC4 are in fact capable of inactivating (66).
Molecular characterization studies indicate that an accessory subunit
similar to the other
-subunits of KV channels confers
inactivation (66). This finding raises the possibility
that BKCa activity could be regulated to modulate the
muscle excitability independently in each esophageal region and
potentially contribute further to functional differences in contraction. For example, Wade et al. (64) have recently
shown that blockers of the KDR and KCa channels
can have different effects on circular esophageal muscle contraction in
regard to resting tone, amplitude, and duration of phasic contractions
and the production of repetitive activity.
In our studies, there is also evidence for a transient KTO
current seen in cat esophageal body regions as previously noted in the
opossum by Akbarali et al. (1). The KTO was
characteristically blocked by 4-AP leaving a small 4-AP-resistant
delayed rectifier component (Fig. 4B), the latter likely
accounting for the small outward current that remains in all muscles
after both IbTX and 4-AP are added (Fig. 3). Together, these
findings suggest that the KTO current may not be
playing a large part in the differences of voltage-activated currents
or RMP in EBC4 and EBC2 cells. We did not explore these aspects, but
they are open to testing. If differences are present, they may relate
more to the time to onset of the action potential and resulting
circular muscle contraction amplitude at each level. Blockade of the
KTO current by 4-AP prolongs the time to onset of the
action potential (1), and more prominent KTO
activity distally, if present, could provide a contribution of
the muscle to the increasing delays of the contraction along the smooth
muscle esophagus.
The regional diversity we have demonstrated in esophageal smooth muscle
has potential functional implications in health and in disease states.
Because the main putative excitatory (ACh) and inhibitory (nitric
oxide) neurotransmitters exert their effects to depolarize or
hyperpolarize through actions on ion channels (32, 60),
regional differences provide a basis for a muscle contribution to the
nature of the peristaltic contraction. We, therefore, propose that
muscle properties resident in regional ion channel diversity act to
regulate the responses to the excitatory and inhibitory innervation and
therefore contribute to the nature of the contraction and its orderly
peristaltic progression. Information about these ion channel
differences and responses opens the door to directing therapeutic
agents at more specific regional targets, such as KV
channels (30), that can regulate contraction features such
as amplitude, duration, and delay to onset.
 |
ACKNOWLEDGEMENTS |
We thank Drs. P. Backx and M. B. Wheeler for kind donation of dendrotoxin.
 |
FOOTNOTES |
This work was supported by a grant from the Medical Research Council of
Canada and ASTRA Pharma PA-13527.
Address for reprint requests and other correspondence: N. E. Diamant, Univ. Health Network (Western Division), 399 Bathurst St.,
Rm. 12-419 McLaughlin Pavilion, Toronto, Ontario M5T 2S8, Canada
(E-mail: ndiamant{at}uhnres.utronto.ca).
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.
10.1152/ajpgi.00124.2001
Received 22 March 2001; accepted in final form 24 September 2001.
 |
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