Departments of Medicine and Physiology, University of Toronto, and Playfair Neuroscience Unit, Toronto Western Hospital, Toronto, Ontario, Canada M5T 2S8
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ABSTRACT |
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The whole cell patch-clamp technique was used to
investigate whether there were inwardly rectifying K+
(Kir) channels in the longitudinal muscle of cat esophagus.
Inward currents were observable on membrane hyperpolarization negative to the K+ equilibrium potential (Ek)
in freshly isolated esophageal longitudinal muscle cells. The
current-voltage relationship exhibited strong inward rectification with
a reversal potential (Erev) of 76.5 mV.
Elevation of external K+ increased the inward current
amplitude and positively shifted its Erev after
the Ek, suggesting that potassium ions carry
this current. External Ba2+ and Cs+ inhibited
this inward current, with hyperpolarization remarkably increasing the
inhibition. The IC50 for Ba2+ and
Cs+ at
60 mV was 2.9 and 1.6 mM, respectively.
Furthermore, external Ba2+ of 10 µM moderately
depolarized the resting membrane potential of the longitudinal muscle
cells by 6.3 mV while inhibiting the inward rectification. We conclude
that Kir channels are present in the longitudinal muscle of
cat esophagus, where they contribute to its resting membrane potential.
esophageal longitudinal muscle; patch clamp; barium; cesium
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INTRODUCTION |
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SMOOTH MUSCLE OF THE LONGITUDINAL layer of the esophageal body differs from the muscle in the circular layer in a number of ways. The esophageal circular muscle demonstrates descending inhibition that precedes its peristaltic contraction and provides for ready propulsion of the bolus along the esophagus. On the other hand, the function of the longitudinal muscle depends solely on its contraction. Its contraction would serve to shorten the esophagus, especially in the region in advance of the peristaltic circular muscle contraction, thereby expanding the lumen and stabilizing the wall in this region and facilitating distal propulsion (22). The mechanical activities of the muscles are dictated by ion channels on the plasma membrane. Several ion channel currents have been identified and characterized in esophageal circular muscle from different species, including delayed-rectifier K+ currents, transient outward K+ currents, Ca2+-activated K+ currents, and L- and T-type Ca2+ channel currents (37, 1, 29). In contrast, much less information is obtained from esophageal longitudinal smooth muscle cells. To our knowledge, only voltage-gated K+ outward currents have been found in human esophageal longitudinal muscle (37).
The resting membrane potential of cat esophageal longitudinal muscle cells is constant and relatively negative along the esophagus, as opposed to that of circular muscle cells, which becomes less negative distally (27). A relative stable resting membrane potential resulting from the activity of ion channels may play a role in modulating physiological function of the esophageal longitudinal smooth muscle. Inwardly rectifying K+ (Kir) channels conduct K+ more readily at membrane potentials negative to the K+ equilibrium potential (Ek) and therefore play a role in the maintenance of resting membrane potential. Kir channels have been identified in a variety of tissue types (13), including cardiac (26), vascular (6), and skeletal muscle (8). In light of these findings, a similar kind of channel that acts to stabilize the resting membrane potential would be expected to exist in the esophageal longitudinal smooth muscle.
In the current study, we applied the whole cell patch-clamp technique to investigate whether there were Kir channels in smooth muscle cells freshly isolated from the longitudinal layer of cat esophagus. We recorded an inward rectifier K+ current in these cells on membrane hyperpolarization negative to the Ek. This inward current was highly K+ selective and was inhibited by extracellular cations such as Ba2+ and Cs+ in a voltage-dependent manner. External Ba2+ depolarized the membrane potential most likely because of inhibition of the outward component of the inward rectification. These results indicate the existence of Kir channels in the esophageal longitudinal muscle and their contribution to the resting membrane potential of the muscle.
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METHODS |
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Cell isolation. Adult cats of either sex were killed by intravenous injection of pentobarbital sodium (0.5 mg/kg) according to the protocol approved by The Toronto Hospital Animal Care Committee. Each esophagus was quickly excised and placed in modified Krebs solution composed of (in mM) 115 NaCl, 4.6 KCl, 1.2 NaH2PO4, 1.2 MgSO4, 22 NaHCO3, 2.5 CaCl2, and 11 glucose and bubbled with 95% O2-5% CO2. After the mucosa and the circular muscle were stripped off, the exposed longitudinal muscle was dissected out and cut into squares of ~2 mm2. These squares were placed in a test tube with 1 ml of dissociation solution composed of (in mM) 125 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, 2.5 EDTA, and 10 glucose (pH 7.2). Papain (2 mg/ml) as well as collagenase blend F (1.3 mg/ml), 1,4-dithio-L-threitol (154 µg/ml), and BSA (1 mg/ml) were added to the test tube, which was then incubated at 37°C for tissue digestion (28). After 45 min, tissues were washed with enzyme-free dissociation solution three times and gently agitated with a plastic transfer pipette. Spindle-shaped single smooth muscle cells were dispersed and used for patch-clamp study within the following 5 h.
Whole cell patch-clamp recording.
Isolated cells in dissociation solution were placed in a 1-ml
glass-bottom dish mounted on the stage of an inverted microscope and
allowed to adhere to the bottom for 30 min. These cells were perfused
with standard external solution containing (in mM) 140 NaCl, 5 KCl, 2.5 CaCl2, 1 MgCl2, 10 HEPES, and 5.5 glucose (pH 7.4). Membrane currents from longitudinal muscle cells were recorded in
the voltage-clamp mode. Pipettes were made from borosilicated, thin-walled glass capillary tubings (OD 1.5 mm, ID 1.10 mm; Sutter Instrument, Novato, CA) with a two-stage microelectrode puller (MF-83,
Narishige, Tokyo, Japan). The reference electrode made from Ag-AgCl
wire was directly connected to the bath. The resistance of a pipette
tip was ~3-6 M after being filled with standard pipette
solution composed of (in mM) 140 KCl, 0.5 CaCl2, 1 MgCl2, 5 EGTA, 10 HEPES, and 5 Na2ATP (pH 7.2).
Recordings were performed using an Axopatch 1D amplifier (Axon
Instruments, Foster City, CA). The junction potential between the
pipette and the external solution was adjusted to 0 at the beginning of
the experiment. A tight seal (>1 G
) was established between the
cell membrane and the pipette tip by gentle suction, and then the
membrane was ruptured by a further suction. The voltage-clamp protocols
were generated by pClamp 6 software (Axon Instruments). Data were
filtered at 1 kHz by an on-board eight-pole Bessel filter before
digitization with a DigiData 1200 analog-to-digital converter (Axon
Instruments). Values for cell capacitance were determined by transient
cancellation of capacitive transients. Data were analyzed using pClamp
6-Clampfit software (Axon Instruments). Leak current was subtracted
from original current during analysis. The formula applied by Clampfit to estimate leak current is stimulus waveform/(CF × resistance), where CF is the correction factor (0.001 in our experimental
situation). Recordings were commenced 5 min after the formation of the
whole cell configuration to obtain stable currents. All experiments were performed at a room temperature of 20-22°C.
Solutions and drugs used. External solutions with 30, 70, and 140 mM K+ were made by increasing the amount of KCl to respective concentrations while decreasing the amount of NaCl to 115, 75, and 0 mM in standard external solution, respectively. Therefore, the total solute osmolarity of external solutions was preserved. Na+-free external solution was prepared by replacing the excessive NaCl with N-methyl-D-glucamine (Sigma Chemical, St. Louis, MO). Mg2+-free pipette solution was made by omitting 1 mM MgCl2 and 0.5 mM CaCl2, and 5 mM EGTA was replaced by 5 mM EDTA in the standard pipette solution. Tetraethylammonium chloride (TEA) (Sigma Chemical) was dissolved in distilled water. Stock solutions of glibenclamide (Sigma Chemical) and 4-aminopyridine (4-AP) (Sigma Chemical) were made in 10% DMSO that was further diluted 100-fold in the final concentration.
Statistical analysis. Data are presented as means ± SE, and n indicates the number of cells, each of which came from a different animal. Unless otherwise stated, unpaired Student's t-test (2-tail) was used to compare data from different groups. P < 0.05 was considered to be significantly different.
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RESULTS |
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Freshly isolated esophageal longitudinal muscle cells were
spindle-shaped and relaxed. Whole cell capacitance was 46.0 ± 1.8 pF averaged from 75 cells. The mean input resistance of these cells was
3.3 ± 0.2 G (n = 30). Reversible contractions
could be observed when the cells were exposed to
Ca2+-containing external solution, indicating that they
have retained contractile function after enzymatic digestion.
Whole cell inward rectifier currents.
Esophageal longitudinal muscle cells were bathed in external solution
with 5 mM K+ and dialyzed with pipette solution containing
140 mM K+. To investigate the membrane conductance in the
inward direction, a series of test pulses from 30 to
150 mV at
20-mV steps were applied for 400 ms from a holding potential of
50
mV. To prevent gigaseal breakdown, the maximal hyperpolarization
voltage was limited to
150 mV, and the step voltage protocol was
always run from +30 to
150 mV. Figure
1A shows that inward currents
were rapidly activated at voltages negative to
70 mV. The inward
current amplitudes progressively increased with hyperpolarization, and the current waveforms became noisier at membrane potentials negative to
130 mV. During the 400-ms test pulses, the inward currents did not
exhibit obvious inactivation (Fig. 1A). Figure 1B
displays the current-voltage (I-V) relationship
obtained by plotting current amplitudes at the end of test pulses
against membrane potentials. In the I-V curve
shown in Fig. 1B, the inward current was rectified at about
70 mV. However, the outward current between
70 and
30 mV was very
small, being no more than 3 pA at
70 and
50 mV and 5 pA at
30 mV
from that cell. Hence, the inward current exhibited a strong inward
rectification. To determine the reversal potential of the inward
rectification, tail currents were obtained by voltage steps ranging
from
130 to
60 mV (Fig. 1C) after a hyperpolarization
voltage to
130 mV for 1 s that fully activated the inward
rectifier current. As shown in Fig. 1D, the
I-V relationship of tail currents was generated
by plotting the instantaneous tail current amplitudes at each test
potential. It also demonstrated inward rectification. From this
I-V relationship, the extrapolated reversal
potential of the inward rectification was
76.5 mV (n = 4, Fig. 1D), which was close to the
Ek of
83.9 mV with 140 mM internal
K+ and 5 mM external K+. In this situation, the
Na+ and Cl
equilibrium potentials were +66.9
and
1.5 mV, respectively. Therefore, the inward rectifier current is
primarily K+ selective.
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Effect of external K+.
Kir channels are highly selective for K+. An
important property of these channels is the positive shift and
accentuation of the inward rectification with the elevation of external
K+ (6, 25, 23, 34). Therefore, we examined the
effects of raising external K+ concentration
([K+]o) on the inward rectifier current. As
shown in Fig. 4A, exposure of
longitudinal muscle cells to external solutions containing more
K+ shifted the reversal potential of the inward
rectification in the depolarizing direction and increased the inward
current amplitude in a concentration-dependent manner. The values for
reversal potential were extrapolated from I-V
relationships plotted by the ramp voltage-induced currents against
voltages applied. Thus the reversal potentials were actually
zero-current membrane potentials. The Ek was
calculated according to the Nernst equation:
Ek = (RT/F) ln
([K+]o/[K+]i),
where R is the gas constant, T is the temperature
in Kelvin, F is Faraday's constant, and
[K+]i is the internal K+
concentration. The values for reversal potential at 5, 30, 70, and 140 mM [K+]o were 77.8 ± 2.3 (n = 6),
33.8 ± 1.9 (n = 6),
18.5 ± 1.0 (n = 5), and
1.3 ± 0.3 mV
(n = 4), respectively, whereas the theoretical
Ek values were
83.9,
38.8,
17.5, and 0 mV,
respectively. Therefore, the reversal potential of the inward
rectification shifted positively following the
Ek as external K+ was raised. With a
standard [K+]o and
[K+]i of 5 and 140 mM, respectively, the
reversal potential extrapolated from zero-current membrane potential
was
77.8 mV, close to
76.5 mV estimated from tail inward currents
(Fig. 1D). The reversal potential was depolarized by 54 mV
for each 10-fold increase in external K+ (Fig.
4B), similar to the theoretical value of 58.0 mV calculated from the Nernst equation.
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Inhibitory effect of Ba2+.
The voltage-dependent block of the Kir current by external
Ba2+ is an important characteristic of the channel. To
observe the Ba2+ action more efficiently, we recorded the
inward current at high conductance by increasing
[K+]o to 140 mM, which was the same as
[K+]i. Under this condition, the inward
rectifier currents were evoked by 5-s hyperpolarization potentials from
10 to
110 mV in a 10-mV increment. The holding potential was
clamped at 0 mV, which was equal to the resting membrane potential when
external and internal K+ were the same. Figure
5A shows representative inward
currents at voltage steps to
60 and
100 mV in the presence of
external Ba2+ with various concentrations. As
expected, Ba2+ caused a dose-dependent inhibition of the
inward current. For example, the inward current amplitude at the end of
the test pulse was blocked by 24.9% by 1 µM Ba2+ at
60
mV, whereas the block was increased to 51.6% at 3 µM
Ba2+ and 78.6% at 10 µM Ba2+, respectively
(Fig. 5, A and B). More importantly, the
Ba2+-induced inhibition was voltage dependent. At a higher
hyperpolarization step, the block was also enhanced. When 3 µM
Ba2+ was applied, the inward current was only inhibited by
51.6% at
60 mV but by 77.6% at
100 mV (Fig. 5, A and
B). Furthermore, Ba2+ caused faster decay of the
inward current when the cell membrane was hyperpolarized to more
negative potentials (Fig. 5A). The time constant of the
decay was determined by fitting the inward current with a single
exponential. With exposure to 10 µM Ba2+, the mean time
constants were 396.8 ± 64.4 ms at
30 mV, 168.1 ± 16.0 ms
at
60 mV, and 41.3 ± 11.2 ms at
90 mV (n = 5-7). The voltage-dependent inhibition caused by external
Ba2+ is theoretically interpreted by assuming that
Ba2+ binds in a voltage-dependent manner to a site within
the channel pore to prevent K+ movement through the channel
(10). If the binding site is within the membrane potential
field, the membrane hyperpolarization will facilitate a positively
charged Ba2+ to bind to the site. This assumption
can be described by the equation
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(1) |
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(2) |
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Inhibitory effect of Cs+.
External Cs+ is also a voltage-dependent blocker of
Kir channels in skeletal muscle, vascular smooth muscle,
and blood cells, although a higher concentration of Cs+ is
required for this inhibition compared with Ba2+ inhibition
(8, 25, 34). We therefore examined the effect of
extracellular application of Cs+ on the inward rectifier
current in esophageal longitudinal muscle cells bathed in external
solution with 140 mM K+. The inward currents were evoked by
5-s hyperpolarizing test pulses from a holding potential of 0 mV. As
shown in Fig. 6A, external
Cs+ (300 µM) inhibited the inward current, with more
hyperpolarization increasing this inhibition. For instance, when
exposed to 300 µM Cs+, the inward current amplitude
measured at the end of the test pulse was blocked by 14% at 60 mV
and by 57.5% at
80 mV (Fig. 6, A and B). In
contrast to Ba2+ (Fig. 5B), Cs+
demonstrated a steeper block of the inward current with membrane hyperpolarization in the I-V relationship (Fig.
6B). As external Cs+ was raised, the block of
the inward current began to appear at less negative membrane potentials
(Fig. 6B). In addition, Cs+-induced inhibition
was also concentration dependent. Figure 6C summarizes the
concentration-dependent Cs+ inhibition from six cells at
different membrane potentials. The solid lines were best fitted with
Eq. 1 and nonlinear least-squares optimization.
At
90 mV, the inward current was inhibited by 14.2%, 49.0%, and
74.2% at 30, 100, and 300 µM external Cs+, respectively
(n = 6, Fig. 6C). Using the same equations,
the Kd values for Cs+ block were
calculated at
30,
60, and
90 mV to be 2,800, 1,570, and 142 µM,
respectively (n = 6). Therefore, the
Kd was greatly decreased with membrane
hyperpolarization. The estimated value for
K0 from Eq. 2 was 192 mM
with µ of 2.0.
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Effect of external Na+ on inward current.
In those experiments with higher [K+]o,
external K+ was increased in the solutions by equimolar
replacement of Na+. External Na+ is known to
cause inactivation of Kir channels. Therefore, we next
tested whether the inward rectifier current was affected by
Na+-free external solution. Na+-free external
solution was made by replacing equimolar Na+ with
N-methyl-D-glucamine. The step voltage protocol
was initially applied when the cells were bathed in normal solution
(external Na+ concentration = 140 mM) and then
repeated in Na+-free solution. As seen in Fig.
7, Na+-free solution only
slightly increased the inward current by 9.3% at 150 mV and by
12.9% at
130 mV (n = 5) with no statistical significance, suggesting that external Na+ is not an
effective regulator of the inward current in the voltage range used and
furthermore that this current is not carried by sodium ions.
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Effect of internal Mg2+ on inward current.
Intracellular cations such as Mg2+ and polyamines have been
found to modify gating properties of the Kir channel by
decreasing the outward component of the Kir channel current
(32, 18). We next examined whether intracellular
Mg2+ could contribute to the inward rectification seen in
esophageal longitudinal muscle cells. The pipette solution contained
140 mM K+ and 5 mM EDTA but was free of Mg2+
and Ca2+. Under these conditions, internal Mg2+
was reduced to a minimal level. As shown in Fig.
8, dialysis of the cytoplasm with
Mg2+-free pipette solution did not significantly alter the
outward current. Although the inward current at 150 mV was increased by 11% (n = 5) in Mg2+-free pipette
solution, this change failed to reach statistical significance. This
lack of a significant effect of low internal Mg2+
concentration on Kir current has also been reported in
vascular smooth muscle cells (25).
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Contribution of Kir channel to resting membrane
potential.
To study whether Kir channels could play a role in
stabilizing the resting membrane potential of the esophageal
longitudinal muscle, low-dose Ba2+ (10 µM) was added to
the standard bath solution with 5 mM K+. The experiments
were designed to alternatively record membrane currents and membrane
potentials in the voltage- and current-clamp modes, respectively.
Figure 9B shows that external
Ba2+ (10 µM) induced a depolarizing change in membrane
potential, which reached a plateau in 20 s. In eight longitudinal
muscle cells, the resting membrane potential was 51.3 ± 3.0 mV,
in agreement with our (27) previous results. In the
presence of 10 µM Ba2+, the cell membrane was depolarized
by 6.3 ± 0.8 mV (n = 8). In the voltage-clamp
mode, the inward rectifier current evoked by a ramp voltage was shown
to be greatly inhibited by 10 µM Ba2+ (Fig. 9,
A and C), indicating that the
Ba2+-induced depolarization is related to the block of the
Kir channel. These results suggest that the Kir
channel is involved in the maintenance of the resting membrane
potential of the longitudinal muscle and the mechanism is likely
mediated by a small outward K+ current of the channel at
rest.
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DISCUSSION |
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The present study provides, for the first time, strong evidence that Kir channels exist in the longitudinal muscle of cat esophagus. This evidence is based on the following major findings: 1) strong inward rectification near the Ek; 2) the shift of the reversal potential with the Ek and the increase in current magnitude when external K+ was raised; and 3) the voltage-dependent block of the inward rectifier current by external Ba2+ of low concentration and Cs+. Furthermore, the inward rectifier current is insensitive to blockers of Ca2+-activated K+ channels, delayed rectifier K+ channels, and ATP-sensitive K+ channels. Together, these data suggest that the inward rectifier current seen in the esophageal longitudinal muscle results from K+ ion flow through a Kir channel.
An ATP-sensitive K+ channel current has been found in
rabbit esophageal smooth muscle cells from muscularis mucosae
(12). This channel current shows weak inward rectification
and is blocked by glibenclamide. The Kir current observed
in our study can be distinguished from the ATP-sensitive K+
channel current because it displayed strong inward rectification and
was not inhibited by 105 M glibenclamide (Fig.
3B). In addition, 5 mM ATP was routinely included in the
pipette solution to minimize the contamination of our recordings from
ATP-sensitive K+ channel currents. A
hyperpolarization-activated cation inward current has been reported in
smooth muscle cells from different tissues (3, 9). This
current is slowly activated, nonselectively permeable to potassium and
sodium ions, and reversed at approximately
25 to
30 mV. These
characteristics are quite different from the Kir current in
our preparation. Voltage-gated K+ channels that show weak
inward rectification (19) have been found to exist in the
longitudinal muscle of cat (27) and human esophagus
(37). However, TEA and 4-AP did not inhibit the
Kir current from esophageal longitudinal muscle cells,
ruling out its origin from voltage-gated K+ channels (Fig.
3A). Human eag-related gene (HERG) also encodes an inwardly rectifying channel that exhibits rapid inactivation and
slow deactivation (35, 30). The HERG current is
voltage-independently blocked by Ba2+ with an
IC50 of 0.6 mM (35). All of these
electrophysiological and pharmacological characteristics are absent for
the Kir current recorded in our study.
The I-V relationship of the Kir
current from esophageal longitudinal muscle cells exhibited strong
inward rectification with a reversal potential of 76.5 mV, which is
close to the Ek (Fig. 1). The reversal potential
of the inward rectification shifted to more depolarizing potentials
following the Ek as external K+
increased (Fig. 4A). The reversal potential changed by 54 mV for a 10-fold alteration in external K+, near the estimated
value of 58.0 mV from the Nernst equation. Therefore, the
channel responsible for the Kir current acts as a
K+ electrode when external K+ is altered. The
slope conductance of the Kir current increased with
external K+ in a nonlinear manner. It was proportional to
[K+]or with r of
0.46, close to values for r between 0.42 and 0.47 in other
cell types (25, 34).
A voltage-dependent block of the Kir channel by external
Ba2+ of low concentration and Cs+ is the most
prominent property of this channel. Ba2+ is a potent
blocker of the Kir channel with relative high affinity. The
Kd for Ba2+ at 60 mV is 2.1 µM
in vascular smooth muscle cells (25), 14 µM in skeletal
muscle (
65 mV) (31), and 13.7 µM in starfish oocytes
(11). In our preparation, the
Kd for Ba2+ was 2.9 µM at
60 mV
(Fig. 5C), comparable with the Kd
values in other cell types mentioned above. This voltage-dependent
block caused by Ba2+ can be explained by a model in which
the fraction of the applied electric field (µ) acting on the
Ba2+ binding site is 0.4. This value is comparable with
that of chromaffin cells (0.25) (15), smooth muscle cells
(0.51) (25), and oocytes (0.6-0.7) (10).
In addition, the Ba2+ inhibition developed in a
time-dependent manner, with more hyperpolarization causing faster decay
of the Kir current (Fig. 5A). This phenomenon has also been noted for the Kir current in egg cells
(11), skeletal muscle (31), cardiac Purkinje
fibers (4), and smooth muscle from resistance-sized
cerebral arteries (23). In contrast to its highly
sensitive block of the Kir channel, external
Ba2+ was less effective in blocking ATP-sensitive
K+ channels in skeletal muscle (Kd
100 µM at
60 mV) (24), Ca2+-activated
K+ channels in rabbit skeletal muscle
(Kd 335 µM at
60 mV) (36), and
voltage-dependent K+ channels
(Kd > 1 mM) in smooth muscle
(20). Thus Ba2+ can be employed as a selective
blocker of the Kir channel in the esophageal longitudinal muscle.
Cs+ is also an effective blocker of the Kir channel but less potent than Ba2+ in our preparation and other cell types. In contrast to Ba2+, the Cs+ block displayed greater voltage dependence because the I-V relationship in the presence of Cs+ was steeper (Fig. 6B) than that in the presence of Ba2+ (Fig. 5B). The mean value of µ for Cs+ was 2.0, >0.4 for Ba2+, indicating higher sensitivity of Cs+ to the applied electric field, which was consistent with the stronger voltage dependence of the Cs+ block. On the other hand, the Cs+ block of the Kir current was instantaneous (Fig. 6A), unlike the time-dependent decay of the Kir current seen in the Ba2+ block (Fig. 5A). Although Cs+ and Ba2+ are assumed to share a common mode of action by blocking the Kir channel, the discrepancy shown in our preparation and other cell types reflects some slight difference in the molecular mechanisms by which these ions affect the Kir channel.
It is well established that the gating property of the Kir channel is regulated by intracellular Mg2+ and polyamines (21). Using Mg2+-free pipette solution with 5 mM EDTA to minimize the internal Mg2+, we saw Mg2+ produce a small but insignificant alteration of the inward rectification (Fig. 8). A similar result has also been reported elsewhere in coronary artery muscle cells (25). Presumably, in esophageal longitudinal smooth muscle internal polyamines are sufficient to regulate the gating property of the Kir channel without a major role for Mg2+. Polyamines are metabolites of amino acids and found in almost all cells (33). Application of polyamines to inside-out patches containing IRK1 (Kir2) channels in the absence of Mg2+ restores all of the essential features of intrinsic rectification, suggesting that they may function as physiological blockers of the Kir channel (7). We did not assess the role of polyamines in our study.
Several types of Kir channels have been cloned and found to
be expressed in different tissues, including the strongly
rectifying Kir2.1 (IRK1) (17) and
Kir3.1 (GIRK1) (5) and the weakly rectifying
Kir1.1a (ROMK1) (14) and Kir6
(rcKATP-1) (2). Unlike voltage-gated
K+ channels, Kir channels have only two
transmembrane domains (17). The Kir current
observed in our study likely falls into the strong rectification group
because little outward current was seen at positive potentials to the
Ek between 70 and
30 mV (Figs. 1A and 2). Moreover, the cloned IRK1 increases its conductance in a
proportion to 0.47 power of external K+ and is also highly
sensitive to Ba2+ (Kd < 30 µM) (17). These data are comparable with the
observations for the Kir current from the esophageal
longitudinal muscle. Further study such as RT-PCR is needed to
determine the Kir channel subtype in our preparation.
Although some important features of the Kir current in the
esophageal longitudinal muscle are similar to those of the classic Kir current (e.g., strong inward rectification,
K+-mediated current, and high sensitivity to
Ba2+ and Cs+), there are also some distinct
differences. First, inactivation of the classical Kir
current is usually seen at high hyperpolarization levels (e.g., 150
mV). However, this was not seen in the Kir current of
longitudinal muscle cells at hyperpolarization of
150 mV. Second,
Na+ removal does not significantly increase the
Kir current at
150 mV in our preparation. Third, the
Kir current in longitudinal muscle cells is noisy at
hyperpolarization levels from
130 to
150 mV, which is usually not
observed in other preparations. A possible explanation for the first
and second differences may be that the hyperpolarization level was not
negative enough to show the inactivation characteristics
(34). However, these differences require further study.
The Kir channel conducts more efficiently at membrane
potentials negative to the Ek, thus permitting
K+ to pass more readily into the cell than out of it.
However, the small outward current through the Kir channel
should also be considered because it is likely to play a physiological
role over the normal range of membrane potentials. The average resting
membrane potential for esophageal longitudinal muscle cells was 51.3
mV, positive to the Ek. Low-dose
Ba2+ (10 µM) caused a moderate membrane depolarization
with an average level of 6.3 mV and inhibited the Kir
current, suggesting that Ba2+-induced depolarization is
associated with the block of the Kir channel. Because the
outward current component through the Kir channel was only
1 or 2 pA (25), it was difficult to accurately and
consistently measure it in macroscopic recording. However, at a
concentration as low as 10 µM, Ba2+ has not been found to
efficiently affect any known K+ channels except the
Kir channel. Therefore, the Ba2+-induced mild
membrane depolarization indicates that a small outward current through
the Kir channel may contribute to the membrane potential at
rest. A similar result has been obtained by a previous study performed
on vascular smooth muscle (6). The inward current component, the major portion of the Kir channel, is assumed
to maintain a stable resting membrane potential during membrane
hyperpolarization. It may modulate the degree of membrane
hyperpolarization of esophageal smooth muscle as in the situation of
the inhibitory junction potential. Direct evidence for this assumption
should be further investigated.
In conclusion, the longitudinal muscle cells of cat esophagus have Kir channels. External Ba2+ is an efficient and selective blocker of this channel. One physiological role of the Kir channel is likely to maintain more negative resting membrane potential, which results from outflow of K+ through this channel at rest. Its role in modulating the hyperpolarization of the inhibitory junction potential is unknown.
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ACKNOWLEDGEMENTS |
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This study was supported by a grant from Astra Pharmaceutical and Medical Research Council of Canada Grant PA-13527.
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FOOTNOTES |
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Portions of this work have been published previously in abstract form (16).
Address for reprint requests and other correspondence: N. E. Diamant, 12-419 Playfair Neuroscience Unit, Toronto Western Hospital, 399 Bathurst St., Toronto, ON, Canada M5T 2S8.
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 12 January 2000; accepted in final form 24 May 2000.
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