Characterization of outward K+
currents in isolated smooth muscle cells from sheep urethra
M. A.
Hollywood,
K. D.
McCloskey,
N. G.
McHale, and
K. D.
Thornbury
Smooth Muscle Group, Department of Physiology, The Queen's
University of Belfast, Belfast BT9 7BL, Northern Ireland, United
Kingdom
 |
ABSTRACT |
The perforated-patch technique
was used to measure membrane currents in smooth muscle cells from sheep
urethra. Depolarizing pulses evoked large transient outward currents
and several components of sustained current. The transient current and
a component of sustained current were blocked by iberiotoxin, penitrem
A, and nifedipine but were unaffected by apamin or 4-aminopyridine,
suggesting that they were mediated by large-conductance
Ca2+-activated K+ (BK) channels. When the BK
current was blocked by exposure to penitrem A (100 nM) and
Ca2+-free bath solution, there remained a voltage-sensitive
K+ current that was moderately sensitive to blockade with
tetraethylammonium (TEA; half-maximal effective dose = 3.0 ± 0.8 mM) but not 4-aminopyridine. Penitrem A (100 nM) increased
the spike amplitude and plateau potential in slow waves evoked in
single cells, whereas addition of TEA (10 mM) further increased the
plateau potential and duration. In conclusion, both
Ca2+-activated and voltage-dependent K+
currents were found in urethral myocytes. Both of these currents are
capable of contributing to the slow wave in these cells, suggesting that they are likely to influence urethral tone under certain conditions.
calcium-activated potassium current; delayed rectifier; urethral
smooth muscle
 |
INTRODUCTION |
MOST
RESEARCHERS NOW AGREE that urethral smooth muscle plays an
essential role in maintaining urinary continence
(12). However, though it is clear that the smooth
muscle generates spontaneous tone (2, 12,
28), the basis of this tone is poorly understood, largely
because of a lack of electrophysiological data. Nevertheless, several studies with intracellular microelectrodes have clearly demonstrated that the urethra can generate electrical events that are
coupled to contraction. These events have variously been described as
"action potentials" or "slow waves" and consist of a spike of
variable amplitude, followed by a prominent plateau (10, 13, 14, 17). Sucrose gap
recordings suggest that the force of contraction depends on the
amplitude and duration of the slow wave plateau (17),
factors that are likely to affect the degree of Ca2+ influx
into the smooth muscle cells.
Despite the likely importance of the slow wave in tone generation,
there have been few attempts to identify the ionic conductances present
in urethral myocytes. We have recently begun to do this using the
patch-clamp technique and have demonstrated an L-type Ca2+
current and a prominent Ca2+-activated Cl
current in sheep cells (9). Both currents contributed to
the generation of spontaneous slow waves in isolated cells, similar in
configuration to those found in whole tissue recordings
(10, 13, 14, 17).
However, with the exception of one group that has described an
ATP-sensitive K+ channel in the pig
(25-27), so far there has been no attempt to study
the K+ currents present in the urethra. This is an
important omission, because the shape of the action potential in other
smooth muscles is modified by both voltage-dependent and
Ca2+-dependent K+ currents that can act to
attenuate the spike and shorten the plateau and, therefore, reduce
contraction amplitude (21, 29, 33).
The aim of the present study was, therefore, to identify and
characterize the K+ currents in isolated sheep urethral
myocytes by using the patch-clamp technique. We have also examined the
effects of blockers of these currents under current clamp to gain some
insight into their potential role during the slow wave.
 |
METHODS |
Urethras of sheep of either sex were obtained from an abattoir
~15 min after slaughter and transported to the laboratory in Krebs
solution at room temperature. The most proximal 1 cm of the urethra was
removed, and 0.5-cm strips of smooth muscle were dissected free and cut
into 1-mm3 pieces. The method for cell isolation was
similar to that described previously (9). The tissue
pieces were stored in Hanks' Ca2+-free solution for 30 min, after which they were incubated in an enzyme medium containing
(per 5 ml of Hanks' Ca2+-free solution) 15 mg of
collagenase (414 U/mg; type 1a, Sigma), 1 mg of protease (10 U/mg; type
XXIV, Sigma), 10 mg of BSA (Sigma), and 10 mg of trypsin inhibitor
(Sigma) for ~40 min at 37°C. They were then placed in Hanks'
Ca2+-free solution and stirred for a further 15-30 min
to release single relaxed smooth muscle cells. These were plated in
petri dishes containing Hanks' solution (100 µM Ca2+)
and stored at 4°C for use within 8 h. The solutions used were of
the following composition (in mM): 1) Hanks'
Ca2+-free solution: 141 Na+, 5.8 K+, 130.3 Cl
, 15.5 HCO3
,
0.34 HPO42
, 0.44 H2PO4
,
10 dextrose, 2.9 sucrose, and 10 HEPES, pH adjusted to 7.4 with NaOH;
2) PSS: 130 Na+, 5.8 K+, 135 Cl
, 4.16 HCO3
, 0.34 HPO42
, 0.44 H2PO4
, 1.8 Ca2+, 0.9 Mg2+, 0.4 SO42
, 10 dextrose, 2.9 sucrose, and 10 HEPES, pH adjusted to 7.4 with NaOH; and
3) pipette solution: 133 K+, 1 Mg2+,
55 Cl
, 80 gluconate, 0.5 EGTA, and 10 HEPES, pH adjusted
to 7.2 with KOH.
Recordings were made using the amphotericin B perforated-patch
method as described previously (9). Briefly, this
consisted of dipping the tips of the patch pipettes in
amphotericin-free pipette solution for a few seconds and then
backfilling with pipette solution containing 0.6 mg/ml amphotericin B
(Sigma). After gigaseals were obtained, the series resistance fell over
a 10- to 15-min period to 10-15 M
and remained stable for up to
1 h. Series resistance was partially compensated by the circuitry
provided in the patch-clamp amplifier, voltage-clamp commands were
delivered with an Axopatch 1D patch-clamp amplifier (Axon Instruments),
and currents were recorded by means of a 12-bit
analog-to-digital/digital-to-analog converter (Labmaster, Scientific
Solutions) interfaced to an AT-type computer running pCLAMP software
(Axon Instruments). The junction potential between the pipette solution
and the bath was found to be less than
3 mV and was uncorrected.
During experiments the dish containing the cells was superfused with
bath solution. In addition, the cell under study was continuously
superfused by means of a close delivery system consisting of a pipette
(tip diameter 200 µm) placed ~300 µm away. This could be
switched, with a dead space time of <5 s, to a solution containing a drug.
In the single-channel experiments, voltage commands were applied using
pCLAMP ramped potentials. This allowed more efficient measurement of
channel activation by intracellular Ca2+ concentration
([Ca2+]i) and voltage than by conventional
step depolarizations (6). Activation curves were
calculated by averaging current responses to 15 potential ramps and
dividing each data point of the averaged current by the single-channel
amplitude at that holding potential after leakage current correction.
The rate of change of the applied ramp potentials was sufficiently slow
(100 mV/s) so that the activation curves were not distorted by the time
constants of activation or deactivation (6). Both
single-channel and whole cell experiments were carried out at 37°C.
The following drugs were used: nifedipine (Bayer), apamin,
tetraethylammonium (TEA), 4-aminopyridine (4-AP), penitrem A, and iberiotoxin (all from Sigma). Data are presented as means ± SE, and statistical differences were compared using a paired or unpaired t-test, as appropriate, with the P < 0.05 level taken as significant.
 |
RESULTS |
Ca2+-activated
K+ current.
Voltage-clamp experiments were performed using K+-filled
pipettes to characterize the K+ currents evoked by
depolarization. In the typical examples shown in Fig.
1, families of currents were evoked by
holding at
60 mV and stepping to potentials ranging from
80 to +40
mV for 500 ms. This resulted in large transient outward currents at
potentials positive to
30 mV, and these were followed by noisy,
sustained current that declined slowly during the 500-ms pulse (Fig. 1, A-C). In the majority of cells, repolarization back to
60 mV resulted an inward tail current (Fig. 1B and Fig.
2), which we have previously shown to be
due to Ca2+-activated Cl
current
(9). We next set out to identify the other components of
current evoked during the 500-ms depolarization steps. The fast
activation and inactivation of the transient current (full activation,
10 ms; inactivation, 50-100 ms) at first suggested that this might
be an "A" current similar to that described in the ureter
(20). To test this idea, we examined the effect of 4-AP (1 mM) on the transient current. As the example in Fig. 1A shows, this blocker had very little effect on the transient current, thus excluding the possibility that it was an A current. In seven cells
the transient current evoked by a step to 0 mV was 334 ± 76 pA
before and 316 ± 45 pA after 4-AP (1 mM; P = 0.59). In contrast, the transient current was reduced by nifedipine (1 µM) from 452 ± 92 to 209 ± 40 pA (n = 6, P < 0.01) as typified by the example shown in Fig.
1B. This result suggested the possibility that the transient
current was another Ca2+-dependent current. The two most
obvious candidates seemed to be small-conductance (SK) and
large-conductance Ca2+-activated K+ (BK)
currents, because both of these have been demonstrated in renal pelvis
smooth muscle (21). To test whether SK channels were
involved, we examined the effect of apamin (1 µM), and an example is
shown in Fig. 1C. Despite its high concentration, apamin had
almost no effect on the amplitude of the current, suggesting that it
was not mediated by SK channels (however, see Ref. 16). In four cells
the transient current evoked by a step to 0 mV was 390 ± 70 pA
before compared with 418 ± 95 pA after apamin (P = 0.36).

View larger version (32K):
[in this window]
[in a new window]
|
Fig. 1.
The effect of 4-aminopyridine (4-AP), nifedipine, and
apamin on net membrane currents. A: control currents were
evoked (left) in cells by holding the cell at 60 mV and
stepping to test potentials ranging from 80 to +40 mV. This evoked
both transient and sustained components of outward current, followed by
inward Cl tail currents on repolarization to 60 mV.
Addition of 4-AP (right) produced little effect on the
transient current. B: nifedipine greatly reduced the
transient current. C: apamin had little effect on the
transient current.
|
|

View larger version (25K):
[in this window]
[in a new window]
|
Fig. 2.
The effect of iberiotoxin and penitrem A on net membrane currents.
A: control currents were evoked (left) in cells
by holding the cell at 60 mV and stepping to test potentials ranging
from 80 to +50 mV. This evoked both transient and sustained
components of outward current, followed by inward Cl tail
currents on repolarization to 60 mV. The protocol was repeated in the
presence of iberiotoxin (300 nM), and currents recorded under these
conditions (middle) were subtracted from the controls to
give the iberiotoxin-sensitive difference currents (right).
B: a protocol, similar to that in A, in which
penitrem A (100 nM) was used instead of iberiotoxin. C:
summary of the current-voltage relationship of the penitrem A-sensitive
difference current in 6 cells. I, current;
Vm, membrane potential.
|
|
Evidence that a major part of the current was mediated
by BK channels is illustrated by the experiments shown in Fig. 2, in which the effects of supramaximal concentrations of two potent BK
channel blockers, iberiotoxin (11) and penitrem A
(19), were examined. In Fig. 2, A and
B, families of currents were evoked by stepping from
60 mV
to potentials ranging from
80 to +50 mV. Control currents
resembled those described in Fig. 1 in having an early transient
component followed by a sustained current. Addition of iberiotoxin (300 nM; Fig. 2A) or penitrem A (100 nM; Fig. 2B)
completely blocked the transient current and unmasked an initial L-type
Ca2+ current. These substances also reduced the sustained
current, the remainder of which turned out to be a combination of
Ca2+-activated Cl
current and delayed
rectifier potassium current (see Voltage-dependent K+
currents). The BK current was dissected
out from the other currents by examining difference currents
obtained by subtracting the currents in the presence of
blocker from the control currents (Fig. 2, A and
B). Here the transient nature of the BK current may be seen clearly, with the current activating fully within 10 ms and then decaying within 50 ms to a steady level of 20-30% of peak.
Penitrem A-sensitive difference currents from six cells are
summarized in Fig. 2C, which shows that both the
transient and sustained currents activated at steps to
30 mV or more
positive. Results similar to these were obtained with iberiotoxin (300 nM) in three cells (data not shown).
Single Ca2+-activated K+ channels.
Experiments were carried out to determine whether BK channels could be
demonstrated in excised patches. Inside-out patches were studied with
symmetrical 140 mM K+ solutions at a temperature of 37°C.
Under these conditions openings of high-conductance single channels
could be seen (Fig. 3A). The single-channel conductance was estimated by stepping to potentials of
20, 40, 60, 80, and 100 mV and plotting unitary currents against the
appropriate potentials. The current-voltage relationship was fitted
with a straight line to give a mean conductance of 309 ± 3 pS
(n = 6) and reversal potential of
5 ± 1 mV,
typical of BK channels in smooth muscle. Penitrem A has previously been
shown to be an effective blocker of BK channels when applied to the inside of the membrane (8, 19), thus
conferring an advantage over iberiotoxin, which is ineffective when
applied to inside-out patches. The single-channel openings were
completely and irreversibly blocked by penitrem A (100 nM) in six
experiments. An example is shown in Fig. 3A where frequent
openings of a single large-conductance channel could be seen during the
control period. When penitrem A (100 nM) was added to the bath, the
channel activity ceased after 25 s. Penitrem A also appeared to
transiently activate the channels before completely blocking them. This
was a consistent finding and is similar to a previous observation in
lymphatic myocytes (8).

View larger version (36K):
[in this window]
[in a new window]
|
Fig. 3.
Penitrem A, voltage, and Ca2+ sensitivity of
single large-conductance Ca2+-activated K+ (BK)
channels in inside-out patches. A: continuous single-channel
recordings made in an inside-out patch at +50 mV using symmetrical 140 mM KCl solutions [intracellular Ca2+ concentration
([Ca2+]i) = 0.1 µM, temperature
(T) = 37°C]. Channel openings are shown in the upward
direction. Penitrem A (100 nM) was added to the bath at the point
indicated by the arrow. B: Ca2+ and voltage
dependence of single-channel open probability
(NPo). Activation curves were derived from an
inside-out patch where the holding potential was ramped from 100 to
+100 mV. [Ca2+]i was buffered to 0.1, 0.25, and 0.5 µM as indicated. Each data point represents the averaged
response from 15 ramps. The continuous lines show Boltzmann fits of the
data, giving values for the voltage at half-activation
(V1/2) of 89, 46, and 7 mV.
|
|
The voltage and Ca2+ sensitivities of channel activation
were examined simultaneously using voltage-ramp protocols as described in METHODS. The inner surface of the patch was exposed to
different [Ca2+]i, with at least 15 ramps
being recorded at each concentration. The averaged data points derived
from such an experiment are shown in Fig. 3B where
[Ca2+]i was 0.1, 0.25, and 0.5 µM. This
procedure produced activation curves that were then fitted with the
Boltzmann function
where N is the number of channels in the patch,
Po is the single-channel open probability,
n is the maximal NPo,
K
1 is the steepness of the voltage-dependent
activation (change in potential necessary to cause an e-fold
increase in activation), and V1/2 is the voltage
at which there is half-maximal activation. In this experiment (Fig.
3B) V1/2 = +89 mV when
[Ca2+]i = 0.1 µM, and this value
decreased to +46 and
7 mV as [Ca2+]i was
increased to 0.25 and 0.5 µM, respectively. Data derived from four to
eight patches for each value of [Ca2+]i are
summarized in Table 1. The results
indicate that, whereas increasing [Ca2+]i
shifted V1/2 in the hyperpolarizing direction,
there was no obvious pattern to the changes in
K
1, and in most cases the shifts in the
activation curve were essentially parallel. The shift in
V1/2 per 10-fold change in
[Ca2+]i was over 120 mV, ranking these among
the most Ca2+-sensitive BK channels (5).
The whole cell experiments showed that the BK current was transient in
nature, suggesting that the channels may inactivate (32).
To test whether urethral BK channels demonstrate inactivation, we
performed step protocols in excised inside-out patches. Figure 4A shows an example of a patch
stepped from
100 mV to +50 mV for 500 ms. There was little evidence
to suggest that the BK channels had a higher Po
at the beginning of the depolarizing step. The ensemble average trace
from 15 sweeps in Fig. 4B confirms this finding, showing a
current that developed over 50 ms and then remained constant for the
remainder of the depolarizing step. Figure 4C shows a
summary of ensemble average currents in five patches, where current was
plotted at 10 ms and then 50, 100, and 150 ms, etc. These data show
that when [Ca2+]i was clamped (to 0.5 µM),
the BK current did not inactivate.

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 4.
BK channels do not inactivate in off-cell (inside-out)
patches. A: single-channel currents evoked by a step from
100 to +50 mV (symmetrical 140 mM KCl solutions,
[Ca2+]i = 0.5 µM, T = 37°C).
B: ensemble average currents from the same patch used in
A (15 sweeps). C: summary of the ensemble average
currents (as in B) in 5 patches.
|
|
Voltage-dependent K+ currents.
We studied the remaining K+ current after blockade of the
BK current and the Cl
current with a combination of
penitrem A (100 nM) and Ca2+-free bath solution
(Mg2+ substituted, 5 mM EGTA). Penitrem A was preferred to
iberiotoxin because it was the more effective blocker of whole cell BK
current (compare the remaining noise in Fig. 2, A and
B, middle). Under these conditions another
smaller component of K+ current was revealed (Fig.
5A). This current activated
rapidly and then slowly inactivated, reaching a steady state by the end of the 500-ms pulse (Fig. 5A). A summary current-voltage
plot for six experiments in which this current was recorded under these conditions is shown in Fig. 5B. This current activated at
around
50 mV and developed an 8- to 20-pA sustained current in the
range of potentials (
40 to
20 mV) corresponding to the slow wave
plateau.

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 5.
Unmasking the voltage-sensitive K+ current.
A: family of currents recorded in the presence of penitrem A
to block the BK current (left) and then in
Ca2+-free solution to block the Ca2+-current
and the Ca2+-activated Cl current
(right). B: mean current-voltage relationship of
the delayed rectifier K+ current in 6 cells. Protocol was
the same as in A, inset. Data points indicate the
mean ± SE of the peak current measured near the beginning of the
500-ms test potential.
|
|
The time-dependent relaxation of the current was examined in these six
experiments, stepping from a holding potential of
60 mV. At test
potentials below 10 mV, the decay was variable and generally difficult
to fit. At test potentials of +10, +20, +30, +40, and +50 mV, the
decay could be well fitted with single exponentials (R > 0.97), giving mean time constants of 60 ± 9, 66 ± 7, 69 ± 11, 59 ± 9, and 75 ± 8 ms, respectively,
indicating that there was little or no voltage dependence of the
time-dependent inactivation. However, this current did demonstrate the
property of voltage-dependent inactivation over more negative
potentials. This was studied by holding the cell at conditioning
potentials ranging from
100 to +10 mV for 2s, before stepping to a
test potential of +40 mV (Fig.
6A; every second step was
omitted for clarity). As the cell was progressively held at more
positive conditioning potentials, the outward current evoked at +40 mV
was reduced. Figure 6B shows the voltage-dependent
inactivation curve obtained by plotting the normalized peak current
(I/Imax) evoked at +40 mV against the
previous conditioning potential in five cells. These data were fitted
with a Boltzmann function of the form
which gives a V1/2 of
55 ± 7 mV, a
slope factor of
14 ± 4 mV, and a residual (noninactivating)
fraction of current (C) of 30 ± 6%.

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 6.
Voltage-dependent inactivation of the voltage-dependent
K+ current. A: recordings made in penitrem A
(100 nM), Ca2+-free solution. The cell was held at
conditioning potentials ranging from 100 to +10 mV for 2 s
before stepping to a test potential of +40 mV. Every second step was
omitted for clarity. Traces show currents recorded at the +40-mV test
potential following conditioning steps of 100, 80, 60, 40,
20, and 0 mV. B: the mean voltage-dependent inactivation
curve for 5 cells. The data points represent the mean ± SE of the
normalized peak current (I/Imax)
evoked following each conditioning potential. The solid line shows a
fit using the Boltzmann equation, giving a V1/2
of 55 ± 7 mV and a slope factor of 14 ± 4 mV.
|
|
The voltage-dependent K+ current was quite insensitive to
4-AP, with a concentration of 10 mM only producing an inhibition between 5 and 15% (n = 4), but was moderately
sensitive to blockade with TEA. An example of the response to
increasing concentrations of TEA is shown in Fig.
7A, where the cell was
repeatedly stepped to +40 mV from a holding potential of
80 mV. The
effect of TEA was to depress the current in a concentration-dependent
manner. A summary concentration-effect curve from four cells in which this protocol was applied is shown in Fig. 7B. The data
points are means of the peak current recorded during the appropriate concentration of TEA (Idrug) normalized to the
peak control current (Icontrol). These data were
fitted with a Langmuir equation of the form
where ED50 is the half-maximal effective dose and
C is the residual current at supramaximal
concentrations. This gave an ED50 of 3.0 ± 0.8 mM and a C value 13 ± 5% of control.

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 7.
Dose-response relationship of tetraethylammonium (TEA) on
voltage-dependent K+ current. A: the cell was
stepped repeatedly to +40 mV from a holding potential of 80 mV and
exposed to increasing doses of TEA. Recordings were made in penitrem A
(100 nM), Ca2+-free solution. B: summary data
from 4 cells in which the dose-response protocol shown in A
was followed. The current (I) recorded during each dose was
normalized by expressing it as a fraction of the current in the absence
of TEA (Imax). Data points are means ± SE,
and the solid line represents a fit of the data using the Langmuir
equation, giving an ED50 of 3.0 ± 0.8 mM.
|
|
Current-clamp experiments.
To investigate slow waves, cells were studied in current-clamp mode,
and under the conditions of these experiments [K+-filled
pipettes; Cl
equilibrium potential
(ECl) =
24 mV] they had a mean resting potential of
30 ± 3 mV. This was more depolarized than the
value reported for whole tissue urethral recordings, where the resting potential was found to be near
60 mV (13,
14). For reasons not clearly understood, the resting
potential of single smooth muscle cells may be more depolarized than
that measured in whole tissue (30). Therefore, we injected
a small background hyperpolarizing current (2-20 pA) to bring the
resting potential of the cells close to
60 mV. Cells were then
stimulated by injecting 40-ms depolarizing currents of 50-100 pA.
In cells that developed an initial net inward current under voltage
clamp, this procedure evoked slow waves similar to those illustrated in
Fig. 8A. These consisted of
1) a rapid spike, 2) an initial repolarization,
and 3) an afterdepolarization (or "plateau") of variable
duration and amplitude. In 11 cells the spike amplitude was 65 ± 4 mV, the peak plateau potential was
38 ± 4 mV, and the initial
repolarization reached a value of
48 ± 3 mV. Plateau duration,
measured from the initial repolarization to the point at which the cell
had repolarized by 80%, was variable, ranging from 250 ms to 5 s, with a mean of 1,266 ± 397 ms. All of these features were
completely blocked with nifedipine (1 µM; n = 7; not
shown) to leave a passive membrane response, indicating that the evoked
slow wave was dependent on L-type Ca2+ current. The role of
the BK current was assessed by examining the effect of penitrem A on
the slow waves. A typical example is shown in Fig. 8B where
slow waves were evoked at 20-s intervals. Wash in of penitrem A
increased the spike amplitude (from 80 to 100 mV), slowed and reduced
the initial repolarization, and increased the plateau amplitude and
duration. These results were typical of five cells in which the spike
amplitude increased from 60 ± 6 to 87 ± 9 mV
(P < 0.05) and the plateau potential increased from
40 ± 6 to
24 ± 6 mV (P < 0.05).
Plateau duration was also increased in all five cells, but, possibly
because of the large variability in the controls, this was not
statistically significant.

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 8.
Effect of penitrem A and TEA on evoked action potentials
in an isolated urethral myocyte. A: an action potential
evoked in a single cell under current clamp consisted of a spike,
initial repolarization, and a plateau. B: action potentials
were evoked at 20-s intervals, and penitrem A was added to the bathing
solution at the time indicated by the arrow. After 60 s it blocked
the transient repolarization and increased the amplitude of the spike
and plateau. C: control action potential was evoked with
penitrem A (100 nM) present in the bath to block the BK current.
Addition of TEA clearly increased the amplitude and duration of the
plateau, and this effect was reversed following a 20-s wash.
|
|
Investigation of the role of the voltage-activated K+
current on the slow wave was hampered by a lack of a specific blocker because, although millimolar concentrations of TEA reduced this current, micromolar concentrations have been shown to block the BK
current (5). Therefore, the effect of TEA (10 mM) was
examined on slow waves in cells in which the BK current had already
been blocked by penitrem A. An example is shown in Fig. 8C
where TEA reversibly enhanced the amplitude and duration of the plateau and slightly increased the spike. This example was typical of three
cells in which, in each case, the duration and amplitude of the plateau
increased, suggesting that the voltage-activated K+
channels also contribute to the shape of the slow wave.
 |
DISCUSSION |
Though the mechanism of urethral smooth muscle tone generation is
poorly understood, it is now generally believed to contribute significantly to urinary continence (for discussion, see Ref. 12).
Although it is highly likely that ion channels in the membranes of the
smooth muscle cells modulate the contractile state of the cells, there
have been remarkably few studies of isolated urethral myocytes under
voltage clamp. In the present study we have, for the first time,
systematically studied the voltage-sensitive and Ca2+-dependent K+ currents in sheep urethral
myocytes. Because there are similarities in the behavior of human and
sheep urethras at the whole tissue level such as the development of
tone, the presence of a dual adrenergic and cholinergic excitatory
innervation, and relaxation produced by nitrergic nerves
(28), it is reasonable to use sheep cells as a basis to
provide basic background knowledge of the behavior of urethral myocytes
in general before carrying out necessarily much more difficult human
studies in the future. However, because so far there have been no
electrophysiological studies of the human urethra, the validity of
different animal models remains to be established. The only other
single studies that have been carried out were in the pig, in which an
ATP-sensitive K+ current has been found
(25-27). There is evidence that this current may
contribute to the resting membrane conductance (27).
However, because this current was neither voltage nor Ca2+
sensitive, it is unlikely to play a part in determining the shape of
the slow wave, which in any case has not been demonstrated in the pig
(26).
We have now characterized two further components of outward
K+ current in the urethra. One component was
Ca2+ and voltage sensitive and had characteristics in
common with BK currents found in other smooth muscle preparations
(5), whereas the other component was a voltage-activated,
Ca2+-insensitive current similar to delayed rectifier (DK)
current in other smooth muscles. The BK current consisted of a large, rapid transient phase followed by a sustained phase that lasted throughout the 500-ms depolarizing step. In some cells (e.g., Fig.
1B) fluctuations were observed on the sustained component of
current, resembling the STOCs (spontaneous transient outward currents)
described in other smooth muscles (5). At first we thought
that the transient phase was an example of the A current found in a
wide variety of preparations including several types of smooth muscle
(1, 20). However, the transient component was
identified as a BK current on the basis of its insensitivity to 4-AP
(5), complete blockade by penitrem A and iberiotoxin, and
dependence on Ca2+. Also, there was little contribution
from apamin-sensitive SK channels (Fig. 1C), although a
contribution from apamin-insensitive channels (e.g., SK1) cannot be
excluded (16). Our findings in whole cell experiments were
supported by data from excised patches, which showed that the cells
expressed a high density of large-conductance (~300 pS)
Ca2+- and voltage-activated channels that were blocked by
penitrem A.
The transient BK current of the present study contrasts with that
recorded in a pig urethral myocyte (see Fig. 4 of Ref. 25), where only
a nondecaying component was evident. Although this current was not
characterized further, it had some of the features of BK current,
including superimposed STOCs. The time courses of BK currents reported
in other smooth muscle preparations were also enormously variable,
ranging from fairly slowly activating sustained currents in dog colon
and mouse gallbladder (7, 18) to transient
currents in rabbit ileum (24), guinea pig vas deferens, and guinea pig bladder (15). It is not clear whether these
represent genuine species- or tissue-specific differences or reflect
variations in experimental conditions. Inactivating BK channels have
been described in adrenal chromaffin cells, insulinoma tumor cells, and
hippocampal neurons (see Ref. 32). The current in chromaffin cells
superficially resembled that described in the present study in that it
followed a similar time course of inactivation. Recently this behavior
was attributed to the presence of a newly described
3-subunit that not only confers the property of
inactivation but also enhances the Ca2+ sensitivity and
decreases the charybdotoxin sensitivity (32). However, the
fact that the single BK channels of the present study do not inactivate
when studied in off-cell patches, where
[Ca2+]i was clamped, suggests that these
channels do not demonstrate true inactivation but, rather, that the
transient time course in whole cell experiments reflects changes in
subsarcolemmal [Ca2+]i. This is in marked
contrast to the BK channels of adrenal chromaffin cells, which do
inactivate in off-cell patches (32). Thus it seems
unlikely that a specialized
3-subunit was contributing to the behavior of the BK channels in urethral myocytes.
In the present study, several methodological factors may have
contributed to the rapid time course of the BK current. First, recordings were made using the perforated patch so that no exogenous Ca2+ buffers were present intracellularly. This is in
contrast to the study in pig urethra (25), in which cells
were dialyzed with 5 mM EGTA. Buffers could potentially distort the
time course of the current, resulting in either suppression or
enhancement of the various phases of the current depending on the
characteristics of the buffers and the coupling of the BK channels to
their source of Ca2+ (22, 23). A
second factor that may have increased the initial phase of the BK
current in our experiments was the recording temperature of 37°C.
Recording at physiological temperatures has been shown to greatly
increase the size of the L-type Ca2+ current
(31), and this may have been reflected by the BK current. Finally, in our experiments a more accurate time resolution was facilitated by examining difference currents, thereby avoiding contamination by L-type Ca2+ current, which would "drag
down" the BK current.
The other K+ current described in the present study was
voltage sensitive, but Ca2+ insensitive, and was only
clearly visible when both the BK and Cl
currents were
blocked by a combination of Ca2+-free conditions and
penitrem A. Voltage-activated K+ currents in smooth muscle
fall into two broad types: 1) a small group of transient,
highly 4-AP-sensitive, TEA-insensitive A currents (1,
20), and 2) a larger group of DK currents. The
latter are distinguished by slower inactivation kinetics, making them appear less "transient," and are usually sensitive to millimolar concentrations of TEA but also variably sensitive to 4-AP
(3, 18, 29, 33).
The current described in the present study may be classified with the
latter group on the basis of its sensitivity to TEA and relatively slow
inactivation kinetics. However, in several smooth muscles the DK
current has been successfully subdivided into two or more components,
suggesting that its classification is complex (3,
8). Because of the lack of specific blockers, no further
attempt was made to resolve the urethra DK current into possible
subcomponents in the present study, although it is possible that it was
mediated by more than one type of K+ channel.
Though the K+ current can be divided into BK and DK
components in many other smooth muscles, the relative size, activation range, and time course of each component varies considerably, suggesting that the relative contribution of each component to excitability may vary according to muscle type. In the colon and gallbladder, blockade of the DK current with 4-AP resulted in prolongation of the slow wave plateau, but BK blockers had little effect (4, 29, 33). In these
tissues a model was put forward whereby the plateau represented a
quasi-stable membrane potential that resulted from a balance of
sustained inward Ca2+ current and outward DK current with
little proposed role for either a BK current (at least under resting
conditions; see Ref. 4) or a Cl
current. The situation,
however, appears to be quite different in urinary tract muscle, where
blockade of BK current with charybdotoxin prolonged the action
potential in the renal pelvis (21) and Ca2+-activated Cl
current seemed to
"clamp" the membrane potential near ECl
during the slow wave plateau in the urethra (9,
13, 14).
In the present study, isolated urethral cells fired slow waves very
similar to the ones observed in whole tissue (10,
13, 14). These were modulated by both
penitrem A and TEA, suggesting that both the BK and the DK current
contributed to the slow wave. Penitrem A had the effect of increasing
the spike amplitude and blocking the early repolarization. This is
consistent with a role for the transient BK current during the early
phase of the slow wave. Also, the sustained current seemed to
contribute significantly to the overall membrane conductance during the
plateau of the slow wave because the plateau was significantly enhanced
by penitrem A. Though it was more difficult to assess the role of the
DK current because of the lack of a selective blocker, TEA further
enhanced the spike and plateau after the BK current had been blocked
with penitrem A. This suggests that the DK current can also contribute to conductance changes during the slow wave. However, we still require
much more detailed information regarding all the ionic conductances
during the slow wave in the urethra; therefore, further analysis was
not attempted at this stage.
In conclusion, the two K+ currents described in the
present study have voltage- and time-dependent characteristics that
suggest that they oppose depolarization during both the spike and
plateau phases of the slow wave. Such effects are likely to modulate
Ca2+ entry and may therefore play a part in regulating
urethral tone.
 |
ACKNOWLEDGEMENTS |
We thank The Wellcome Trust for financial support and John Robinson
and Sons for supplying sheep urethras. Mark Hollywood holds a Northern
Ireland Development of Research Lectureship.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: K. D. Thornbury, Smooth Muscle Group, Dept. of Physiology, The
Queen's Univ. of Belfast, 97 Lisburn Rd., Belfast BT9 7BL, Northern
Ireland, UK (E-mail: k.thornbury{at}qub.ac.uk).
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.
Received 15 November 1999; accepted in final form 8 March 2000.
 |
REFERENCES |
1.
Beech, DJ,
and
Bolton TB.
A voltage-dependent outward current with fast kinetics in single smooth muscle cells isolated from the rabbit portal vein.
J Physiol (Lond)
412:
375-395,
1989[Abstract].
2.
Bridgewater, M,
McNeil HF,
and
Brading AF.
Regulation of tone in pig urethral smooth muscle.
J Urol
150:
223-228,
1993[ISI][Medline].
3.
Carl, A.
Multiple components of delayed rectifier K+ current in canine colonic smooth muscle.
J Physiol (Lond)
482:
339-353,
1995.
4.
Carl, A,
Bayguinov O,
Shuttleworth CWR,
Ward SM,
and
Sanders KM.
Role of Ca2+-activated K+ channels in electrical activity of longitudinal and circular muscle layers of canine colon.
Am J Physiol Cell Physiol
268:
C619-C627,
1995[Abstract/Free Full Text].
5.
Carl, A,
Lee HK,
and
Sanders KM.
Regulation of ion channels in smooth muscles by calcium.
Am J Physiol Cell Physiol
271:
C9-C34,
1996[Abstract/Free Full Text].
6.
Carl, A,
and
Sanders KM.
Ca2+-activated K+ channels of canine colonic myocytes.
Am J Physiol Cell Physiol
257:
C470-C480,
1989[Abstract/Free Full Text].
7.
Cole, WC,
and
Sanders KM.
Characterization of macroscopic outward currents of canine colonic myocytes.
Am J Physiol Cell Physiol
257:
C461-C469,
1989[Abstract/Free Full Text].
8.
Cotton, KD,
Hollywood MA,
McHale NG,
and
Thornbury KD.
Outward currents in smooth muscle cells isolated from sheep mesenteric lymphatics.
J Physiol (Lond)
503:
1-12,
1997[Abstract].
9.
Cotton, KD,
Hollywood MA,
McHale NG,
and
Thornbury KD.
Ca2+-current and Ca2+-activated chloride current in isolated smooth muscle cells of the sheep urethra.
J Physiol (Lond)
505:
121-131,
1997[Abstract].
10.
Creed, KE,
Oike M,
and
Ito Y.
The electrical properties and responses to nerve stimulation of the proximal urethra of the male rabbit.
Br J Urol
79:
541-553,
1997.
11.
Garcia, ML,
Galvez A,
Garcia-Calvo M,
King VK,
Vazquez J,
and
Kaczorowski GJ.
Use of toxins to study potassium channels.
J Bioenerg Biomembr
23:
615-646,
1991[ISI][Medline].
12.
Greenland, JE,
Dass N,
and
Brading AF.
Intrinsic urethral closure mechanisms in the female pig.
Scand J Urol Nephrol Suppl
179:
75-80,
1996[Medline].
13.
Hashitani, H,
and
Edwards FR.
Spontaneous and neurally activated depolarizations in smooth muscle cells of the guinea-pig urethra.
J Physiol (Lond)
514:
459-470,
1999[Abstract/Free Full Text].
14.
Hashitani, H,
Van Helden DF,
and
Suzuki H.
Properties of depolarizations in circular smooth muscle cells of rabbit urethra.
Br J Pharmacol
118:
1627-1632,
1996[Abstract].
15.
Imaizumi, Y,
Torii Y,
Ohi Y,
Nagano N,
Atsuki K,
Yamamura H,
Muraki K,
Watanabe M,
and
Bolton TB.
Ca2+ images and K+ current during depolarization in smooth muscle of the guinea-pig vas deferens and urinary bladder.
J Physiol (Lond)
510:
705-719,
1998[Abstract/Free Full Text].
16.
Ishii, TM,
Maylie J,
and
Adelman JP.
Determinants of apamin and d-tubocurarine block in SK potassium channels.
J Biol Chem
272:
23195-23200,
1997[Abstract/Free Full Text].
17.
Ito, Y,
and
Kimoto Y.
The neural and non-neural mechanisms involved in urethral activity in rabbits.
J Physiol (Lond)
367:
57-72,
1985[Abstract].
18.
Jagger, JH,
Mawe GM,
and
Nelson MT.
Voltage-dependent K+ currents in smooth muscle cells from mouse gallbladder.
Am J Physiol Gastrointest Liver Physiol
274:
G687-G693,
1998[Abstract/Free Full Text].
19.
Knaus, H-G,
McManus OB,
Lee SH,
Schmalhofer WA,
Garcia-Calvo M,
Helms LMH,
Sanchez M,
Giangiacomo K,
Reuben JP,
Smith AB,
Kaczorowski GJ,
and
Garcia ML.
Tremorgenic indole alkaloids potently inhibit smooth muscle high-conductance calcium-activated potassium channels.
Biochemistry
33:
5819-5828,
1994[ISI][Medline].
20.
Lang, RJ.
Identification of the major membrane currents in freshly dispersed single smooth muscle cells of the guinea-pig ureter.
J Physiol (Lond)
412:
397-414,
1989[Abstract].
21.
Lang, RJ,
and
Zhang Y.
The effects of K+ channel blockers on the spontaneous electrical and contractile activity in the proximal renal pelvis of the guinea-pig.
J Urol
155:
332-336,
1996[ISI][Medline].
22.
Naraghi, M,
and
Neher E.
Linearized buffered Ca2+ diffusion in microdomains and its implications for calculation at the mouth of a calcium channel.
J Neurosci
17:
6961-6973,
1997[Abstract/Free Full Text].
23.
Prakriya, M,
Solaro CR,
and
Lingle CJ.
[Ca2+]i elevations detected by BK channels during Ca2+ influx and muscarinic-mediated release of Ca2+ from the intracellular stores in rat chromaffin cells.
J Neurosci
16:
4344-4359,
1996[Abstract/Free Full Text].
24.
Sakai, T,
Terada K,
Kitamura K,
and
Kuriyama H.
Ryanodine inhibits the Ca-dependent K-current after depletion of Ca stored in smooth-muscle cells of the rabbit ileal longitudinal muscle.
Br J Pharmacol
95:
1089-1100,
1988[Abstract].
25.
Teramoto, N,
and
Brading AF.
Activation by levcromakalim and metabolic inhibition of glibenclamide-sensitive K channels in smooth muscle cells of pig proximal urethra.
Br J Pharmacol
118:
635-642,
1996[Abstract].
26.
Teramoto, N,
Creed KE,
and
Brading AF.
Activity of glibenclamide-sensitive K+ channels under unstimulated conditions in smooth muscle cells of the pig proximal urethra.
Naunyn Schmiedebergs Arch Pharmacol
356:
418-424,
1997[ISI][Medline].
27.
Teramoto, N,
McMurray G,
and
Brading AF.
Effects of levcromakalim and nucleoside diphosphates on glibenclamide-sensitive K channels in pig urethral myocytes.
Br J Pharmacol
120:
1229-1240,
1997[Abstract].
28.
Thornbury, KD,
Hollywood MA,
and
McHale NG.
Mediation by nitric oxide of neurogenic relaxation of the urinary bladder neck muscle in sheep.
J Physiol (Lond)
451:
133-144,
1992[Abstract].
29.
Thornbury, KD,
Ward SM,
and
Sanders KM.
Participation of fast-activating, voltage-dependent K currents in electrical slow waves of colonic circular muscle.
Am J Physiol Cell Physiol
263:
C226-C236,
1992[Abstract/Free Full Text].
30.
Vogalis, F,
and
Sanders KM.
Characterization of ionic currents of circular smooth muscle cells of the canine pyloric sphincter.
J Physiol (Lond)
436:
75-92,
1991[Abstract].
31.
Ward, SM,
and
Sanders KM.
Upstroke component of electrical slow waves in canine colonic smooth muscle due to nifedipine-resistant calcium current.
J Physiol (Lond)
455:
321-337,
1992[Abstract].
32.
Xia, X-M,
Ding JP,
and
Lingle CJ.
Molecular basis for the inactivation of Ca2+- and voltage-dependent BK channels in adrenal chromaffin cells and rat insulinoma tumor cells.
J Neurosci
19:
5255-5264,
1999[Abstract/Free Full Text].
33.
Zhang, L,
Bonev AD,
Nelson MT,
and
Mawe GM.
Ionic basis of the action potential of guinea pig gallbladder smooth muscle cells.
Am J Physiol Cell Physiol
265:
C1552-C1561,
1993[Abstract/Free Full Text].
Am J Physiol Cell Physiol 279(2):C420-C428
0363-6143/00 $5.00
Copyright © 2000 the American Physiological Society