Ca2+ dependence and
pharmacology of large-conductance
K+ channels in nonlabor and
labor human uterine myocytes
Raheela N.
Khan1,2,
Stephen K.
Smith2,
J. J.
Morrison2, and
Michael L. J.
Ashford1
1 Department of Pharmacology,
University of Cambridge, Cambridge CB2 1QJ;
and 2 Department of Obstetrics
and Gynaecology, University of Cambridge, The Rosie Maternity
Hospital, Cambridge CB2 2SW, United Kingdom
 |
ABSTRACT |
Two populations,
Ca2+-dependent
(BKCa) and
Ca2+-independent
K+ (BK) channels of large
conductance were identified in inside-out patches of nonlabor and labor
freshly dispersed human pregnant myometrial cells, respectively.
Cell-attached recordings from nonlabor myometrial cells frequently
displayed BKCa channel openings characterized by a relatively low open-state probability, whereas similar recordings from labor tissue displayed either no channel openings or consistently high levels of channel activity that often
exhibited clear, oscillatory activity. In inside-out patch recordings,
Ba2+ (2-10 mM),
4-aminopyridine (0.1-1 mM), and
Shaker B inactivating peptide
("ball peptide") blocked the
BKCa channel but were much less
effective on BK channels. Application of tetraethylammonium to
inside-out membrane patches reduced unitary current amplitude of
BKCa and BK channels, with
dissociation constants of 46 mM and 53 µM, respectively.
Tetraethylammonium applied to outside-out patches decreased the unitary
conductance of BKCa and BK
channels, with dissociation constants of 423 and 395 µM,
respectively. These results demonstrate that the properties of human
myometrial large-conductance K+
channels in myocytes isolated from laboring patients are significantly different from those isolated from nonlaboring patients.
calcium-activated potassium channels; human myometrium; pregnancy; potassium channel blockers; tetraethylammonium; barium; ball peptide; charybdotoxin
 |
INTRODUCTION |
POTASSIUM CHANNELS CONSIST of a diverse group of
proteins with disparate structural features and controlling mechanisms.
Large-conductance K+ channels
activated by raised intracellular
Ca2+ levels
(BKCa channels) are a feature of
many smooth muscle cell types, playing a central role in the control of
cellular excitability. The presence of
BKCa channels in nonpregnant human
(24), pregnant human (1, 8), and pregnant rat (9), rabbit, or pig
myometrium (22) has been clearly demonstrated. Furthermore, macroscopic Ca2+-sensitive outward
K+ currents have been observed in
rat myometrium at estrus (20) and during pregnancy (18).
The properties of BKCa channels of
neurons (21), skeletal muscle (3), and smooth muscle (7, 15) share many
features, including a high single channel conductance (200-250
pS), voltage-dependent activation, and a similar pharmacology. Thus
intracellular Ba2+ blocks the
BKCa channel of neurons (21),
rabbit T-tubules (25), and smooth muscle (2, 15) at submillimolar
concentrations, whereas tetraethylammonium ion (TEA) block of most
BKCa channels is more potent when
this quaternary ammonium ion is applied to the extracellular rather
than the internal membrane surface (2, 26, 30). We reported previously
(8) the presence of a Ca2+- and
voltage-sensitive K+-selective ion
channel in pregnant, human nonlabor myometrium that has the
characteristics of the classical
BKCa channel, in that activation
of the channel is a function of membrane potential and intracellular
Ca2+ concentration
([Ca2+]i),
and it is sensitive to block by internal
Ba2+ but less so by TEA. In
contrast, the electrophysiological properties of the channel most
frequently recorded from human myometrium after the onset of labor are
considerably different from those of the
BKCa channel, in that the former
lacks Ca2+ dependence, has a high
open-state probability
(Po) in the
absence of internal Ca2+, and
exhibits little voltage dependence. In addition, this channel (which we
have termed BK to indicate large conductance and
Ca2+ independence) does not
display the typical pharmacology of
BKCa channels, in that TEA
sensitivity is enhanced and Ba2+
sensitivity is reduced in inside-out patches (8). A large-conductance Ca2+-insensitive
K+ channel with similar features
has also been reported in bovine tracheal myocytes (7). Therefore, the
aim of the present study was to investigate further the differences in
sensitivity of BKCa and BK
channels in human myometrium to relatively specific and nonspecific
K+ channel blockers.
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METHODS |
Tissue collection.
Myometrial tissue was collected from women undergoing elective or
emergency cesarean section under regional analgesia (spinal or epidural
anesthesia for emergency cesarean sections, spinal only for elective
cesarean sections) at full-term gestation (38-42 wk) after patient
consent with Local Ethical Committee approval (Cambridge District
Health Authority). Patients were defined as being in labor if they had
regular uterine contractions at a frequency greater than one every 5 min. Biopsy tissue from the upper section of the lower uterine segment
was obtained from the area immediately below the reflection of the
visceral peritoneum from nonlabor and labor patients. The use of this
site as a marker for tissue collection also ensured that the cervix was
not mistaken for the myometrium. Furthermore, visual inspection of the
biopsy confirmed the muscular nature of the tissue, and there appeared
to be no obvious morphological differences in myometrium obtained from nonlabor or labor patients. Tissue was kept for up to 12 h in Ham's
F-12 supplemented with 100 U/ml penicillin and 100 µg/ml streptomycin.
Cell isolation.
Freshly isolated myometrial cells were obtained by enzymatic
disaggregation of finely minced myometrium with 2 mg/ml collagenase (type IA, 300-400 U/mg; Sigma Chemical) in Hanks' buffered salt solution. The incubation with enzyme was performed at 37°C for 2 h
followed by centrifugation (800-1,000 revolutions/min) in 60%
Percoll for 10 min; then the pellicle was removed, washed, and spun in
physiological solution composed of (mM) 135 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, and 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES, pH 7.2). Single cells were plated onto 35-mm petri dishes
(Falcon) that had been pretreated with lectin; thus 1 mg/ml concanavalin A in physiological solution was added to the dish for 2 min; then the dish was rinsed with physiological solution before the
cells were plated, and experiments were begun immediately. The
pretreatment of plates with concanavalin A improved cell adhesion to
the plastic substrate but had no noticeable effect on
channel activity.
Electrophysiological recordings and data analysis.
Inside-out and outside-out patches were utilized for this study. Patch
pipettes with resistances of 8-12 M
when filled with electrolyte were fabricated from borosilicate glass on a PB-7 puller
(Narashige). Single channel recordings were made with an Axopatch 200 patch-clamp amplifier (Axon Instruments), collected on digital
audiotapes through a digital audiotape recorder (model DTC 1000ES,
Sony), and replayed for illustration onto a Gould RS 3200 chart
recorder. Voltages were measured with respect to an AgCl reference
electrode and are expressed according to the usual sign convention,
i.e., inside negative.
Single channel recordings were analyzed off-line. Data (60- to 120-s
duration) were filtered at 1 kHz with an eight-pole Bessel filter
(
3 dB) and digitized at 5 kHz with a 12-bit analog-to-digital converter (Data Translation). Current amplitude, single channel Po, and channel
activity (NPo)
were determined by computer analysis (model 4DX33, Viglen) as described
previously (8) using the patch-clamp analysis program PAT V6.2 (kindly
provided by Dr. J. Dempster, Strathclyde, UK). In patches containing
more than one channel, the total current at a constant voltage was
measured by integration of the current signal, and the result was
expressed as NPo.
The mean single channel
Po could then be
calculated according to the following equation
where
I is the total current,
i is the single channel current at a
constant voltage, and N is the maximum
number of simultaneously active channels observed in the patch at a
membrane potential of +50 mV. This method of calculating
Po was accurate
for multichannel patches containing up to five active channels in the
patch and was used to compare channel activity in the absence and
presence of blockers. For outside-out patches, where channel number
often exceeded five, channel activity was expressed as
NPo, inasmuch as
it was difficult to determine absolute channel number with any degree
of certainty.
In experiments where the blocking effect of internal TEA was studied,
the linearized form of the Woodhull (29) equation (Eq. 1) was used. Woodhull analysis has been used to
describe the kind of fast block, e.g., with TEA, that results in a
reduction in single channel current amplitude, eventually rendering the block and the closed state indistinguishable from each other. From
Eq. 1, it is possible to obtain values
for the dissociation constant
(Kd) of TEA to
its blocking site and the location of the blocking site within the
membrane electric field (
)
|
(1)
|
where
i and
iTEA are the
single channel current amplitudes in the absence and presence of
internal TEA, respectively; [TEA] is the TEA concentration;
F, R, and
T represent Faraday's constant, the
gas constant, and absolute temperature, respectively; and z is the blocker valency.
The Kd for TEA
applied at the external or internal aspect of the patch membrane was
obtained from Eq. 2, assuming a 1:1
drug-to-receptor binding scheme
|
(2)
|
where
iC the single
channel current amplitude in the absence of TEA.
Drugs and solutions.
For inside-out patches the electrode contained in (mM) 140 KCl, 1 CaCl2, 1 MgCl2, and 10 HEPES (pH 7.2). The
bath intracellular solution consisted of (mM) 140 KCl, 0.35-0.9
Ca2+, 1 potassium ethylene
glycol-bis(
-aminoethyl
ether)-N,N,N',N'-tetraacetic acid (EGTA), 1 MgCl2, and 10 HEPES
(pH 7.2). These solutions were reversed for outside-out patch
recordings. The free Ca2+
concentrations in the bathing solution were calculated as described previously (8). Potassium-EGTA was held constant at 1 mM, and CaCl2 was added as calculated.
Thus 50 nM Ca2+ was obtained by
the addition of 0.35 mM CaCl2, 0.5 µM Ca2+ with 0.85 mM
CaCl2, and 0.8 µM
Ca2+ with 0.9 mM
CaCl2. Physiological solution was
used to bathe the cells before experiments.
The effects of TEA, 4-aminopyridine (4-AP), and
Ba2+ were tested by diluting 1 M
stock solutions of these compounds in physiological solution or
high-K+ solution as required.
Charybdotoxin (ChTX; Alomone Labs) was prepared as a 1 µM stock
solution in physiological saline to which bovine serum albumin (0.1%)
had been added to prevent nonspecific binding of the toxin to plastic-
or glassware. Drugs were bath applied by a gravity-feed superfusion
system at a flow rate of 10 ml/min, with complete solution exchange
within 2 min. Tetraethylammonium chloride (TEA),
BaCl2, 4-AP, Hanks' buffered salt
solution, Ham's F-12, penicillin, streptomycin, Percoll, concanavalin
A, collagenase (type IA), and Analar grade chemicals were purchased
from Sigma Chemical (Poole, Dorset, UK). Ball peptide was kindly
donated by Dr. B. Robertson (Dept. of Biochemistry, Imperial College of Science and Technology, London, UK). All experiments were conducted at
room temperature (21-25°C). Results are expressed as means ± SE. Statistical analyses were performed using unpaired Student's t-test. All graphical plots were
fitted by either linear regression or iterative curve-fitting routines
(when fitting to equations) using Kaleidagraph (V3.0.5, Abelbeck
Software).
 |
RESULTS |
Cell-attached recordings.
Cell-attached recordings demonstrated a clear difference in channel
activity, depending on whether the cells were obtained before or after
the onset of established labor. Cell-attached recordings from labor
uterine myocytes were characterized by an absence of channel activity
(n = 7) or displayed spontaneous
rhythmic activity (12 of 19 patches). For example, with physiological
saline (5 mM K+) in the bath and
140 mM K+ in the electrode and no
applied voltage, oscillations in membrane current on which bursts of
action currents were superimposed could be recorded under voltage-clamp
conditions (Fig. 1A, top
trace). These persisted for the duration
of the recording. When cell-attached patches were made from labor
myometrium with physiological solution (135 Na+, 5 mM
K+) in the bath and high (140 mM) K+ in the electrode, cyclical
channel activity was frequently observed; i.e., the cell would undergo
periods of intense activity involving several simultaneously active
channels characterized by a high average
Po (0.62 ± 0.11, n = 5). This is
illustrated in Fig. 1A (bottom trace) at an applied pipette
potential of
80 mV, which corresponds to a membrane potential of
+40 mV, assuming a mean myometrial cell resting membrane potential of
42 mV for labor cells recorded in the whole cell configuration
(data not shown). In marked contrast, cell-attached patches of nonlabor
myometrium rarely exhibited spontaneous activity (3 of 29 patches).
Single BKCa channel activity in
nonlabor myometrium was characterized by a low
Po (0.16 ± 0.04, n = 8) in the absence of any
applied membrane voltage. However, as pipette potential was made more negative (i.e., depolarized in the cell-attached configuration), channel openings underwent an increase in
Po (0.36 ± 0.08, n = 5 at pipette potential of
80 mV) and conversely on hyperpolarization. Representative
single BKCa channel activity from
a cell-attached patch at a membrane potential of +40 mV (assuming a
mean membrane potential of
47 mV for nonlabor cells, data not
shown) is illustrated in Fig. 1B. The
clearly distinct patterns of channel behavior recorded in cells
obtained before and after the onset of labor imply distinct
physiological controlling mechanisms operating in the intact cells.

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Fig. 1.
A: Labor myometrium.
Top trace: cell-attached recordings
showing spontaneous rhythmic activity with action currents superimposed recorded at rest (0 voltage). Electrode contained 140 mM
K+; bathing solution was
physiological solution. Bottom trace:
cell-attached recording showing cyclical behavior of single
K+ channels at a membrane
potential of +40 mV. Membrane potential in cell-attached configuration
was obtained by assuming a cell resting membrane potential of 42
mV and an applied potential of 80 mV. Open-state probability
(Po) for this
patch is 0.46. B: representative
cell-attached recording from nonlabor myometrium at a membrane
potential of +40 mV. Activity is low compared with labor myometrium.
Membrane potential in cell-attached configuration was obtained by
assuming a cell resting membrane potential of 47 mV and an
applied potential of 80 mV. Channel
Po is 0.13. Solutions as in A. Arrowheads, closed
state of channel; inward current is shown as downward deflections.
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Inside-out recordings.
The identification of myometrial large-conductance
K+ channels was verified by
increasing the Ca2+ concentration
from 50 nM to 0.5 µM at the intracellular surface of inside-out
patches. Channels were identified as
BKCa if this procedure resulted in
marked enhancement of BKCa channel
activity or as BK if channel activity remained high on lowering or
raising Ca2+ concentration.
Representative recordings of BKCa
and BK channel activity are shown in Fig.
2.
BKCa channels are characterized by
little activity at 50 nM Ca2+,
whereas at 0.5 µM Ca2+ there was
a substantial increase in activity, and at both
Ca2+ concentrations this channel
type displayed marked voltage dependence, with depolarization causing
an increase in activity (Fig. 2A). In contrast, BK channels are characterized by high levels of channel activity independent of membrane voltage (
60 to +60 mV) or
Ca2+ concentration (from 50 nM to
0.5 µM; Fig. 2B). In this study the single channel conductance of BK and
BKCa channels in the inside-out
configuration was 229 ± 13 (n = 7) and 219 ± 16 pS (n = 24), respectively
(P > 0.05). These values are in
close agreement (221 and 212 pS for BK and
BKCa, respectively) with those
reported previously (8). Furthermore, in accord with our previous
study, only BKCa channels were
detected in nonlabor (n = 34) and BK
channels in labor (n = 17) myometrium.
To verify the presence of the BK channel in both configurations,
patches from labor myocytes were excised from the same cell; the
inside-out patch always possessed Ca2+- and voltage-independent BK
channel activity, whereas the outside-out patch always contained
voltage-dependent BK-like channels.

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Fig. 2.
Ca2+ dependence of human
myometrial Ca2+-dependent
(BKCa) and
Ca2+-independent large-conductance
K+ (BK) channels. Single channel
currents were recorded from inside-out patches excised into symmetrical
K+ conditions.
A:
BKCa channels (nonlabor) recorded
in presence of 50 nM Ca2+
(left) and 0.5 µM free
Ca2+
(right).
Po were as
follows: in 50 nM Ca2+, 0.09 at
+40 mV, 0.02 at +20 mV, 0.001 at 20 mV, and 0.00 at 40
mV; in 0.5 µM Ca2+, 0.56 at +40
mV, 0.47 at +20 mV, 0.17 at 20 mV, and 0.14 at 40 mV.
B: BK channels (labor) recorded in
presence of 50 nM Ca2+
(left) and 0.5 µM
Ca2+ (right).
Po were as
follows: in 50 nM Ca2+, 0.76 at
+40 mV, 0.68 at +20 mV, 0.73 at 20 mV, and 0.81 at 40 mV;
in 0.5 µM Ca2+, 0.67 at +40 mV,
0.58 at +20 mV, 0.71 at 20 mV, and 0.84 at 40 mV.
Arrowheads, closed state of channel; inward current is shown as
downward deflections.
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Outside-out recordings.
Recordings from outside-out patches obtained from nonlabor and labor
myometrial cells did not display such marked differences between the
channel types. In both cases, large-conductance
K+ channels exhibited strong
voltage dependence, such that depolarization resulted in an increase in
BK (labor) and BKCa (nonlabor)
channel activity compared with channel activity at negative membrane
potentials (Fig. 3). In this patch
configuration, BK and BKCa
channels are characterized by unitary conductances of 224 ± 9.2 (n = 6) and 217 ± 8.7 pS
(n = 9), respectively (Fig.
3B), and are not significantly different (P > 0.05) from the
conductances reported from inside-out patch recordings (see above).
However, although both channel types display voltage dependence, there
was a marked difference in their sensitivity to
Ca2+ and voltage. This is clearly
seen in Fig. 3, A and
C, where, at a
[Ca2+]i
of 50 nM, BK channels recorded from labor tissue are more active at
negative voltages, and the increase in activity on depolarization is
much more marked than for the corresponding
BKCa channels from nonlabor
tissue. In addition, under this recording configuration, consistently
higher numbers of channels were observed in patches obtained from labor
myometrium. Analysis of
NPo at this
concentration of Ca2+ indicated a
significant difference between BK and
BKCa channels at negative and
positive membrane potentials. For example, at a membrane potential of
20 mV, mean values for
NPo were 0.31 ± 0.14 (n = 4) and 0.02 ± 0.001 (n = 6) for BK and
BKCa channels, respectively
(P < 0.05), and at +20 mV the
corresponding NPo
values were significantly greater for BK channels (1.47 ± 0.34, n = 4) than for
BKCa channels (0.16 ± 0.03, n = 6, P < 0.05).

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Fig. 3.
BK and BKCa channel activity
recorded from outside-out patches. A:
representative records of BKCa
(left) and BK (right)
channel activity in presence of 50 nM
Ca2+. Arrowheads, closed state of
channel; inward current is shown as downward deflections.
B: single channel current-voltage
(I-V) relationships for
BKCa ( ) and BK ( ) channels.
Conductances of these channels determined from slope of line fitted to
data by linear regression were 207 and 214 pS, respectively.
C: channel activity
(NPo)-V
relationship. Activity increased dramatically after patch
depolarization for BK channel ( ) compared with
BKCa channel ( ).
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Effects of intracellular
Ba2+.
Application of Ba2+ (2-10 mM)
to the intracellular aspect of inside-out membrane patches obtained
from nonlabor myometrial cells (n = 7)
resulted in blockade of BKCa
channel activity at depolarized membrane potentials (Fig.
4A)
characterized by the appearance of long-lived closed periods of several
seconds duration. The block by
Ba2+ is strongly voltage
dependent, with only brief channel openings at positive potentials,
whereas considerable channel activity is apparent on hyperpolarization,
although this is accompanied by a slight, but insignificant, reduction
in unitary current amplitude at all voltages in the presence of 10 mM
Ba2+ (Fig.
4B). A plot of
Po vs. voltage in
the absence and presence of 10 mM
Ba2+ shows that
Ba2+ caused an inversion of this
relationship (Fig. 4C). Thus few channel openings occurred at +50 mV in the presence of
Ba2+
[Po = 0.02 ± 0.002 (n = 4)
compared with Po = 0.42 ± 0.33 in the absence of
Ba2+], but
Po increased as
the patch was hyperpolarized to
50 mV [Po = 0.33 ± 0.27 (n = 4) compared with
Po = 0.04 ± 0.002 (n = 4) in the absence of
Ba2+]. The effects of
Ba2+ were only partially
reversible on washing (data not shown). Although 2 mM
Ba2+ caused a reduction in channel
Po, it did not
result in the inversion of the
Po-voltage
relationship (n = 3; data not shown).
BK channels recorded from inside-out patches obtained from labor
myometrium were much less sensitive to block by internal
Ba2+. Neither single channel
amplitude nor Po
of the BK channel was affected by 5 mM
Ba2+
(n = 5); however, at 10 mM
Ba2+, depression of unitary
current amplitude (n = 3) was evident (Fig.
5A).
This was observed only at depolarized potentials where inward
rectification was induced, characterized by a reduction in single
channel conductance (over the range 0 to +50 mV) from 233 pS in the
absence of Ba2+ to 136 pS in the
presence of 10 mM Ba2+ (Fig.
5B). In addition, 10 mM
Ba2+ induced a flickery block of
BK channel activity at depolarized potentials (Fig.
5A), and this was associated with a
reduction in Po,
whereas there was no significant effect of 10 mM
Ba2+ at negative membrane
potential (Fig. 5C). The effects of
external Ba2+ were not studied on
either channel.

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Fig. 4.
Effects of intracellular Ba2+ on
single BKCa currents from nonlabor
myocytes. A: representative record of
BKCa channel activity recorded
from an inside-out patch in 50 nM
Ca2+; channel activity increased
as membrane potential was made positive. Po were as
follows: 0.34 at +40 mV, 0.23 at +20 mV, 0.05 at 20 mV, and 0.02 at 40 mV. Ba2+ (5 mM)
blocked BKCa channels in a
voltage-dependent manner. Note long closed states at positive voltages
in presence of Ba2+.
Po were as
follows: 0.03 at +40 mV, 0.06 at +20 mV, 0.14 at 20 mV, and 0.29 at 40 mV. Data shown without and with
Ba2+ were recorded from the same
patch. Arrowheads, closed state of channel; inward current is shown as
downward deflections. B: single channel I-V relationship for
BKCa channel of pregnant human
uterine myocytes. Note reduction in single channel current with 10 mM Ba2+ ( ) compared with control
( ) recorded from the same patch. C: Po-V
relationship in presence of 50 nM intracellular
Ca2+ for
BKCa with ( ) and without 10 mM
Ba2+ ( ).
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Fig. 5.
Effects of Ba2+ on BK channels
from labor myocytes. A: representative
records of BK channel activity from an inside-out patch in presence of
50 nM Ca2+ without
(top) and with
(bottom) 10 mM
Ba2+.
Po were as
follows: 0.74 at +40 mV and 0.64 at 40 mV without Ba2+ (control) and 0.27 at +40 mV
and 0.61 at 40 mV with
Ba2+. Arrowheads, closed state of
channel; inward current is shown as downward deflections.
B: single channel
I-V relationship for BK channel shows
that 10 mM Ba2+ ( ) decreased
unitary current amplitude at positive but not negative voltages
compared with control ( ). C:
Po-V
relationship for BK channel without
Ba2+ ( ) and with 5 mM ( ) and
10 mM Ba2+ ( ).
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4-AP sensitivity of BK and BKCa channels.
In inside-out patch recordings from nonlabor myometrial cells, with 0.5 µM Ca2+ in the bathing solution
to maintain a high level of channel activity, application of 0.1 mM
4-AP was without effect on BKCa
current amplitude (data not shown) but slightly decreased
Po
(n = 5; Fig. 6A).
Raising the concentration of 4-AP to 1 mM resulted in a significant reduction in the
Po of the
BKCa channel at all voltages
examined (n = 4 of 7; Fig.
6A). In contrast, 4-AP (0.1-1
mM) applied directly to the intracellular aspect of patches excised
from labor myometrial cells had no effect on the single channel current
amplitude or Po
of BK channels (n = 4; Fig.
6B).

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Fig. 6.
Effects of 4-aminopyridine (4-AP) on
BKCa and BK channels.
A, top: representative records of
BKCa channel activity from an inside-out patch at a membrane potential of +50 mV with 0.5 µM Ca2+. Top
trace, control data
(Po = 0.61);
bottom trace, with 1 mM 4-AP
(Po = 0.14). 4-AP
(1 mM) had no effect on single channel amplitude. A,
bottom:
Po-V
relationship with 0.1 ( ) and 1 mM ( ) 4-AP compared with control
( ). B, top: representative records of BK channel activity from an inside-out patch under conditions in A. 4-AP (1 mM) had no effect
on BK channel current amplitude or
Po in labor
myometrium. Po
were as follows: 0.87 and 0.84 for control and 4-AP, respectively.
B, bottom:
Po-V
relationship with 1 mM 4-AP ( ) compared with control ( ).
Arrowheads, closed state of channel; inward current is shown as
downward deflections.
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Effect of intracellular TEA.
Figure 7 illustrates the block of
BKCa channels when TEA was applied
to the cytoplasmic aspect of inside-out patches in the presence of 50 nM Ca2+ from nonlabor myometrium.
This ion had little effect at concentrations between 1 and 5 mM on
BKCa channels
(n = 10; data not shown), but
10-100 mM TEA (n = 4) decreased
unitary current amplitude of BKCa
channels in a concentration-dependent manner (Fig.
7A). The block was voltage dependent
and resulted in the appearance of inward rectification of the
current-voltage relationship at voltages positive to +20 mV. There was
no effect of TEA on single channel current amplitude at hyperpolarized
voltages. A plot of iTEA/iC
vs. [TEA] resulted in a
Kd of 45.7 ± 2.4 mM (Fig. 7B). Analysis, using
the Woodhull (29) method, of the reduction in current amplitude by TEA
of the BKCa channel (Fig.
7C) resulted in values of 64.8 ± 3.6 mM (n = 3) for
Kd(0 mV)
and 0.27 ± 0.03 (n = 3) for
,
suggesting that the TEA binding site experiences 27% of the electric
field and so is not strongly voltage dependent. An additional feature
of TEA action on BKCa channels was
observed at high (>20 mM) concentrations, where a noticeable increase
in channel activity (Fig. 7A) was
accompanied by an increase in the Po
(n = 4); however, this was not
investigated further in this study.

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Fig. 7.
Intracellular tetraethylammonium (TEA) sensitivity of
BKCa channel currents.
A: representative single channel
current records of BKCa channel
activity from an inside-out patch at a membrane potential of +40 mV in
presence of 50 nM intracellular
Ca2+. Actions of an ascending
series of TEA concentrations are shown. Arrowheads, closed state of
channel; inward current is shown as downward deflections.
B: relationship between percent
inhibition of single channel current amplitude
(iTEA/ic,
where iTEA and
ic are single
channel currents with and without TEA, respectively) and concentration
of applied TEA ([TEA]). Values are means ± SE of
3-5 separate patches. Estimated
Kd from this
analysis is 46 mM. C:
ln(i/iTEA 1)-V relationship at 50 mM
intracellular TEA for 3 separate inside-out patches. Slope gives a
value of 0.27 for location of blocking site within membrane electric
field ( ), and y-intercept gives a
value of 64.8 mM for
Kd at 0 mV
[Kd(0 mV)].
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In contrast, internal TEA blocked BK channels at much lower
concentrations (Fig. 8,
A and
B). In inside-out patches from labor myometrium, application of TEA (50 µM-2 mM) induced a
concentration-dependent reduction in single channel current amplitude
that was apparent at all voltages examined
(n = 11). The block was rapid and
reversible, characterized by a decrease in single channel current
amplitude, which was associated with an increase in open channel noise
indicative of rapid open channel block (2). The
Kd of internal
TEA block of the channel obtained from Fig.
8B is 53.0 ± 4.9 µM. Analysis of
the TEA block of BK currents (n = 3)
yielded an estimated
Kd(0 mV) of 163.9 ± 3.4 µM and a value for
of 0.013% (Fig.
8C), illustrating the voltage
independence of TEA block of the labor channel. This Kd(0 mV)
corresponds to an ~400-fold increase in the sensitivity of the BK
channel to internal TEA compared with its nonlabor counterpart BKCa
(P < 0.05). The
Kd(0 mV) of
163.9 ± 3.4 µM for the BK channel is significantly different
(P < 0.05) from the
Kd of 53.0 ± 4.9 µM, as are the
Kd(0 mV)
(65 mM) and Kd
(46 mM) for the BKCa channel.

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|
Fig. 8.
Intracellular TEA sensitivity of BK channel currents.
A: representative single channel
current records of BK channel activity from an inside-out patch at a
membrane potential of +40 mV in presence of 50 nM intracellular
Ca2+. Actions of an ascending
series of TEA concentrations are shown. Arrowheads, closed state of
channel; inward current is shown as downward deflections.
B: relationship between percent
inhibition of single channel current amplitude and concentration of
applied TEA. Values are means ± SE from 3 separate patches; where
no error bars are apparent, SE is within symbol. Estimated
Kd from this analysis is 53 µM. C:
ln(i/iTEA 1)-V relationship with 100 µM intracellular TEA. Slope gives a value of 0.013 for , and
y-intercept gives a value of 163 µM
for
Kd(0 mV).
|
|
Effect of extracellular TEA.
TEA inhibited current flow through
BKCa
(n = 9; Fig.
9A) and
BK (n = 6; Fig.
9B) channels in outside-out patches
from nonlabor and labor myometrial cells, respectively, at much lower
concentrations (100 µM-5 mM) than those required to block
BKCa channel activity from the
internal membrane surface. The inhibition produced by extracellular TEA
was flickery in nature and accompanied by increased channel noise in
the open state and, therefore, typical of fast block, which is observed
as a reduction in unitary current. The block was not strongly voltage
dependent and was fully reversible. Figure
9C shows fractional inhibition of
channel activity for BKCa and BK
channels in the presence of TEA plotted as a function of the
extracellular [TEA]. It is clear from these data that the TEA block of both channel types is identical. The estimated
Kd values for
BKCa and BK channels were 423.0 ± 65.1 and 395.3 ± 58.4 µM
(P > 0.05), respectively, obtained
by fitting the curves to Eq. 2.

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|
Fig. 9.
Extracellular TEA sensitivity of
BKCa and BK channel currents.
Single channel current records are representative of outside-out patches obtained at a membrane potential of +30 mV and with 50 nM
Ca2+ bathing intracellular aspect
of membrane. A: recordings of
BKCa channel activity without TEA
(control, top trace;
Po = 0.17), with
100 µM TEA (middle trace;
Po = 0.15), and
with 1 mM TEA (bottom trace;
Po = 0.09).
B: recordings of BK channel activity
without TEA (control, top trace;
Po = 0.41), with
100 µM TEA (middle trace; Po = 0.47), and
with 1 mM TEA (bottom trace;
Po = 0.36).
Arrowheads, closed state of channel; inward current is shown as
downward deflections. C: relationship
between percent inhibition of single channel current amplitude and
concentration of applied TEA for
BKCa ( ) and BK ( ) channels.
Values are means ± SE from 4 separate patches; where no error bars
are apparent, SE is within symbol. Estimated
Kd from this
analysis are 423 and 395 µM for
BKCa and BK channels, respectively.
|
|
Effect of ball peptide.
A synthetic peptide of 20 amino acids found at the amino terminus of
the Shaker B
K+ channel causes block of
BKCa channels when applied
intracellularly to inside-out patches of rat brain (6) and pig coronary
smooth muscle (23). In view of the observed differences in TEA and Ca2+ sensitivity between the two
channel types described in this study, the effect of the synthetic
20-mer ball peptide was tested, using inside-out patches, on BK and
BKCa channel activity. Application of 50 µM ball peptide inhibited the activity of single
BKCa channels, and this was
observed as a reduction in channel
Po (Table
1). An analysis of the channel dwell times
demonstrated that the channel's closed state was best fitted by two
exponentials. The mean closed times were significantly increased from
4.63 ± 1.41 to 9.98 ± 1.73 ms at +30 mV
(n = 3, P < 0.05) and from 14.55 ± 3.31 to 24.65 ± 3.48 ms at
30 mV
(P < 0.05). In some recordings of
longer duration, closures of BKCa
channels lasting several seconds were observed, which is in agreement
with the findings of Toro et al. (23) and Foster et al. (6) for this
peptide on smooth and skeletal muscle
BKCa channels, respectively. A
greater degree of blockade of BKCa
channels by ball peptide was observed on depolarization than on
hyperpolarization, confirming the voltage-dependent nature of the
block. Ball peptide had no effect on the single channel amplitude,
Po, or closed
times of the BK channel of labor myometrium (n = 2; data not shown).
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|
Table 1.
Effect of ball peptide on Po and channel closed time of
BKCa channel recorded from inside-out patches in
symmetrical (140 mM) K+
|
|
Effect of ChTX.
ChTX (50-100 nM) applied to the external aspect of outside-out
patches (n = 9) from nonlabor and
labor myometrial cells reduced BKCa and BK channel openings,
respectively (Table 2), whereas single
channel current amplitude remained essentially unaltered (data not
shown). The block did not exhibit voltage dependence for either channel
type. ChTX (100 nM) had no effect when applied to the intracellular
aspect of an inside-out patch having BK
(n = 2) or
BKCa
(n = 2) channels.
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|
Table 2.
Effect of ChTX on single large-conductance K+
channels recorded from outside-out patches from nonlabor and labor
myocytes
|
|
 |
DISCUSSION |
The observed changes in the Ca2+
and voltage sensitivity in addition to the altered sensitivity to
internal Ba2+, TEA, ball peptide,
and 4-AP, but not external TEA and ChTX, indicate modifications to the
intracellular control mechanisms of human myometrial
BKCa and BK channels during
pregnancy and labor. The voltage-dependent reduction in
BKCa channel
Po at positive membrane potentials by internal
Ba2+ has been reported for smooth
muscle (2, 15) and neuronal (21)
BKCa channels. It has been
suggested that Ba2+ blocks
BKCa channels by a slow block
mechanism (17), where Ba2+
entering the channel pore bind to a site located 80-95% of the way through the membrane (2, 17, 25). The inversion of the
Po-voltage
relationship of the myometrial
BKCa channel leads us to suggest
that an additional mechanism may be operating in which
Ba2+ may substitute for
Ca2+ in promoting
BKCa channel activity. However,
previous studies suggest that Ba2+
cannot mimic Ca2+ in activating
the channel (25), and this appears to be true for the BK channel, where
a small reduction in unitary current amplitude was apparent.
The marked contrast in 4-AP sensitivity between
BKCa (nonlabor tissue) and BK
(labor) channels may indicate some variation in the structure of these
large-conductance myometrial K+
channels in the region of the cytoplasmic aspect of S6, since studies
on cloned Kv channels and mutants have led to the suggestion that 4-AP
likely associates with regions of the putative cytoplasmic ends of
transmembrane segments S5 and S6, with the intracellular portion of S6
providing the binding site (10). Alternatively, 4-AP no longer has
access to its blocking site on the
-subunit of the channel, thereby
accounting for its lack of effect on the BK channel and suggesting a
change in channel architecture associated with labor.
The Kd values for
internal TEA inhibition of the
BKCa channel are 20-70 mM
compared with 100-300 µM for external TEA (13) and are mediated
by clearly distinct external and internal TEA binding sites. The
characteristics of external TEA block of both channels in human
myometrium are similar to those characterized in vascular myocytes
(12), chromaffin cells (30), and skeletal muscle (3, 26), in which the
flickering associated with the open state represents rapid association
and dissociation of TEA from its binding site.
The values obtained for block by internal TEA,
Kd(0 mV) of
65 mM and
of 0.27 for the myometrial
BKCa channel, are in close agreement with those for BKCa
channels of cultured rat muscle [Kd(0 mV) = 60 mM,
= 0.26 (3)] and chromaffin cells
[Kd(0 mV) = 24 mM,
= 0.10 (30)] and imply that TEA sensitivity is
conserved among certain BKCa
channels. The mechanism of internal TEA block of
BKCa channels is consistent with
the "quiet" fast block of these channels in rat skeletal muscle
(26). In contrast, the Kd of 53 µM and
Kd(0 mV) of
163 µM for internal TEA blockade of the BK channel of human labor
myometrial cells are closer to values obtained for
BKCa channels of cerebral artery
smooth muscle (0.83 mM) (27), rat synaptosomes (0.80 mM) (5), and
clonal anterior pituitary cells (0.08 mM) (28). Furthermore, the
voltage-independent nature of the block of BK channels (
= 0.013)
indicates that this TEA binding site is located outside the membrane
electric field. The observed block of
BKCa by ball peptide is in
accordance with findings supporting the existence of discrete yet
distinct receptor sites, which bind this peptide to produce short or
long blocks (6, 23). However, the apparent enhanced TEA sensitivity and
insensitivity to ball peptide of the BK channel points to the
involvement of alternative mechanisms by which this channel senses
intracellular cationic blockers.
In view of the clear pharmacological and physiological differences
between the BKCa and BK channel
described in the present study, we can only speculate as to the nature
of this variability. It is possible that association and dissociation
of regulatory proteins, e.g.,
-subunits (11), which can alter the
Ca2+ sensitivity of the
BKCa channel when expressed with
the
-subunit (16), maybe strong candidates in altering channel
properties. Changes in phosphorylation states may be possible
alternative mechanisms whereby
BKCa channel behavior can be
profoundly altered, as demonstrated in neurons (14) and cloned channels
(4); indeed, in the myometrium, protein kinase A can inhibit or
increase the activity of the BKCa
channel, depending on whether the tissue is from a pregnant or a
nonpregnant source (19).
In the third trimester of human pregnancy the maternal uterine
physiology begins to change to prepare itself for childbirth. Inasmuch
as myometrial contractions are
Ca2+ mediated, in view of the
extremely high sensitivity of the uterine BKCa channel to
[Ca2+]i
(8), any rise in
[Ca2+]i
would subdue excitability by the activation of the
BKCa channel conductance. However,
if the Ca2+ and voltage dependence
of the BKCa channel were uncoupled
(i.e., BK channel), the myometrium could still undergo relaxation but would be free from
[Ca2+]i
changes, thereby effectively dissociating
BKCa channel activation and
[Ca2+]i.
A high level of BK channel activity would tend to hyperpolarize the
cell, making it less excitable. During labor, however, the uterus is
required to undergo periods of intense relaxation followed by powerful
contractions. The likelihood that the BK channel by virtue of its large
conductance and Ca2+ insensitivity
may fulfill this role by providing a strong hyperpolarizing influence
contributing to the relaxation phases cannot be excluded. This is an
exciting phenomenon, since the BK channel does not appear to be
directly controlled by changes in
[Ca2+]i
once labor has commenced, as seen in cell-attached patches, but by some
unknown intrinsic factors. The switch in myometrial K+ channel properties, closely
associated with the progression to labor, is a dramatic and clear
illustration of the dynamic nature of ion channel regulation in
response to physiological adaptations.
 |
ACKNOWLEDGEMENTS |
We are grateful to the staff at The Rosie Maternity Hospital for
tissue collection.
 |
FOOTNOTES |
This work was supported by the Medical Research Council and Wellcome
Trust Grant 040806.
Present addresses: M. L. J. Ashford, Dept. of Biomedical Sciences,
University of Aberdeen, Institute of Medical Sciences, Foresterhill,
Aberdeen AB25 2ZD, UK; J. J. Morrison, Dept. of Obstetrics and
Gynaecology, University College London Medical School, 86-96 Chenies
Mews, London WC1E 6HX, UK.
Address for reprint requests: R. N. Khan, University Dept. of
Pharmacology, The University of Oxford, Mansfield Rd., Oxford OX1 3QT,
UK.
Received 18 March 1997; accepted in final form 8 July 1997.
 |
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