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
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Abstract
Introduction
Methods
Results
Discussion
References

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
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Abstract
Introduction
Methods
Results
Discussion
References

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.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

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 MOmega 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
<IT>P</IT><SUB>o</SUB> = <IT>I</IT>/(<IT>N</IT> ⋅ <IT>i</IT>)
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 (partial )
ln[(<IT>i</IT>/<IT>i</IT><SUB>TEA</SUB>) − 1] = (−<IT>z&dgr;FV</IT>/<IT>RT</IT>) + ln([TEA]/<IT>K</IT><SUB>d(0 mV)</SUB>) (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
<IT>i</IT><SUB>TEA</SUB> = <IT>i</IT><SUB>C</SUB>/1 + [TEA]/<IT>K</IT><SUB>d</SUB> (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(beta -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
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Abstract
Introduction
Methods
Results
Discussion
References

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.

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.

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 (open circle ) and BK (black-lozenge ) 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 (black-lozenge ) compared with BKCa channel (open circle ).

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+ (black-square) compared with control (bullet ) recorded from the same patch. C: Po-V relationship in presence of 50 nM intracellular Ca2+ for BKCa with (black-triangle) and without 10 mM Ba2+ (bullet ).


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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+ (black-square) decreased unitary current amplitude at positive but not negative voltages compared with control (bullet ). C: Po-V relationship for BK channel without Ba2+ (bullet ) and with 5 mM (black-triangle) and 10 mM Ba2+ (black-square).

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 (black-down-triangle ) and 1 mM (black-lozenge ) 4-AP compared with control (bullet ). 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 (black-lozenge ) compared with control (bullet ). Arrowheads, closed state of channel; inward current is shown as downward deflections.

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 partial , 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 (partial ), and y-intercept gives a value of 64.8 mM for Kd at 0 mV [Kd(0 mV)].

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 partial  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 partial , 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 (open circle ) and BK (black-triangle) 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
Top
Abstract
Introduction
Methods
Results
Discussion
References

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 alpha -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 partial  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, partial  = 0.26 (3)] and chromaffin cells [Kd(0 mV) = 24 mM, partial  = 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 (partial  = 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., beta -subunits (11), which can alter the Ca2+ sensitivity of the BKCa channel when expressed with the alpha -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.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

1.   Anwer, K., L. Toro, C. Oberti, E. Stefani, E., and B. M. Sanborn. Ca2+-activated K+ channels in pregnant rat myometrium: modulation by a beta -adrenergic agent. Am. J. Physiol. 263 (Cell Physiol. 32): C1049-C1056, 1992[Abstract/Free Full Text].

2.   Benham, C. D., T. B. Bolton, R. J. Lang, and T. Takewaki. The mechanism of action of Ba2+ and TEA on single Ca2+-activated K+ channels in arterial and intestinal smooth muscle cell membranes. Pflügers Arch. 403: 120-127, 1985[Medline].

3.   Blatz, A. L., and K. L. Magleby. Ion conductance and selectivity of single calcium-activated potassium channels in cultured rat skeletal muscle. J. Gen. Physiol. 84: 1-23, 1984[Abstract].

4.   Esguerra, M., J. Wang, C. D. Foster, J. P. Adelman, R. A. North, and I. B. Levitan. Cloned Ca2+-dependent K+ channel modulated by a functionally associated protein kinase. Nature 369: 563-565, 1994[Medline].

5.   Farley, J., and B. Rudy. Multiple types of voltage-dependent Ca-activated potassium channels of large conductance in rat brain synaptosomal membranes. Biophys. J. 53: 919-934, 1988[Abstract].

6.   Foster, C. D., S. Chung, W. N. Zagotta, R. W. Aldrich, and I. B. Levitan. A peptide derived from the Shaker B K+ channel produces short and long blocks of reconstituted Ca2+-dependent K+ channels. Neuron 9: 229-236, 1992[Medline].

7.   Green, K. A., R. W. Foster, and R. C. Small. A patch-clamp study of K+-channel activity in bovine isolated tracheal smooth muscle cells. Br. J. Pharmacol. 102: 871-878, 1991[Abstract].

8.   Khan, R. N., S. K. Smith, J. J. Morrison, and M. L. J. Ashford. Properties of large-conductance K+ channels in human myometrium during pregnancy and labor. Proc. R. Soc. Lond. B 251: 9-15, 1993[Medline].

9.   Kihira, M., K. Matsuzawa, H. Tokuno, and T. Tomita. Effects of calmodulin antagonists on calcium-activated potassium channels in pregnant rat myometrium. Br. J. Pharmacol. 100: 353-359, 1990[Abstract].

10.   Kirsch, G. E., and J. A. Drewe. Gating-dependent mechanism of 4-aminopyridine block in two related potassium channels. J. Gen. Physiol. 102: 797-816, 1993[Abstract].

11.   Knaus, H.-G., K. Folander, M. Garcia-Calvo, M.-L. Garcia, G. J. Kaczorowski, M. Smith, and R. Swanson. Primary sequence and immunological characterization of beta -subunit of high-conductance Ca2+-activated K+ channel from smooth muscle. J. Biol. Chem. 269: 17274-17278, 1994[Abstract/Free Full Text].

12.   Langton, P. D., M. T. Nelson, Y. Huang, and N. B. Standen. Block of calcium-activated potassium channels in mammalian arterial myocytes by tetraethylammonium ions. Am. J. Physiol. 260 (Heart Circ. Physiol. 29): H927-H934, 1991[Abstract/Free Full Text].

13.   Latorre, R., A. Oberhauser, P. Labarca, and O. Alvarez. Varieties of calcium-activated potassium channels. Annu. Rev. Physiol. 51: 385-399, 1989[Medline].

14.   Lee, K., I. C. M. Rowe, and M. L. J. Ashford. Characterization of an ATP-modulated large conductance Ca2+-activated K+ channel present in rat cortical neurones. J. Physiol. (Lond.) 488: 319-337, 1995[Abstract].

15.   McCann, J. D., and M. J. Welsh. Calcium-activated potassium channels in canine airway smooth muscle. J. Physiol. (Lond.) 372: 113-127, 1986[Abstract].

16.   McManus, O. B., L. M. H. Helms, L. Pallanck, B. Ganetzky, R. Swanson, and R. J. Leonard. Functional role of the beta -subunit of high conductance calcium-activated potassium channels. Neuron 14: 645-650, 1995[Medline].

17.   Miller, C., R. Latorre, and I. Reisin. Coupling of voltage-dependent gating and Ba++ block in the high-conductance, Ca++-activated K+ channel. J. Gen. Physiol. 90: 427-449, 1987[Abstract].

18.   Mirroneau, J., and J. P. Savineau. Effects of calcium ions on outward currents in rat uterine smooth muscle. J. Physiol. (Lond.) 302: 411-425, 1980[Abstract].

19.   Perez, G., and L. Toro. Differential modulation of large-conductance KCa channels by PKA in pregnant and nonpregnant myometrium. Am. J. Physiol. 266 (Cell Physiol. 35): C1459-C1463, 1994[Abstract/Free Full Text].

20.   Piedras-Renteria, E., E. Stefani, and L. Toro. Potassium currents in freshly dispersed myometrial cells. Am. J. Physiol. 261 (Cell Physiol. 30): C278-C284, 1991[Abstract/Free Full Text].

21.   Smart, T. G. Single calcium-activated potassium channels recorded from cultured rat sympathetic neurones. J. Physiol. (Lond.) 389: 337-360, 1987[Abstract].

22.   Toro, L., J. Ramos-Franco, and E. Stefani. GTP-dependent regulation of myometrial KCa channels incorporated into lipid bilayers. J. Gen. Physiol. 96: 373-394, 1990[Abstract].

23.   Toro, L., E. Stefani, and R. Latorre. Internal blockade of a Ca2+-activated K+ channel by Shaker B inactivating ball peptide. Neuron 9: 237-245, 1992[Medline].

24.   Tritthart, H. A., W. Mahnert, W., A. Fleischhacker, and N. Adelwohrer. Potassium channels and modulating factors of channel functions in the human myometrium. Z. Kardiol. 80: 29-33, 1991[Medline].

25.   Vergara, C., and R. Latorre. Kinetics of Ca2+-activated K+ channels from rabbit muscle incorporated into planar lipid bilayers---evidence for Ca2+ and Ba2+ blockade. J. Gen. Physiol. 82: 543-568, 1983[Abstract].

26.   Villaroel, A., O. Alvarez, A. Oberhauser, and R. Latorre. Probing a Ca2+-activated K+ channel with quaternary ammonium ions. Pflügers Arch. 413: 118-126, 1988[Medline].

27.   Wang, Y., and D. A. Mathers. Ca2+-dependent K+ channels of high conductance in smooth muscle cells from rat cerebral arteries. J. Physiol. (Lond.) 462: 529-545, 1993[Abstract].

28.   Wong, B. S., and M. Adler. Tetraethylammonium blockade of calcium-activated potassium channels in clonal anterior pituitary cells. Pflügers Arch. 407: 279-284, 1986[Medline].

29.   Woodhull, A. M. Ionic blockage of sodium channels in nerve. J. Gen. Physiol. 61: 687-708, 1973[Abstract/Free Full Text].

30.   Yellen, G. Ionic permeation and blockade in Ca2+-activated K+ channels of bovine chromaffin cells. J. Gen. Physiol. 84: 157-186, 1984[Abstract].


AJP Cell Physiol 273(5):C1721-C1731
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