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Journal of Physiology (2001), 537.2, pp. 443-452
© Copyright 2001 The Physiological Society

1-Subunit of the Ca²⁺-activated K⁺ channel regulates contractile activity of mouse urinary bladder smooth muscle

Georgi V. Petkov, Adrian D. Bonev, Thomas J. Heppner, Robert Brenner *, Richard W. Aldrich * and Mark T. Nelson

	ABSTRACT

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1. The large-conductance calcium-activated potassium (BK) channel plays an important role in controlling membrane potential and contractility of urinary bladder smooth muscle (UBSM). These channels are composed of a pore-forming -subunit and an accessory, smooth muscle-specific, 1-subunit.
2. Our aim was to determine the functional role of the 1-subunit of the BK channel in controlling the contractions of UBSM by using BK channel 1-subunit 'knock-out' (KO) mice.
3. The -galactosidase reporter (lacZ gene) was targeted to the 1 locus, which provided the opportunity to examine the expression of the 1-subunit in UBSM. Based on this approach, the 1-subunit is highly expressed in UBSM.
4. BK channels lacking 1-subunits have reduced activity, consistent with a shift in BK channel voltage/Ca²⁺ sensitivity.
5. Iberiotoxin, an inhibitor of BK channels, increased the amplitude and decreased the frequency of phasic contractions of UBSM strips from control mice.
6. The effects of the 1-subunit deletion on contractions were similar to the effect of iberiotoxin on control mice. The UBSM strips from 1-subunit KO mice had elevated phasic contraction amplitude and decreased frequency when compared to control UBSM strips.
7. Iberiotoxin increased the amplitude and frequency of phasic contractions, and UBSM tone of UBSM strips from 1-subunit KO mice, suggesting that BK channels still regulate contractions in the absence of the 1-subunit.
8. The results indicate that the 1-subunit, by modulating BK channel activity, plays a significant role in the regulation of phasic contractions of the urinary bladder.

	INTRODUCTION

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Large-conductance calcium-activated K⁺ (BK) channels are present in many types of smooth muscle, including urinary bladder smooth muscle (UBSM) (Klöckner & Isenberg, 1985; Brading, 1992; Markwardt & Isenberg, 1992; Heppner et al. 1997; Herrera et al. 2000). These channels are activated by membrane depolarization and by intracellular Ca²⁺, and are blocked with high affinity by the scorpion venom toxin, iberiotoxin (Galvez et al. 1990; Suarez-Kurtz et al. 1991). As the only member of the voltage-dependent potassium channel family that is activated by both voltage and Ca²⁺, the BK channel is uniquely suited to serve as a Ca²⁺-voltage signal integrator in the modulation of membrane excitability. In UBSM, BK channels have been demonstrated to play an important role in controlling membrane potential and excitability (Heppner et al. 1997), and to regulate contraction and relaxation (Suarez-Kurtz et al. 1991; Herrera et al. 2000).

UBSM exhibits spontaneous action potentials (Brading, 1992; Heppner et al. 1997; Hashitani et al. 2001), which determine the phasic nature of the contractions in this tissue. Ca²⁺ entry through dihydropyridine-sensitive, voltage-dependent Ca²⁺ channels (VDCCs) (Klöckner & Isenberg, 1985; Brading, 1992; Nakayama & Brading, 1993; Hashitani et al. 2001) is responsible for the upstroke of the action potential, and gives rise to phasic contractions in UBSM. The repolarization phase of the UBSM action potential is mediated in part by BK channels (Heppner et al. 1997). Blocking BK channels with iberiotoxin prolongs the action potential, causes membrane potential depolarization (Heppner et al. 1997), and increases the amplitude but decreases the frequency of phasic contractions of guinea-pig UBSM strips (Herrera et al. 2000). Iberiotoxin also increases the average force in these preparations (Suarez-Kurtz et al. 1991; Herrera et al. 2000). Thus, BK channels, by modulating membrane excitability and Ca²⁺ entry through VDCCs, play an important role in regulating UBSM contractions.

The BK channel is composed of a pore-forming -subunit (Knaus et al. 1994b; Garcia-Calvo et al. 1994) and, in some tissues, of an accessory -subunit (Knaus et al. 1994a; Tanaka et al. 1997). Four different -subunits with tissue-specific distribution have been identified (Knaus et al. 1994a; McManus et al. 1995; Riazi et al. 1999; Wallner et al. 1999; Behrens et al. 2000; Brenner et al. 2000a; Meera et al. 2000). The smooth muscle-specific 1-subunit was first identified by co-purification with the -subunit in bovine tracheal and aortic smooth muscle (Garcia-Calvo et al. 1994; Knaus et al. 1994a,b). Subsequent characterization of the interactions between 1- and -subunits showed that the 1-subunit affects the gating kinetics and the apparent Ca²⁺/voltage sensitivity of the BK channel (McManus et al. 1995; Nimigean & Magleby, 1999, 2000; Cox & Aldrich, 2000; Brenner et al. 2000a,b).

The role of the 1-subunit of the BK channel in UBSM is unknown. Since the 1-subunit elevates the Ca²⁺/voltage sensitivity of BK channels in other systems, the absence of the 1-subunit should decrease channel activity. Gene-targeting techniques have made it possible to engineer a strain of mice that lacks the 1-subunit of the BK channel (Brenner et al. 2000b). Deletion of the 1-subunit gene causes a decrease in the apparent Ca²⁺ sensitivity of BK channels in arterial myocytes, an increase in arterial tone and an elevation in blood pressure (Brenner et al. 2000b; see also Plüger et al. 2000). Unlike vascular smooth muscle, UBSM exhibits action potentials and phasic contractions. In UBSM, small changes in BK channel activity are likely to have significant effects on action potentials and related phasic contractions. We hypothesized that 'knocking out' the smooth muscle 1-subunit gene would decrease the apparent Ca²⁺/voltage sensitivity of BK channels in UBSM, thereby decreasing BK channel activity. Since BK channels per se would remain functional even in the absence of the 1-subunit, removing the 1-subunit may have an effect similar to partially blocking BK channels with iberiotoxin.

We found that the 1-subunit is highly expressed in UBSM. BK channels in UBSM cells from 1-subunit 'knock-out' (KO) mice have a reduced open probability compared to control mice, consistent with measurements from arterial smooth muscle cells (Brenner et al. 2000b). The amplitude of phasic contractions is elevated and contraction frequency is lower in UBSM strips from 1-subunit KO mice compared to control mice, and this effect is similar to that of iberiotoxin on UBSM strips from control mice. Iberiotoxin increased phasic contractions and tone of UBSM strips from 1-subunit KO mice, indicating that BK channels still contribute to the regulation of UBSM function in the absence of the 1-subunit. The results indicate that the 1-subunit, by modulating BK channel activity, plays a significant role in the regulation of phasic contractions of the UBSM, and therefore could be a potential drug target for modulating urinary bladder function.

	METHODS

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Generation of 1-subunit KO mice

To create a null allele of the 1 gene locus, the gene-targeting vector was constructed to delete the first coding exon, which encodes the first transmembrane domain of the 1 protein. The targeting vector was designed to insert a -galactosidase reporter (lacZ gene) in translational frame with the 1-subunit translation initiation site, and thus report transcription from the 1 gene promoter. An antisense RNA probe encompassing the amino terminal coding region was used as a probe against RNA extracted from 1 knockout and control tissue to confirm the disruption of the 1 gene. The 1-subunit KO mice generation is described in detail elsewhere (Brenner et al. 2000b).

Tissue preparation

Control mice (obtained from strains 129svj and C57BL) and 1-subunit KO mice were used in the experiments. Adult mice of either sex (25-35 g) were killed with pentobarbitone sodium overdose (0.2 ml), followed by exsanguination. This procedure was reviewed and approved by the Office of Animal Care Management at the University of Vermont.

The entire urinary bladder was removed and placed in ice-cold physiological saline solution (PSS, see later for composition). Following this rinse in PSS, the bladder was pinned to the bottom of a Sylgard-coated Petri dish containing dissection solution (see later for composition). After removing the surrounding adipose and connective tissue, the bladder was cut open with a longitudinal incision beginning from the urethral orifice. The mucosal surface of the bladder was removed and the bladder pinned serosal side up for dissection.

Contractility studies

Small muscle strips (100-300 µm wide and 1-3 mm long) were cut free from the bladder wall and transferred to a small Petri dish containing dissection solution. Miniature aluminium clips were placed at each end of the muscle strip to allow mounting of the strip in a tissue bath. Individual strips were placed in thermostatically controlled (37 °C) tissue baths (2 ml volume). One end of the strip was attached to a stationary metal hook while the other end was connected to a force-displacement transducer (model BG-10G; Kulite Semiconductor Products, Inc., Leonia, NJ, USA) for isometric tension recording. The force generation by the muscle strips was recorded on a computer-based data acquisition system (Axotape; Axon Instruments, Union City, CA, USA), and a chart recorder. The strips were suspended under a 1 mN tension. These procedures were carried out in a nominally Ca²⁺-free dissociation solution. Five minutes later the bath solution was replaced with a Ca²⁺-containing PSS to initiate contractions. There was a 60-90 min equilibration period. During this period the bath solution was changed every 5-10 min.

Cell isolation

To dissociate UBSM cells, three to four tissue strips were initially placed in 2 ml of dissociation solution (see later for composition) containing 1 mg ml^-1 papain (Worthington, Lakewood, NJ, USA), 1 mg ml^-1 dithioerythreitol and 1 mg ml^-1 bovine serum albumin (Sigma). The tissue pieces were incubated at 37 °C for 25 min, after which they were transferred to a solution containing 1 mg ml^-1 collagenase XI (Sigma), 1 mg ml^-1 bovine serum albumin, 1 mg ml^-1 trypsin inhibitor and 100 µM Ca²⁺, for 6-7 min. Following the incubation, the digested tissue was washed several times in dissociation medium and then was gently triturated to yield single smooth muscle cells. Several drops of the solution containing the dissociated cells were then placed in a recording chamber. Cells were allowed to adhere to the bottom of the recording chamber for 20-30 min prior to recording. Most cells were elongated and had a bright, shiny appearance when examined using phase-contrast microscopy.

Electrophysiological measurements

Single BK channel currents were recorded in inside-out patches using the patch-clamp technique (Hamill et al. 1981). The patches were exposed to a 10 µM Ca²⁺ buffered solution. We used 10 µM Ca²⁺ to approximate [Ca²⁺]_i levels during the peak of the action potential and during a Ca²⁺ spark. Single-channel currents were recorded over 2-10 min at steady potentials of -80 to +100 mV at room temperature (22 °C). Pipettes were pulled from borosilicate glass (Sutter Instruments, Novato, CA, USA) using a Narishige PP-83 vertical puller, coated with sticky dental wax to reduce capacitance, and polished with a Narishige MF-83 fire polisher to give a final tip resistance of approximately 5-8 M. Currents were measured using an Axopatch 200 amplifier (Axon Instruments), and filtered using an eight-pole Bessel filter. Single-channel currents were low-pass filtered at 2 kHz and digitized at 10 kHz. Data were acquired using Axotape software and further analysed by pCLAMP software (Axon Instruments).

Histochemical staining of UBSM

Whole urinary bladders were fixed in phosphate-buffered saline (PBS) with 0.2 % glutaraldehyde, 2 mM MgSO₄ and 2.5 mM EGTA, for 5 min, and then washed twice for 5 min in PBS. The tissues were then incubated overnight (12 h) at 37 °C in 5-bromo-4-chloro-3-indolyl--D-galactosidase (X-gal) staining solution and stained for indolyl--D-galactosidase activity as described elsewhere (Hogan et al. 1994).

Solutions and drugs

PSS was made daily and contained (mM): 119 NaCl, 4.7 KCl, 24 NaHCO₃, 1.2 KH₂PO₄, 2.5 CaCl₂, 1.2 MgSO₄ and 11 glucose, and aerated with 95 % O₂-5 % CO₂ to obtain pH 7.4. The dissection/ dissociation solution was Ca²⁺ free and contained (mM): 80 monosodium glutamate, 55 NaCl, 6 KCl, 10 glucose, 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid (Hepes) and 2 MgCl₂, pH adjusted to 7.3 with NaOH. The bath and pipette solution for inside-out patches contained (mM): 140 KCl, 10 Hepes, 5.047 MgCl₂ (1 Mg²⁺ free), 5-hydroxyethylethylene-diaminetriacetic acid (HEDTA) and 0.687 CaCl₂ (10 µM Ca²⁺-free), pH adjusted to 7.2 with NaOH. Atropine, carbachol, iberiotoxin, propranolol, phentolamine, suramin and tetrodotoxin were purchased from Sigma.

Statistics

Summary data are presented as means ± S.E.M. for n, the number of separate preparations or cells isolated from different animals. The last 5 min before iberiotoxin application (for the control) and the period from the 15th to the 20th minute after exposure to iberiotoxin were taken as the analysis periods. To compare the amplitude of phasic contractions, data were normalized to the 60 mM K⁺-induced response. Average force was determined by integrating the force generated by the phasic contractions over a 5 min period. Relative increase in tone was determined by dividing steady force in the presence of 100 nM iberiotoxin and 20 mM K⁺ or 1 µM carbachol by the steady force induced by 20 mM K⁺ or 1 µM carbachol. The data were assessed for statistical significance using Student's t test (paired or unpaired). A P value less than 0.05 was considered significant.

	RESULTS

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1-Subunit of the BK channels is enriched in UBSM

Having the lacZ gene targeted to the 1 locus provided the opportunity to examine directly the expression of the 1-subunit in UBSM (n = 3). Figure 1 illustrates intense lacZ staining in an isolated urinary bladder from a 1-subunit KO mouse, indicating that the 1-subunit is highly expressed in UBSM. Based on this approach, 1-subunit gene expression appeared to be restricted to smooth muscle (Brenner et al. 2000b; see also Garcia-Calvo et al. 1994; Knaus et al. 1994a; Jiang et al. 1999; Behrens et al. 2000; Brenner et al. 2000a). The body weight/ bladder weight ratio was not significantly different in the controls and 1-subunit KO mice (1080 ± 294 in controls, n = 6 and 1162 ± 192 in 1-subunit KO mice, n = 11; P > 0.05).

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Figure 1. Detection of lacZ gene expression from 1 gene-targeted mice Whole urinary bladders isolated from 1-subunit KO and control mice were stained with X-gal staining solution to identify lacZ gene expression (blue colour). The smooth muscle layer of the bladder from the 1-subunit KO mouse shows intense and uniform lacZ expression.

BK channels from 1-subunit KO mice have a reduced open probability

The 1-subunit increases the apparent Ca²⁺/voltage sensitivity of BK channels in heterologous expression systems (McManus et al. 1995; Nimigean & Magleby, 1999, 2000; Cox & Aldrich, 2000; Brenner et al. 2000a) and in cerebral artery smooth muscle (Brenner et al. 2000b). To determine how the 1-subunit affects BK channels in UBSM, the activity (open probability, P_o) of BK channels was examined in inside-out patches at a physiological resting membrane potential (-40 mV) for UBSM (Heppner et al. 1997; Hashitani et al. 2001) in myocytes freshly isolated from control and 1-subunit KO mice.

Figure 2 compares recordings of single BK channel currents in excised UBSM patches from 1-subunit KO and control animals, bathed in symmetrical 140 mM K⁺ solution. At -40 mV, BK channels from UBSM cells of control mice have a P_o of 0.128 ± 0.032 (n = 9) in the presence of 10 µM Ca²⁺. In contrast, BK channels from UBSM cells of 1-subunit KO animals had an open probability at least 40-fold lower at -40 mV (P_o = 0.00286 ± 0.00146; n = 9; P < 0.005). Membrane potential depolarization to +40 mV increased the open probability of BK channels from both control and KO mice, but the control BK channels still have a significantly higher open probability than the BK channels from 1-subunit KO mice (control, P_o = 0.648 ± 0.043, n = 9; KO, P_o = 0.410 ± 0.067, n = 11; P < 0.05; Fig. 2B).

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Figure 2. Voltage dependence of BK channel activity in UBSM myocytes from control and 1-subunit KO mice A, single-channel recordings from inside-out patches held at -40 and +40 mV in 10 µM free Ca2+. Arrows indicate the closed state of the channels. B, BK channel open probability in control and 1-subunit KO animals at two different voltages (-40 and +40 mV). Values are means ± S.E.M. Data were obtained from 9-11 cells of each animal group. *P < 0.05, **P < 0.005.

The BK channel unitary conductance was not affected in the 1-subunit KO mice, as determined from the slope of the single-channel current-voltage relationship, measured between -60 and +60 mV in symmetrical 140 mM K⁺ solution (Fig. 3A). The single-channel conductance was 214 ± 6 pS (n = 3-11) in control and 227 ± 3 pS (n = 3-12; P > 0.05) in 1-subunit KO mice. The density of BK channels was estimated in control and 1-subunit KO UBSM cells by determining the number of channels per patch. No significant difference was found between the average number of channels per patch excised from UBSM cells of control and 1-subunit KO mice (Fig. 3B), suggesting that the BK channel density is similar in UBSM cells from control and 1-subunit KO mice.

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Figure 3. Single BK channel conductance and channel density in UBSM cells from 1-subunit KO and control mice A, current-voltage relationship for the single BK channel conductance. Mean amplitude ± S.E.M. of the BK channel unitary current from 1-subunit KO and control mice is plotted versus the membrane potential. The standard error bars are present but are too small to be seen in the figure. B, mean number of channels per patch excised from UBSM cells from control and 1-subunit KO mice (n = 11).

1-Subunit regulates phasic contraction amplitude and frequency

We sought to investigate the basic contractile properties of UBSM strips isolated from 1-subunit KO mice and compare them to those of control animals. It is well known that UBSM contractions are modulated by neurotransmitters, released from autonomic nerves located in the bladder wall. To minimize any possible effects caused by neurotransmitter release, all experiments were performed in PSS containing blockers for known neurotransmitter receptors in the UBSM (atropine, 1 µM, a muscarinic antagonist; phentolamine, 1 µM, an -adrenergic antagonist; propranolol, 1 µM, a -adrenergic antagonist; suramin, 10 µM, a purinergic antagonist; and tetrodotoxin, 1 µM, a neuronal Na⁺ channel blocker; see Herrera et al. 2000). Atropine was excluded in the experiments with carbachol (see following text).

Blocking BK channels with iberiotoxin leads to an increase in the amplitude and a decrease in the frequency of phasic contractions in guinea-pig UBSM (Herrera et al. 2000), which caused an approximately 4-fold increase in average muscle force (Suarez-Kurtz et al. 1991; Herrera et al. 2000). The effect of iberiotoxin (100 nM) on spontaneous phasic contractions of UBSM strips from control mice was similar to the effect on guinea-pig UBSM (Suarez-Kurtz et al. 1991; Herrera et al. 2000), i.e. contraction amplitude increased and contraction frequency decreased (Fig. 4 and Fig. 5). Unlike guinea-pig UBSM, mouse UBSM strips often do not exhibit spontaneous phasic contractions. Therefore, depolarization with 20 mM K⁺ or muscarinic receptor activation with carbachol (1 µM) was used to induce phasic contractions (Fig. 4). Iberiotoxin (100 nM) increased the amplitude but decreased the frequency of phasic contractions (Fig. 4 and Fig. 5). Iberiotoxin (100 nM) also increased average force (force integral) of the 20 mM K⁺-induced phasic contractions 1.70 ± 0.11-fold (n = 5; P < 0.01) and of the carbachol (1 µM)-induced phasic contractions 1.81 ± 0.21-fold (n = 4; P < 0.05). In addition, iberiotoxin (100 nM) further increased the tone of UBSM strips bathed in 20 mM K⁺ or 1 µM carbachol 1.50 ± 0.05-fold (n = 10; P < 0.0001) and 1.28 ± 0.03-fold (n = 16; P < 0.0001), respectively.

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Figure 4. Original recordings of spontaneous, 20 mM K+-induced and 1 µM carbachol-induced phasic contractions of UBSM strips isolated from control mice and the effect of blocking BK channels with iberiotoxin Iberiotoxin (100 nM) increased the amplitude but decreased the frequency of the phasic contractions.

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Figure 5. Differences in phasic contractions of UBSM strips from 1-subunit KO and control mice A, comparison between 20 mM K+-induced phasic contractions of a UBSM strip from a control mouse (in the absence and presence of iberiotoxin) and from a 1-subunit KO mouse. B, differences in the phasic contraction frequency of UBSM strips from control mice, control mice treated with iberiotoxin (100 nM), and 1-subunit KO mice in response to 20 mM K+ and 1 µM carbachol. C, differences in the phasic contraction amplitude of UBSM strips from control mice, control mice treated with iberiotoxin (100 nM), and 1-subunit KO mice in response to 20 mM K+ and 1 µM carbachol. Values are means ± S.E.M. In C, data were normalized to the 60 mM K+-induced response. *P < 0.05, **P < 0.01.

Deletion of the 1-subunit gene lowers the activity of BK channels (see Fig. 2). This should have functional consequences that are similar to partially blocking BK channels with iberiotoxin. Indeed, UBSM strips from 1-subunit KO mice contract at a lower frequency than those from control mice (Fig. 5). Under stimulation either by 20 mM K⁺ or by carbachol (1 µM), the contraction frequency was lower in UBSM strips isolated from 1-subunit KO mice compared to the control mice (Fig. 5, see also Fig. 4 and Fig. 6). In the case of 20 mM K⁺-induced stimulation, the contraction frequency was 5.1 ± 0.7 contractions per minute (contr min^-1) in control mice (n = 5; Fig. 5B), 3.2 ± 0.4 contr min^-1 in control UBSM strips treated with iberiotoxin (100 nM; n = 5; P < 0.05 vs. control; Fig. 5B), and 2.5 ± 0.6 contr min^-1 in UBSM strips from 1-subunit KO mice (n = 6; P < 0.05 vs. control mice; Fig. 5B). The frequency of carbachol (1 µM)-induced phasic contractions was 4.6 ± 0.5 contr min^-1 in control UBSM strips (n = 10), 2.8 ± 0.3 contr min^-1 in control UBSM strips treated with iberiotoxin (100 nM; n = 10; P < 0.05 vs. control; Fig. 5B), and 2.8 ± 0.9 contr min^-1 in 1-subunit KO UBSM strips (n = 4; P < 0.05 vs. control mice; Fig. 5B).

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Figure 6. Original recordings of phasic contractions from UBSM strips isolated from 1-subunit KO mice and the effect of blocking BK channels with iberiotoxin Iberiotoxin (100 nM) increased the amplitude and frequency of the spontaneous 20 mM K+-induced and 1 µM carbachol-induced phasic contractions of UBSM strips isolated from 1-subunit KO mice.

UBSM strips from control and 1-subunit KO mice contract robustly in response to 60 mM K⁺. The amplitudes of the 60 mM K⁺-induced contractions were not significantly different (8.71 ± 0.95 mN, n = 16 in controls and 12.14 ± 1.65 mN, n = 16, P > 0.05, in 1-subunit KO mice). To compare the amplitude of the phasic contractions in control and 1-subunit KO mice, phasic contraction amplitudes were normalized to the 60 mM K⁺-induced contraction. The amplitude of the phasic contractions induced by 20 mM K⁺ and carbachol (1 µM) was significantly greater in 1-subunit KO mice compared to control mice, normalized to the amplitude of a 60 mM K⁺-induced contraction (Fig. 5C). The normalized amplitude of 20 mM K⁺-induced phasic contractions was 0.21 ± 0.04 in UBSM strips from control mice (n = 5) and 0.46 ± 0.08 in UBSM strips from 1-subunit KO mice (n = 6; P < 0.05; Fig. 5C). For comparison, the normalized amplitude of the 20 mM K⁺-induced contractions after iberiotoxin (100 nM) treatment was significantly higher than the control but similar to the amplitude of the 20 mM K⁺-induced contractions in UBSM strips from 1-subunit KO mice (Fig. 5C). In the case of carbachol (1 µM)-induced stimulation, the normalized contraction amplitude was 0.10 ± 0.02 in control mice (n = 7) and 0.45 ± 0.14 in 1-subunit KO mice (n = 4; P < 0.01; Fig. 5C). In the presence of iberiotoxin (100 nM), the normalized amplitude of the carbachol (1 µM)-induced contractions was again significantly higher than the control but similar to the contraction amplitude of UBSM strips from 1-subunit KO mice (Fig. 5C). These results indicate that the 1-subunit of BK channels modulates phasic contractions in UBSM by increasing contraction frequency and decreasing contraction amplitude.

BK channels still regulate UBSM contractility in the absence of the 1-subunit

BK channel activity is greatly reduced at the level of the resting membrane potential (-40 mV) in UBSM cells from 1-subunit KO mice (see Fig. 2). However, membrane depolarization decreased the difference in the open probability of BK channels between control and 1-subunit KO mice (see Fig. 2). Since UBSM undergoes dynamic depolarization-repolarization cycles during action potentials, it is likely that BK channels might still have some influence on phasic contractions of UBSM in the absence of the 1-subunit. To examine this possibility, the effect of iberiotoxin was tested on phasic contractions of UBSM strips from 1-subunit KO mice.

Iberiotoxin (100 nM) increased the amplitude and frequency of spontaneous 20 mM K⁺- and carbachol (1 µM)-induced phasic contractions of UBSM strips from 1-subunit KO mice (Fig. 6). Iberiotoxin (100 nM) increased the frequency of 20 mM K⁺-induced contractions from 2.5 ± 0.6 to 6.8 ± 1.9 contr min^-1 (n = 6; P < 0.05), whereas carbachol (1 µM)-induced contractions were increased from 2.8 ± 0.9 to 6.9 ± 2.1 contr min^-1 (n = 4; P < 0.05). Normalized contraction amplitude in response to 20 mM K⁺-induced stimulation was increased from 0.46 ± 0.08 to 0.66 ± 0.05 (n = 6; P < 0.05) by iberiotoxin (100 nM). Iberiotoxin (100 nM) increased average force (force integral) of the 20 mM K⁺-induced phasic contractions 3.6 ± 0.7-fold (n = 4; P < 0.05) and of the carbachol (1 µM)-induced phasic contractions 1.66 ± 0.11-fold (n = 4; P < 0.05). In 1-subunit KO mice, iberiotoxin (100 nM) further increased the tone of UBSM strips bathed in 20 mM K⁺ or 1 µM carbachol 1.56 ± 0.13-fold (n = 7; P < 0.005) and 1.32 ± 0.10-fold (n = 6; P < 0.05), respectively. These results are consistent with the idea that BK channels still contribute to the regulation of UBSM contractions in the absence of the 1-subunit.

	DISCUSSION

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The 1-subunit of the BK channel appears to be exclusively expressed in smooth muscle (Garcia-Calvo et al. 1994; Knaus et al. 1994a; Tanaka et al. 1997; Brenner et al. 2000a,b). UBSM is no exception, as the intense lacZ staining observed in urinary bladder (Fig. 1) indicates that the 1-subunit is expressed abundantly in this tissue. The 1-subunit appears to enhance the apparent voltage/Ca²⁺ sensitivity of the BK channel (McManus et al. 1995; Nimigean & Magleby, 1999, 2000; Cox & Aldrich, 2000; Brenner et al. 2000b). Consistent with a previous study on vascular smooth muscle (Brenner et al. 2000b), the present results indicate that in UBSM cells, BK channels lacking 1-subunits have a much lower open probability at a given membrane potential and Ca²⁺ concentration. At voltages close to the resting membrane potential of UBSM (-40 mV), BK channel P_o was at least 40-fold lower in UBSM cell membrane patches from 1-subunit KO than control mice (Fig. 2). At voltages close to the peak of the UBSM action potential, the difference in P_o between 1-subunit KO and control mice was much lower (< 2-fold).

The 1-subunit regulates phasic contraction amplitude and frequency of UBSM

A major finding presented here is that the BK channel 1-subunit contributes significantly in shaping the phasic contractions of the UBSM. In UBSM, a phasic contraction reflects an elevation of Ca²⁺ entry through VDCCs caused by a burst of action potentials. The amplitude of a phasic contraction depends on the increase in Ca²⁺ entry caused by membrane depolarization. The frequency of phasic contractions should be a reflection of mechanisms that cause periodic, temporary cessation of action potentials. Blocking BK channels with iberiotoxin increases action potential duration and frequency, and causes membrane potential depolarization (Heppner et al. 1997), which elevates Ca²⁺ entry and increases phasic contraction amplitude (Suarez-Kurtz et al. 1991; Herrera et al. 2000). UBSM strips from 1-subunit KO mice also exhibited an elevation in phasic contraction amplitude, consistent with the idea that the 1-subunit is important for coupling Ca²⁺ signals to activation of BK channels. Thus, the lower P_o of BK channels lacking the 1-subunit results in a functional outcome, which is similar to blocking BK channels with iberiotoxin.

Blocking BK channels with iberiotoxin slows the frequency of UBSM phasic contractions of mouse (this study) and guinea-pig (Herrera et al. 2000). UBSM strips from 1-subunit KO mice exhibit a lower contraction frequency under basal conditions. It was initially surprising that blocking BK channels with iberiotoxin or disabling BK channels by 1-subunit deletion (this study), which should enhance excitability, decreases contraction frequency. Since the elevation of phasic contraction amplitude in response to iberiotoxin in UBSM strips (Heppner et al. 1997; Herrera et al. 2000) presumably reflects elevated Ca²⁺ entry through VDCCs, it is possible that the decrease in contraction frequency is a result of prolonged membrane depolarization or elevated intracellular Ca²⁺ accumulation during the phasic contraction. This could lead to voltage- or Ca²⁺-dependent inactivation of VDCCs and a prolonged quiescent period. Alternatively, prolonged Ca²⁺ accumulation could lead to activation of iberiotoxin-insensitive Ca²⁺-activated K⁺ channels (e.g. small conductance Ca²⁺-activated K⁺ channels or intermediate conductance Ca²⁺-activated K⁺ channels), which could cause a quiescent period, i.e. decreased contraction frequency. In guinea-pig UBSM, the slowing of contraction frequency by iberiotoxin is prevented by blocking the sarcoplasmic reticulum Ca²⁺ release channels with ryanodine (Herrera et al. 2000). This result suggests that the slowing effect on contraction frequency depends on sarcoplasmic reticulum Ca²⁺ loading (Herrera et al. 2000). Thus, it is possible that elevated sarcoplasmic reticulum Ca²⁺ release activates iberiotoxin-insensitive K⁺ channels or inactivates VDCCs directly to cause action potentials to cease and decrease contraction frequency. Nevertheless, the precise mechanism by which iberiotoxin or 1-subunit KO lowers contraction frequency remains to be elucidated.

In contrast to control mice, iberiotoxin increased contraction frequency of UBSM strips from 1-subunit KO mice (Fig. 6). It is possible that the process, which slows the contraction frequency in control strips, is already saturated in UBSM strips from 1-subunit KO mice, since contraction amplitude, and hence Ca²⁺ entry during the contraction, is already elevated. Therefore, further BK channel block with iberiotoxin would not slow contraction frequency in UBSM strips from 1-subunit KO mice.

The slowing effect of iberiotoxin on contraction frequency in guinea-pig and control mouse UBSM strips was less than the induced increase in contraction amplitude such that the force integral increased (Suarez-Kurtz et al. 1991; Herrera et al. 2000; and this study). In the 1-subunit KO mice, blocking BK channels with iberiotoxin increased phasic contraction amplitude and frequency as well as tone. Thus, BK channels associated with 1-subunits have a profound role in determining UBSM contractility.

BK channels still regulate UBSM contractility in the absence of the 1-subunit

Iberiotoxin still had a significant effect on phasic contractions of UBSM strips from 1-subunit KO mice (Fig. 6). In contrast, iberiotoxin had no effect on the diameter of pressurized arteries from 1-subunit KO mice (Brenner et al. 2000b) and on 30 mM K⁺-induced contractions of aortic strips (Plüger et al. 2000). Unlike vascular smooth muscle, UBSM exhibits action potentials and phasic contractions. It is likely that, even in the absence of the 1-subunit, the large membrane depolarization and subsequent elevation of intracellular Ca²⁺ concentration during the action potential activates BK channels to a level that contributes to the action potential repolarization. Thus, BK channels can still regulate UBSM phasic contractions even in the absence of the 1-subunit.

1-Subunit as a target for drugs to treat urinary incontinence

Urinary incontinence adversely affects the lifestyle of millions of people. An unstable or hyperactive bladder is one type of urinary incontinence that is associated with abnormal detrusor contractions resulting in involuntary leakage of urine. The cause of an unstable bladder is thought to lie within the UBSM (Brading, 1997). Current treatments for an unstable bladder are not very effective and have unwanted side effects. An understanding of the molecules involved in regulating UBSM contractions is crucial to developing new drugs for incontinence treatment and perhaps human gene therapies. In recent years, much effort has gone toward increasing our understanding of ion channels, such as ATP-sensitive K⁺ channels and BK channels, that play a significant role in regulating UBSM excitability and contractility (Brading, 1992; Heppner et al. 1997; Herrera et al. 2000; Petkov et al. 2001). It is believed that activation of these ion channels by certain drugs would decrease the excitability of the UBSM and might prove useful in the treatment of a hyperactive bladder. To develop ion channel therapeutics that are effective in controlling incontinence, the relationship between ion channel activation and functional effects must be clearly understood. Here, we used a combination of molecular and physiological approaches to study the role of the BK channel 1-subunit at the single channel level and correlated these findings with functional studies. The present study reveals a key role for the 1-subunit of the BK channels in the control of single-channel activity and related phasic contractions and tone of UBSM. Thus, the 1-subunit, which is highly expressed in the detrusor, could represent a new target of drugs in the treatment of an unstable bladder.

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Acknowledgements

The authors would like to thank Drs Gerald Herrera and David Hill-Eubanks for critical reading of the manuscript. This work was supported by National Institutes of Health Grants DK53832, HL44455 and HL63722 to M.T.N. and the Howard Hughes Medical Institute.

Corresponding author

M. T. Nelson: Department of Pharmacology, Given Building, Room B-303, 89 Beaumont Avenue, University of Vermont, College of Medicine, Burlington, VT 05405-0068, USA.

Email: mtnelson{at}zoo.uvm.edu

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