Differential regulation of SK and BK channels by Ca2+ signals from Ca2+ channels and ryanodine receptors in guinea-pig urinary bladder myocytes

doi:10.1113/jphysiol.2002.017707

Differential regulation of SK and BK channels by Ca²⁺ signals from Ca²⁺ channels and ryanodine receptors in guinea-pig urinary bladder myocytes

Gerald M. Herrera and Mark T. Nelson

Departments of Molecular Physiology and Biophysics and Pharmacology, University of Vermont College of Medicine, Burlington, VT 05405, USA

ABSTRACT

Top
Abstract
Introduction
Methods
Results
Discussion
References

Small-conductance (SK) and large-conductance (BK) Ca²⁺-activated K⁺ channels are key regulators of excitability in urinary bladder smooth muscle (UBSM) of guinea-pig. The overall goal of this study was to define how SK and BK channels respond to Ca²⁺ signals from voltage-dependent Ca²⁺ channels (VDCCs) in the surface membrane and from ryanodine-sensitive Ca²⁺ release channels or ryanodine receptors (RyRs) in the sarcoplasmic reticulum (SR) membrane. To characterize the role of SK channels in UBSM, the effects of the SK channel blocker apamin on phasic contractions were examined. Apamin caused a dose-dependent increase in the amplitude of phasic contractions over a broad concentration range (10^-10 to 10^-6 M). To determine the effects of Ca²⁺ signals from VDCCs and RyRs to SK and BK channels, whole cell membrane current was measured in isolated myocytes bathed in physiological solutions. Depolarization (-70 to +10 mV for 100 ms) of isolated myocytes caused an inward Ca²⁺ current (I_Ca), followed by an outward current. The outward current was reduced in a dose-dependent manner by apamin (10^-10 to 10^-6 M), and designated I_SK. I_SK had a mean amplitude of 53.8 ± 6.1 pA or ~1.4 pA pF^-1 at +10 mV. The amplitude of I_SK correlated with the peak I_Ca. Blocking I_Ca abolished I_SK. In contrast, I_SK was insensitive to the RyR blocker ryanodine (10 µM). These data indicate that Ca²⁺ signals from VDCCs, but not from RyRs, activate SK channels. BK channel currents (I_BK) were isolated from other currents by using the BK channel blockers tetraethylammonium ions (TEA⁺; 1 mM) or iberiotoxin (200 nM). Voltage steps evoked transient and steady-state I_BK components. Transient BK currents have previously been shown to result from BK channel activation by local Ca²⁺ release through RyRs ('Ca²⁺ sparks'). Transient BK currents were inhibited by ryanodine (10 µM), as expected, and had a mean amplitude of 152.6 pA at +10 mV. The mean number of transient BK currents during a voltage step (range 0 to 3) correlated with I_Ca. There was a long delay (52.4 ± 2.7 ms) between activation of I_Ca and the first transient BK current. In contrast, ryanodine did not affect the steady-state BK current (mean amplitude 135.4 pA) during the voltage step. The steady-state BK current was reduced 95 % by inhibition of VDCCs, suggesting that this process depends largely on Ca²⁺ entry through VDCCs and not Ca²⁺ release through RyRs. These results indicate that Ca²⁺ entry through VDCCs activates both BK and SK channels, but Ca²⁺ release (Ca²⁺ sparks) through RyRs activates only BK channels.

(Resubmitted 25 January 2002; accepted after revision 21 February 2002)
Corresponding author M. T. Nelson: Department of Pharmacology, University of Vermont College of Medicine, Burlington, VT 05405, USA. Email: mtnelson{at}zoo.uvm.edu

INTRODUCTION

Top
Abstract
Introduction
Methods
Results
Discussion
References

The urinary bladder generates spontaneous phasic contractions that are triggered by action potentials (Brading, 1992). This myogenic activity provides the basis whereby, once stimulated by parasympathetic nerves, the bladder contracts forcefully to expel urine. The various phases of the urinary bladder smooth muscle (UBSM) action potential are mediated by the coordinated action of distinct conductances. The upstroke of the action potential is attributed to Ca²⁺ entry through dihydropyridine-sensitive, voltage-dependent Ca²⁺ channels (VDCCs). Blocking VDCCs abolishes action potentials and spontaneous contractions in UBSM (Creed et al. 1983; Klöckner & Isenberg, 1985a; Heppner et al. 1997; Herrera et al. 2000). The repolarization phase is mediated by the activity of both voltage-dependent K⁺ (K_V) channels and large-conductance Ca²⁺-dependent K⁺ (K_Ca) channels (BK channels) (Klöckner & Isenberg, 1985a; Heppner et al. 1997). Following the spike and repolarization, the action potential in UBSM displays a prolonged after-hyperpolarization (AHP) when the membrane potential is more negative than the resting potential (Creed et al. 1983; Heppner et al. 1997).

Pharmacological evidence suggests that the AHP is mediated by apamin-sensitive small conductance K_Ca (SK) channels. Apamin, a peptide from bee venom (Habermann, 1984), blocks SK channels with high affinity (Köhler et al. 1996; Shah & Haylett, 2000; Strøbæk et al. 2000). Creed and coworkers (1983) demonstrated that apamin (500 nM) blocked the AHP of action potentials triggered by field stimulation in rabbit UBSM. Fujii and colleagues (1990) reported similar findings that apamin (100 nM) attenuated the AHP in guinea-pig UBSM. Furthermore, apamin (1 µM) augments the amplitude of UBSM contractions evoked by 15 mM extracellular K⁺ (Zografos et al. 1992). Our laboratory has recently shown that a lower concentration of apamin (100 nM) greatly increases the amplitude of phasic contractions of guinea-pig UBSM (Herrera et al. 2000). The effects of apamin suggest that SK channels regulate UBSM function. However, currents through SK channels have not been identified or characterized in UBSM, and their regulation by Ca²⁺ signals is not known.

BK channels are also important in regulating UBSM cell function (Zografos et al. 1992; Heppner et al. 1997; Herrera et al. 2000, 2001). BK channels are activated by membrane potential depolarization and by increases in cytosolic [Ca²⁺] (Barrett et al. 1982; Cox et al. 1997). Thus, BK channels could potentially be activated by Ca²⁺ release from ryanodine receptors (RyRs) in the sarcoplasmic reticulum (SR) membrane in the form of Ca²⁺ sparks, as well as by Ca²⁺ entry through VDCCs. Since they are also Ca²⁺ dependent, RyRs may also be activated by Ca²⁺ entry through VDCCs, as in cardiac muscle (Cannell et al. 1995; Lopez-Lopez et al. 1995). In the present study, we examined the relationship between Ca²⁺ entry through VDCCs and spark-activated BK currents, which is not known.

The goals of this study were to characterize SK channels in UBSM and determine the communication of VDCCs and RyRs to BK and SK channels. Our results are consistent with 'loose coupling' of VDCCs to RyRs (Collier et al. 2000) and local, 'tight coupling' of RyRs to BK channels (Pérez et al. 1999; Herrera et al. 2001). Communication of RyRs to SK channels was not detected. Activation of SK channels and BK channels, both spark-activated and steady-state components were dependent on Ca²⁺ entry through VDCCs. These results support the concept that proximity of ion channel types plays a central role in UBSM physiology.

METHODS

Top
Abstract
Introduction
Methods
Results
Discussion
References

General

All procedures were reviewed and approved by the Office of Animal Care Management at the University of Vermont. Guinea-pigs (250-350 g) were killed by overdose with either halothane or isoflurane followed by exsanguination. The urinary bladder was placed in cold physiological saline solution (PSS, see below for composition). After this initial rinse in PSS, the bladder was placed in a dish containing dissection solution (DS, see below for composition). Small individual bundles of detrusor muscle (100-300 µm wide and 3-5 mm long) were cut away from the serosal surface of the bladder wall and stored in DS.

Isometric tension recording

The effects of the SK channel blocker apamin were assessed on spontaneous phasic contractions in strips of UBSM mounted in tissue baths superfused with aerated PSS (~1 ml volume, 95 % O₂ and 5 % CO₂, 37 °C). All experiments were performed in the presence of a cocktail containing neurotransmitter receptor antagonists and tetrodotoxin, which we have previously shown to inhibit nerve-mediated responses in this preparation (Herrera et al. 2000).

Urinary bladder smooth muscle cell isolation

Muscle bundles (10 to 15) were placed in a vial containing DS supplemented with 1 mg ml^-1 bovine serum albumin (BSA), 1 mg ml^-1 papain (Worthington Biochemical Corporation, Freehold, NJ, USA), and 1 mg ml^-1 dithioerythritol for 20 to 35 min at 37 °C. Next, the tissue was placed in fresh DS (37 °C) containing 1 mg ml^-1 BSA, 1 mg ml^-1 collagenase (either from Fluka, Milwaukee, WI, USA, or type II from Sigma) and 100 µM CaCl₂ for 5-10 min. Following enzyme treatment, the tissue was washed repeatedly with fresh BSA-containing DS, and then stored in this solution on ice. Individual cells were freed from the tissue by passing tissue bundles through the tip of a fire-polished Pasteur pipette.

Whole-cell voltage clamp recording

Smooth muscle cells prepared as above were kept in BSA-containing DS on ice until use, usually within 6 h. Cells plated in an experimental chamber were washed with fresh extracellular solution (see below for composition). The amphotericin-perforated patch technique was used to measure whole-cell currents (see Horn & Marty, 1988). For the cells used in the present study, resting membrane potential was -27.9 ± 1.3 mV, cell capacitance was 39.2 ± 1.9 pF and series resistance was 35.8 ± 3.7 M $Omega$ (n = 35 cells). In a subset of experiments, the standard extracellular solution was replaced by a solution containing an elevated K⁺ concentration, such that the K⁺ equilibrium potential shifted more positive (see below for composition). All experiments were performed at room temperature (22 °C).

Chemicals and solutions

PSS contained (mM): 119 NaCl, 4.7 KCl, 24 NaHCO₃, 1.2 KH₂PO₄, 2.5 CaCl₂, 1.2 MgSO₄, 0.023 ethylenediaminetetraacetic acid (EDTA) and 11 glucose. DS was made up of (mM): 80 monosodium glutamate, 55 NaCl, 6 KCl, 10 glucose, 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes), 2 MgCl₂, pH adjusted to 7.3 with NaOH. The standard extracellular (bath) solution used in electrophysiological recordings contained (mM): 134 NaCl, 6 KCl, 1 MgCl₂, 2 CaCl₂, 10 glucose, 10 Hepes, pH 7.4 with NaOH. The modified bath solution with an elevated K⁺ concentration contained (mM): 63 NaCl, 77 KCl, 1 MgCl₂, 2 CaCl₂, 10 glucose, 10 Hepes, pH 7.4 with NaOH. The intracellular (pipette) solution contained (mM): 110 potassium aspartate, 30 KCl, 10 NaCl, 1 MgCl₂, 10 Hepes, 0.05 ethyleneglycol-bis-N,N,N',N'-tetraacetic acid (EGTA), pH 7.2 with NaOH and supplemented with 200 µg ml^-1 amphotericin B. TEA, apamin, iberiotoxin and diltiazem were from Sigma. Ryanodine was from L.C. Laboratories (Woburn, MA, USA).

Calculations and statistics

Results are summarized as means ± S.E.M. Data were compared using Student's t test or one-way analysis of variance, where appropriate. The Student-Newman-Keuls method was used for all multiple comparisons. A P < 0.05 was considered statistically significant. Apamin dose-response curves were constructed for contraction experiments and electrophysiological recordings of apamin-sensitive currents. The data were fitted to the equation:

where A is either the contraction amplitude or the current amplitude, A_max and A_min are the maximum and minimum contraction or current amplitudes, EC₅₀ is the apamin concentration at half-maximum effect, X is the concentration of apamin and n is the Hill slope.

RESULTS

Top
Abstract
Introduction
Methods
Results
Discussion
References

Apamin-sensitivity of phasic contractions of urinary bladder smooth muscle

Although previous studies have shown that apamin causes an increase in UBSM contraction force (Zografos et al. 1992; Herrera et al. 2000), an evaluation of the apamin sensitivity of UBSM has not been performed. Of the three cloned SK channel subtypes (SK1-3), SK1 and SK3 channels were sensitive to nanomolar concentrations of apamin, while SK2 channels were sensitive to apamin concentrations in the subnanomolar range (see Köhler et al. 1996; Ishii et al. 1997; Vergara et al. 1998; Bond et al. 1999; Shah & Haylett, 2000; Strøbæk et al. 2000). Since the three SK channel isoforms exhibited differential apamin sensitivities, we postulated that the concentration dependence of the apamin response should provide clues as to which SK channel isoforms are present in the bladder.

Strips of UBSM undergoing spontaneous contractions were treated with a range of apamin concentrations to assess the functional importance of SK channels in this tissue (Fig. 1). In the absence of apamin, contraction amplitude was 2.16 ± 0.36 mN (n = 7). Contraction amplitude more than doubled at apamin concentrations above 10 nM (Fig. 1). Figure 1C shows the apamin-induced increase in contraction amplitude for a range of apamin concentrations fitted to obtain an EC₅₀ for apamin (see Methods). The fit yielded an EC₅₀ for apamin of 0.2 nM, with a 95 % confidence interval of 5 pM to 9 nM, and a Hill slope of 0.3. The broad apamin dose-response curve suggested that more than one SK isoform was responsible for the functional effects of apamin.

View larger version
[in this window]
[in a new window]

Figure 1. SK channels regulate contractility in UBSM
A, original recording of phasic contractions from a strip of UBSM. Apamin (10^-12 to 10^-7 M) was administered cumulatively. B, single contractions shown on an expanded time scale to clearly illustrate the augmented contractility following SK channel inhibition with increasing concentrations of apamin. Each contraction trace is the average of seven to nine individual contractions in a given condition. Con, control. The dotted line shows baseline tension (1 mN). C, normalized contraction amplitude at increasing concentrations of apamin (n = 7 preparations). D, summary of the effects of apamin (1 µM) on contraction frequency, duration and area of each contraction. Apamin increased contraction area ~2-fold with little effect on frequency or duration.

The frequency of spontaneous contractions was 1.5 ± 0.2 contractions min^-1 under control conditions (n = 7). At the highest concentration of apamin tested (1 µM), contraction frequency was slightly reduced to 74 % of control (Fig. 1D). Contraction duration was 6.1 ± 0.4 s under control conditions and was unaffected by apamin (Fig. 1D). Contraction integral (area) was 5.84 ± 0.89 mN s under control conditions and increased up to 2-fold in response to apamin (Fig. 1D). Thus, increases in contraction amplitude (and area) were observed over a range of apamin concentrations expected to inhibit SK2 (subnanomolar apamin) and SK1 or SK3 channels (nanomolar to micromolar apamin).

SK currents recorded from urinary bladder smooth muscle

Original recordings of SK currents in native tissue have been sparse (Koh et al. 1997; Vogalis & Goyal, 1997; Marrion & Tavalin, 1998; Hirschberg et al. 1999; Shah & Haylett, 2000). Most information on SK currents is derived from recombinant channels studied in heterologous expression systems (see Köhler et al. 1996; Ishii et al. 1997; Hirschberg et al. 1998; Vergara et al. 1998; Bond et al. 1999; Strøbæk et al. 2000). Recently, there have been several important reports correlating SK currents recorded in neuronal tissue with the molecular expression profile of SK channels in the same tissue (Stocker et al. 1999; Pedarzani et al. 2000; Wolfart et al. 2001). The only reports of SK currents recorded in native smooth muscle have been obtained in gastrointestinal smooth muscle preparations (Koh et al. 1997; Vogalis & Goyal, 1997).

To determine if electrophysiological evidence for SK channel expression could be obtained in UBSM cells, whole-cell membrane currents were recorded in freshly isolated myocytes using the perforated patch-clamp technique. A brief depolarization protocol was used, similar to the depolarization that occurs during the action potential. UBSM cells were held at -70 mV and then stepped to +10 mV for 100 ms (Fig. 2). This depolarization step evoked an inward current followed by an outward current (Fig. 2A 'control'). Much of this outward current was conducted by BK channels and consisted of transient and steady-state components (see Fig. 5 and Fig. 6). To isolate SK currents from BK currents, BK channels were blocked by tetraethylammonium ions (TEA⁺; 1 mM). TEA⁺ (1 mM) is an effective (5-fold higher than IC₅₀; see Langton et al. 1991; Volk et al. 1991), rapid, reversible and cost-effective blocker of BK channels and this concentration should have little effect on SK and K_V channels (Langton et al. 1991; Robertson & Nelson, 1994; Ishii et al. 1997; Jaggar et al. 1998a). To ensure that the current blocked by TEA⁺ was indeed conducted by BK channels and not K_V channels, the effect of 1 mM TEA⁺ was tested in the presence of the highly selective BK channel blocker, iberiotoxin (200 nM; Galvez et al. 1990). The mean outward current during a 100 ms depolarization from -70 to +10 mV in the presence of iberiotoxin was 35.2 ± 4.0 pA (n = 3). In the continued presence of iberiotoxin, TEA⁺ (1 mM) was without further effect, and the mean current during the depolarization pulse was 34.1 ± 4.6 pA (P = 0.86 vs. iberiotoxin). Thus, TEA⁺ at 1 mM reduced outward currents by blocking BK channels.

View larger version
[in this window]
[in a new window]

Figure 2. Apamin-sensitive currents in UBSM cells
A, whole-cell currents were recorded in UBSM cells during 100 ms depolarizations from -70 to +10 mV. Traces are original current recordings from a UBSM cell under control conditions, in the presence of TEA⁺ (1 mM), and apamin (1 nM and 1 µM). Substances were applied cumulatively with TEA⁺ added first, followed by increasing concentrations of apamin. The dotted line indicates zero current level. B, apamin-sensitive component (SK current) from A obtained by subtracting currents in the presence of apamin from current in the presence of TEA⁺. C, summary of the mean SK current, I_SK, obtained by averaging the entire apamin-sensitive current during the depolarization step for increasing apamin concentrations (n = 4 to 13 cells per concentration). D, the whole-cell I_SK reverses at E_K. Extracellular [K⁺] was 77 mM and intracellular [K⁺] was 140 mM (calculated E_K -15 mV). Cells were held at -70 mV, stepped to +10 mV for 50 ms and then stepped to potentials from 0 to -40 mV in 10 mV increments. The inset shows the apamin-sensitive tail currents from one UBSM cell. I_SK was measured ~3 ms after the voltage step and is plotted normalized to cell capacitance. The dotted line in the inset is zero current. n = 3 cells.

In the presence of TEA⁺, the SK channel blocker apamin was applied at concentrations from 0.1 nM to 1 µM (Fig. 2A). Figure 2B shows the apamin-sensitive currents ('SK current') for the recordings shown in Fig. 2A. The apamin-sensitive current was obtained by subtracting the current in the presence of apamin (with TEA⁺ present) from the current in the presence of TEA⁺ alone. The mean amplitude of SK current (I_SK) during the depolarization step was measured and was plotted in Fig. 2C over a range of apamin concentrations. The fit yielded an EC₅₀ for apamin of 10 nM, with a 95 % confidence interval of 40 pM to 3 µM and a Hill slope of 0.4. As with the contraction data, the broad apamin dose-response curve was consistent with more than one SK isoform contributing to the SK current.

K⁺ selectivity of apamin-sensitive currents in urinary bladder smooth muscle

To test the ionic selectivity of apamin-sensitive currents, a tail current protocol was utilized (Fig. 2D). UBSM cells were held at -70 mV and stepped to +10 mV for 50 ms to activate VDCCs and SK channels. Cells were then stepped to a series of test potentials from -100 to +10 mV in 10 mV increments and subsequent tail currents were recorded. BK channels were blocked with either TEA⁺ (1 mM) or iberiotoxin (200 nM). To determine the portion of the resulting tail currents that were conducted by SK channels, apamin (1 µM) was applied. In the standard bath solution containing 6 mM K⁺, the calculated equilibrium potentials for the major ions were +66 mV for Na⁺, -80 mV for K⁺ and -30 mV for Cl^-. Under these conditions, it was not possible to detect a reversal potential for the apamin-sensitive tail current. Apamin-sensitive currents were undetectable at potentials negative to -20 mV. At -10 mV, the amplitude of the apamin-sensitive current was 23.6 ± 4.0 pA (n = 4). However, when external K⁺ was elevated to 77 mM (E_K = -15 mV), apamin-sensitive currents reversed at -16.0 ± 2.6 mV (n = 3) (Fig. 2D). This reversal potential is very near the calculated equilibrium potential for K⁺. These results are consistent with the apamin-sensitive current being through K⁺ channels.

Ca²⁺ signalling requirements of SK currents in urinary bladder smooth muscle

The source of Ca²⁺ required for SK channel activation in UBSM is not known. Two likely sources of Ca²⁺ signals are VDCCs and RyRs. We have previously shown that Ca²⁺ sparks do not cause detectable activation of SK channels in guinea-pig UBSM cells, since transient outward currents are unaffected by the SK channel blocker apamin (Herrera et al. 2001). However, since depolarization could potentially evoke non-spark, ryanodine-sensitive Ca²⁺ release, the effects of blocking ryanodine receptors (RyRs) with ryanodine on I_SK were tested (Fig. 3B). I_SK was recorded after blocking BK channels with TEA⁺ (1 mM) in the absence (control) and presence of the RyR blocker ryanodine (10 µM) using the same voltage clamp protocol as before. In control cells (Fig. 3A), mean I_SK (determined in the presence of 1 µM apamin) at +10 mV was 53.8 ± 6.1 pA or 1.32 ± 0.14 pA pF^-1 (n = 14 cells) (Fig. 4B). Ryanodine at a concentration of 10 µM was applied for 20 min to block RyRs. We have previously shown that application of ryanodine in this manner is sufficient to inhibit Ca²⁺ sparks and associated transient BK currents in UBSM cells (Herrera et al. 2001). Blocking RyRs did not alter the amplitude of I_SK (Fig. 3B and Fig. 4B). To confirm that the results from the ryanodine experiments were not confounded by changes in the amplitude of I_Ca following ryanodine treatment, we measured peak inward current in the presence of 1 mM TEA⁺ (denoted I_Ca) in control cells and in cells treated with ryanodine (10 µM; Fig. 4C). I_Ca was not significantly altered by ryanodine at 10 µM.

View larger version
[in this window]
[in a new window]

Figure 3. SK channel activation requires Ca²⁺ entry through VDCCs
A, upper traces are currents recorded in a control cell (in the absence of Ca²⁺ channel blockers). Whole-cell currents were recorded during a 100 ms test pulse from -70 to +10 mV under control conditions in the presence of TEA⁺ (1 mM) and then following apamin (1 µM) in the continued presence of TEA⁺. SK current is shown in the lower trace. B, family of currents recorded in a cell treated with ryanodine to block RyRs. Ryanodine (10 µM) was added after the control record and then kept in the experimental bath for the duration of the experiment. Note a ryanodine-sensitive transient BK current occurred at the end of the control test pulse. TEA⁺ substantially reduced the outward current in the presence of ryanodine. TEA⁺ and apamin were applied as in A. C, whole-cell currents recorded in a cell treated with the VDCC blocker diltiazem. Diltiazem (50 µM) was added after the control record and the kept in the experimental bath for the remainder of the experiment. Diltiazem eliminated the inward I_Ca. TEA⁺ and apamin were applied as in A. Blocking VDCCs, but not RyRs, abolishes SK current. The dotted lines indicate zero current level.

View larger version
[in this window]
[in a new window]

Figure 4. Relationship between Ca²⁺ currents and I_SK
A, correlation between the mean SK current (I_SK; determined in the presence of 1 µM apamin) and peak Ca²⁺ current (I_Ca; determined in the presence of 1 mM TEA⁺) recorded during 100 ms test pulses from -70 to +10 mV. The relationship between I_Ca and I_SK suggests that Ca²⁺ entry through VDCCs activates SK channels. Blocking RyRs with ryanodine did not substantially alter the relationship between I_Ca and I_SK. n = 22 control cells and 10 cells treated with ryanodine. B, summary data showing mean I_SK under control conditions and in the presence of ryanodine, diltiazem or diltiazem and ryanodine. n = 14 cells, six cells, eight cells and four cells for control, ryanodine, diltiazem, and diltiazem and ryanodine, respectively. C, peak I_Ca at +10 mV was not significantly affected by ryanodine. NS, not significant. * P < 0.05 vs. control. † P< 0.05 vs. ryanodine.

Although blocking RyRs did not affect SK currents, inhibition of VDCCs with the reversible Ca²⁺ channel blocker diltiazem (50 µM, 10-20 min application) completely abolished the SK current (Fig. 3C and Fig. 4B). A correlation between I_Ca and SK current was observed (Fig. 4A). Ryanodine did not affect the relationship between I_Ca and I_SK (Fig. 4A). Furthermore, the delay between the start of the depolarization pulse and the peak of I_SK was 67.9 ± 2.2 ms (n = 14 cells), which may reflect the kinetics of the global Ca²⁺ transient. These observations indicate that SK channels require Ca²⁺ entry through VDCCs, but not Ca²⁺ release through RyRs, for activation.

Communication from VDCCs to RyRs and BK channels in urinary bladder smooth muscle: evidence for long distance signalling

BK channels are activated by both membrane potential depolarization and elevations in [Ca²⁺] (Barrett et al. 1982; Cox et al. 1997). During the UBSM action potential, BK channels could be activated by Ca²⁺ signals from VDCCs, RyRs or both, depending on the relative spatial organization of BK channels, VDCCs and RyRs. Local Ca²⁺ release events (Ca²⁺ sparks) caused by the opening of RyRs are effective activators of BK channels in smooth muscle including UBSM, suggesting that RyRs and BK channels are in very close proximity (Pérez et al. 1999; Jaggar et al. 2000; Herrera et al. 2001).

In contrast to the 'tight coupling' between RyRs (Ca²⁺ sparks) and BK channels in UBSM, recent evidence indicates that RyRs (Ca²⁺ spark sites) are distant from VDCCs in UBSM (Collier et al. 2000). Collier and coworkers (2000) observed that I_Ca evoked a small number of Ca²⁺ sparks, and there was a considerable delay between activation of I_Ca and the first Ca²⁺ spark. Consistent with their observations, we found that I_Ca induced a small number of transient BK currents (between 0 and 3 per 100 ms depolarization pulse) with a lag of 52.4 ± 2.7 ms (n = 56 measurements) between the start of depolarization (activation of I_Ca) and the peak of the first transient BK current (see Fig. 5A). This delay is too long to be attributed to local communication between VDCCs and RyRs, which in cardiac calcium-induced calcium release occurs in a matter of a few milliseconds (Cannell et al. 1995; Lopez-Lopez et al. 1995), and instead suggests that VDCCs and RyRs are spatially separated in UBSM.

View larger version
[in this window]
[in a new window]

Figure 5. Dependence of transient BK currents on Ca²⁺ entry through VDCCs
A, original currents recorded in response to 100 ms steps from -70 to +10 mV under control conditions (top trace) and in the presence the BK channel blocker TEA⁺ (1 mM; middle trace). The lower trace shows the TEA⁺-sensitive portion (BK current). The BK current consists of steady-state and transient Ca²⁺ spark-induced components. The lag between the start of depolarization (activation of VDCCs) and the first transient BK current is indicated. The dotted lines indicate zero current. B, histogram showing that the frequency of transient BK currents increases with larger I_Ca. Cells were grouped into three categories, depending upon the size of I_Ca. The average number of transient BK currents per 100 ms test pulse for each group is shown. * P < 0.05 vs. '0-25 pA'. † P < 0.05 vs. '25-50 pA'.

This type of long-distance communication between VDCCs and RyRs in UBSM was termed 'loose coupling' by Collier and coworkers (2000). These authors observed that the probability of a Ca²⁺ spark depended on the total flux of Ca²⁺ during a voltage step, rather than Ca²⁺ influx through a single VDCC as in heart (Collier et al. 2000). Consistent with this observation, the frequency of transient BK currents, which is an index of Ca²⁺ spark probability, increased with larger I_Ca (Fig. 5B). Furthermore, blocking VDCCs with diltiazem (50 µM, applied for 10-20 min) reduced the frequency of transient BK currents from 1.3 ± 0.1 to 0.5 ± 0.2 transient currents per 100 ms (P < 0.05). These findings suggest that Ca²⁺ spark sites are activated by the elevation in cytosolic [Ca²⁺] or SR [Ca²⁺], which follows Ca²⁺ entry through VDCCs during depolarization.

Dependence of BK channel activity on SR Ca²⁺ release, Ca²⁺ entry through VDCCs and membrane potential depolarization in urinary bladder smooth muscle

To examine the signals that activate BK channels, whole-cell BK currents were recorded under control conditions (n = 18 cells), in the presence of ryanodine (n = 6 cells), diltiazem (n = 8 cells) and both diltiazem and ryanodine (n = 4 cells) (Fig. 6). Under control conditions, BK currents consisted of transient and steady-state components (see Fig. 5A). Inhibiting RyRs with ryanodine (10 µM) blocked only the transient BK currents (Fig. 6A and B; see also Fig. 3B and Fig. 5A), which on average contributed 44.2 ± 4.6 % of the total BK current at +10 mV. A steady-state BK current remained in the presence of ryanodine (Fig. 6A). The combination of the VDCC inhibitor diltiazem (50 µM) and the RyR inhibitor ryanodine (10 µM) reduced the total BK current to 4.2 ± 3.3 % of control (Fig. 6B). Thus, a significant component (about 45 %) of the BK current is activated by RyRs (Ca²⁺ sparks), which is regulated indirectly by Ca²⁺ entry through VDCCs. The remainder of the BK current is activated by Ca²⁺ entry through VDCCs and membrane potential depolarization.

View larger version
[in this window]
[in a new window]

Figure 6. Sources of Ca²⁺ that activate BK channels
A, currents recorded in a cell treated with ryanodine to block RyRs. Whole-cell currents were recorded during a 100 ms test pulse from -70 to +10 mV. Ryanodine (10 µM) was added after the control record and then TEA⁺ (1 mM) was applied. The middle trace shows the ryanodine-sensitive portion of the current. The lower trace shows the TEA⁺-sensitive portion of the current (BK current) remaining in the presence of ryanodine. Blocking RyRs abolishes transient BK currents, but steady-state BK current remains. B, whole-cell currents recorded in a cell treated with the RyR inhibitor ryanodine and the VDCC blocker diltiazem. Ryanodine (10 µM) and diltiazem (50 µM) were added after the control record and then TEA⁺ was applied. The steady-state, ryanodine-insensitive BK current (see panel A) is almost completely abolished by blocking VDCCs. The dotted lines indicate zero current level.

DISCUSSION

Top
Abstract
Introduction
Methods
Results
Discussion
References

SK channels in UBSM

In this study we provide the first characterization of SK currents in UBSM cells, and define the regulation of SK and BK channels by Ca²⁺ signals in UBSM (Fig. 7). A component of a depolarization-induced outward current is blocked by apamin at concentrations expected to block SK channels, providing direct support for the hypothesis that UBSM cells express functional SK channels. This apamin-sensitive current is abolished by the VDCC blocker diltiazem, but not by inhibiting RyRs with ryanodine. We also demonstrate that blocking SK channels with apamin potentiates the amplitude of spontaneous contractions of guinea-pig UBSM. These effects occur over a range of apamin concentrations expected to block SK2 (subnanomolar) and SK1 or SK3 (nanomolar to micromolar) channels. These findings indicate that SK channels are activated by Ca²⁺ influx through VDCCs during an action potential, resulting in the after-hyperpolarization phase of the action potential. In this way, SK channels play an important role in regulating the contractile activity of guinea-pig bladder smooth muscle.

View larger version
[in this window]
[in a new window]

Figure 7. Regulation of SK and BK channel activity in urinary bladder smooth muscle
Schematic representation of the Ca²⁺ signals that influence the activity of BK and SK channels in UBSM to control the membrane potential. Depolarization during an action potential activates VDCCs, resulting in Ca²⁺ influx and a subsequent increase in average cytoplasmic [Ca²⁺]. Local Ca²⁺ signalling from RyRs to BK channels (Ca²⁺ spark) is indicated (Pérez et al. 1999; Herrera et al. 2001). Distant Ca²⁺ signalling from VDCCs to RyRs is also shown (Collier et al. 2000; this study). Activity of BK and SK channels depends on Ca²⁺ entry through VDCCs, although it is not known if this communication is local or distant. Pharmacological compounds used in this study to inhibit each pathway are indicated in italics. Although they were not a focus of the present study and they are not indicated on this diagram, K_V channels also regulate UBSM action potential repolarization.

Our results indicate that SK channels are activated by the rise in [Ca²⁺] which occurs upon Ca²⁺ entry through VDCCs (Fig. 7). A recent study examined the activity of SK channels and L-type VDCCs in single excised membrane patches from hippocampal neurons (Marrion & Tavalin, 1998). These authors demonstrated that SK channel opening is intimately linked to the opening of a single L-type VDCC. Whether this local communication between VDCCs and SK channels occurs in UBSM cells, however, is not known.

In UBSM, average cytosolic [Ca²⁺]_i ranges from resting levels of 100 nM up to 1 µM (Ganitkevich & Isenberg, 1991). Cloned SK channels are half-maximally activated by Ca²⁺ in the 300 to 700 nM range (Köhler et al. 1996; Hirschberg et al. 1998; Xia et al. 1998), which agrees with SK channel Ca²⁺ sensitivities estimated in native tissues (Hirschberg et al. 1999; see Bond et al. 1999 for review). SK channels constitutively interact with the Ca²⁺-binding protein calmodulin (Xia et al. 1998; Keen et al. 1999), and Ca²⁺ binding to the N-lobe of calmodulin activates the SK channel (Bond et al. 1999; Schumacher et al. 2001). These properties of SK channels make them particularly well-suited to sensing global Ca²⁺ levels which vary in the 100 nM to 1 µM range.

The density of SK channels appears to be relatively low in UBSM. Using the mean I_SK at +10 mV of ~50 pA (Fig. 4B) and a unitary current of 1 pA (Köhler et al. 1996), a UBSM cell has at least 50 SK channels, or 0.01 SK channels/µm². For comparison, UBSM cells have at least 2.3 VDCCs/µm² (Klöckner & Isenberg, 1985b; see Rubart et al. 1996 for arterial smooth muscle), and at least two BK channels/µm² (Petkov et al. 2001). The relatively low density of SK channels can explain the lack of effect of ryanodine on SK currents (Fig. 3 and Fig. 4). A Ca²⁺ spark affects approximately 13 µm² of the cell membrane (Herrera et al. 2001), and thereby would activate at the most only one SK channel, which would not be detectable in the present experiments. Both the density and Ca²⁺ sensitivity of SK channels make them well suited as sensors of changes in global Ca²⁺ caused by activation of VDCCs during the UBSM action potential.

On the relationship between VDCCs and BK currents

Transient and steady-state BK currents were detected during voltage steps to +10 mV (Fig. 5 and Fig. 6). The transient BK currents have been shown to be activated by Ca²⁺ sparks (RyRs) in UBSM cells from guinea-pig (Herrera et al. 2001). Analysis of simultaneous measurements of Ca²⁺ sparks and transient BK currents indicated that every spark site in the SR is in close proximity to BK channels in the surface membrane. The communication between Ca²⁺ sparks and BK channels appears local in that micromolar local Ca²⁺ from the sparks is required to cause the observed BK channel activation (Pérez et al. 1999, 2001; Herrera et al. 2001).

Our results support the idea that VDCCs are distant from the Ca²⁺ spark sites (see also Collier et al. 2000). The significant lag (52 ms; see Fig. 5A) between activation of VDCCs and the first transient BK current is consistent with the lag between I_Ca and Ca²⁺ spark initiation observed by Collier and colleagues (2000). This result is in striking contrast to the rapid communication (< 5 ms) of I_Ca to Ca²⁺ spark sites in cardiac muscle (Cannell et al. 1995; Lopez-Lopez et al. 1995). Another interpretation for the prolonged delay in the communication of VDCCs to RyRs in UBSM relative to heart is that RyRs in UBSM cells are less sensitive to Ca²⁺ than RyRs in cardiac muscle. The frequency of transient BK currents did depend on Ca²⁺ entry through VDCCs (Fig. 5B and Fig. 7B). Furthermore, Ca²⁺ spark frequency has been shown to depend on Ca²⁺ entry through VDCCs (Jaggar et al. 1998b; Herrera et al. 2001). This dependency may reflect activation of RyRs by cytoplasmic Ca²⁺ (Rousseau et al. 1987). Furthermore, an increase in cytoplasmic Ca²⁺ will lead to an elevation of SR Ca²⁺ which would also increase Ca²⁺ spark frequency (ZhuGe et al. 1999; see Fig. 7).

Conclusions

SK and BK channels have critical roles in controlling UBSM function. SK channels appear to be responsible for the after-hyperpolarization of the UBSM action potential (Creed et al. 1983; Brading et al. 1992). BK channels regulate the resting potential and the repolarization of the action potential (Heppner et al. 1997). Our results support the concept that SK channel activation absolutely depends on Ca²⁺ entry through VDCCs, but not on Ca²⁺ release through RyRs (Figs 3, 4 and 7). By sensing elevations in global [Ca²⁺]_i arising from Ca²⁺ entry through VDCCs, SK channels limit the basal level of UBSM excitability, and thus contractility. In contrast, BK channel activation depends on both Ca²⁺ entry through VDCCs and Ca²⁺ release through RyRs (Fig. 6 and Fig. 7). Steady-state BK channel activity is highly dependent upon Ca²⁺ entry through VDCCs, and this communication could be local (Fig. 7; cf. Guia et al. 1999). Transient BK currents require local communication from RyRs (Ca²⁺ sparks) to BK channels. Ca²⁺ release from RyRs is modulated by VDCCs (Figs 5, 6 and 7). The communication of Ca²⁺ signals from VDCCs to RyRs is slow (Fig. 7) in contrast to cardiac muscle. These results underscore the importance of spatial localization of Ca²⁺-sensitive targets with respect to their appropriate Ca²⁺ signals (Fig. 7).

REFERENCES

Top
Abstract
Introduction
Methods
Results
Discussion
References

BARRETT, J. N., MAGLEBY, K. L. & PALLOTTA, B. S. (1982). Properties of single calcium-activated potassium channels in cultured rat muscle. Journal of Physiology 331, 211-230 [Medline]

BOND, C. T., MAYLIE, J. & ADELMAN, J. P. (1999). Small-conductance calcium-activated potassium channels. Annals of the New York Academy of Sciences 868, 370-378 [Medline]

BRADING, A. F. (1992). Ion channels and control of contractile activity in urinary bladder smooth muscle. Japanese Journal of Pharmacology 58, 120-127P

CANNELL, M. B., CHENG, H. & LEDERER, W. J. (1995). The control of calcium release in heart muscle. Science 268, 1045-1049

COLLIER, M. L., JI, G., WANG, Y.-X. & KOTLIKOFF, M. I. (2000). Calcium-induced calcium release in smooth muscle: loose coupling between the action potential and calcium release. Journal of General Physiology 115, 653-662 [Abstract/Full Text]

COX, D. H., CUI, J. & ALDRICH, R. W. (1997). Allosteric gating of a large conductance Ca-activated K⁺ channel. Journal of General Physiology 110, 257-281 [Abstract/Full Text]

CREED, K. E., ISHIKAWA, S. & ITO, Y. (1983). Electrical and mechanical activity recorded from rabbit urinary bladder in response to nerve stimulation. Journal of Physiology 338, 149-164 [Abstract]

FUJII, K., FOSTER, C. D., BRADING, A. F. & PAREKH, A. B. (1990). Potassium channel blockers and the effects of cromakalim on the smooth muscle of the guinea-pig bladder. British Journal of Pharmacology 99, 779-785 [Abstract]

GALVEZ, A., GIMENEZ-GALLEGO, G., REUBEN, J. P., ROY-CONTACIN, L., FEIGENBAUM, P., KACZOROWSKI, G. J. & GARCIA, M. L. (1990). Purification and characterization of a unique, potent, peptidyl probe for the high conductance calcium-activated potassium channel from venom of the scorpion Buthus tamulus. Journal of Biological Chemistry 265, 11083-11090 [Abstract]

GANITKEVICH, V. Y. & ISENBERG, G. (1991). Depolarization-mediated intracellular calcium transients in isolated smooth muscle cells of guinea-pig urinary bladder. Journal of Physiology 435, 187-205 [Abstract]

GUIA, A., WAN, X., COURTEMANCHE, M. & LEBLANC, N. (1999). Local Ca²⁺ entry through L-type Ca²⁺ channels activates Ca²⁺-dependent K⁺ channels in rabbit coronary myocytes. Circulation Research 84, 1032-1042 [Abstract/Full Text]

HABERMANN, E. (1984). Apamin. Pharmacological Therapeutics 25, 255-270

HEPPNER, T. J., BONEV, A. D. & NELSON, M. T. (1997). Ca²⁺-activated K⁺ channels regulate action potential repolarization in urinary bladder smooth muscle. American Journal of Physiology 273, C110-117 [Medline]

HERRERA, G. M., HEPPNER, T. J. & NELSON, M. T. (2000). Regulation of urinary bladder smooth muscle contractions by ryanodine receptors and BK and SK channels. American Journal of Physiology - Regulatory, Integrative and Comparative Physiology 279, R60-68 [Medline]

HERRERA, G. M., HEPPNER, T. J. & NELSON, M. T. (2001). Voltage dependence of the coupling of Ca²⁺ sparks to BK_Ca channels in urinary bladder smooth muscle. American Journal of Physiology - Cell Physiology 280, C481-490 [Medline]

HIRSCHBERG, B., MAYLIE, J., ADELMAN, J. P. & MARRION, N. V. (1998). Gating of recombinant small-conductance Ca-activated K⁺ channels by calcium. Journal of General Physiology 111, 565-581 [Abstract/Full Text]

HIRSCHBERG, B., MAYLIE, J., ADELMAN, J. P. & MARRION, N. V. (1999). Gating properties of single SK channels in hippocampal CA1 pyramidal neurons. Biophysical Journal 77, 1905-1913 [Abstract/Full Text]

HORN, R. & MARTY, A. (1988). Muscarinic activation of ionic currents measured by a new whole cell recording method. Journal of General Physiology 92, 145-159 [Abstract]

ISHII, T. M., MAYLIE, J. & ADELMAN, J. P. (1997). Determinants of apamin and d-tubocurarine block in SK potassium channels. Journal of Biological Chemistry 272, 23195-23200 [Abstract/Full Text]

JAGGAR, J. H., MAWE, G. M. & NELSON, M. T. (1998a). Voltage-dependent K⁺ currents in smooth muscle cells from mouse gallbladder. American Journal of Physiology 274, G687-693 [Medline]

JAGGAR, J. H., PORTER, V. A., LEDERER, W. J. & NELSON, M. T. (2000). Calcium sparks in smooth muscle. American Journal of Physiology - Cell Physiology 278, C235-256

JAGGAR, J. H., STEVENSON, A. S. & NELSON, M. T. (1998b). Voltage-dependence of Ca²⁺ sparks in intact cerebral arteries. American Journal of Physiology 274, C1755-1761 [Medline]

KEEN, J. E., KHAWALED, R., FARRENS, D. L., NEELANDS, T., RIVARD, A., BOND, C. T., JANOWSKY, A., FAKLER, B., ADELMAN, J. P. & MAYLIE, J. (1999). Domains responsible for constitutive and Ca²⁺-dependent interactions between calmodulin and small conductance Ca²⁺-activated potassium channels. Journal of Neuroscience 19, 8830-8838 [Abstract/Full Text]

KLÖCKNER, U. & ISENBERG, G. (1985a). Action potentials and net membrane currents of isolated smooth muscle cells (urinary bladder of the guinea-pig). Pflügers Archiv 405, 329-339 [Medline]

KLÖCKNER, U. & ISENBERG, G. (1985b). Calcium currents of cesium loaded isolated smooth muscle cells (urinary bladder of the guinea pig). Pflügers Archiv 405, 340-348 [Medline]

KOH, S. D., DICK, G. M. & SANDERS, K. M. (1997). Small-conductance Ca²⁺-dependent K⁺ channels activated by ATP in murine colonic smooth muscle. American Journal of Physiology 273, C2010-2021 [Medline]

KÖHLER, M., HIRSCHBERG, B., BOND, C. T., KINZIE, J. M., MARRION, N. V., MAYLIE, J. & ADELMAN, J. P. (1996). Small-conductance, calcium-activated potassium channels from mammalian brain. Science 273, 1709-1714

LANGTON, P. D., NELSON, M .T., HUANG, Y. & STANDEN, N. B. (1991). Block of calcium-activated potassium channels in mammalian arterial myocytes by tetraethylammonium ions. American Journal of Physiology 260, H927-934 [Medline]

LOPEZ-LOPEZ, J. R., SHACKLOCK, P. S., BALKE, C. W. & WIER, W. G. (1995). Local calcium transients triggered by single L-type calcium channel currents in cardiac cells. Science 268, 1042-1045

MARRION, N. V. & TAVALIN, S. J. (1998). Selective activation of Ca²⁺-activated K⁺ channels by colocalized Ca²⁺ channels in hippocampal neurons. Nature 395, 900-905 [Medline]

PEDARZANI, P., KULIK, A., MULLER, M., BALLANYI, K. & STOCKER, M. (2000). Molecular determinants of Ca²⁺-dependent K⁺ channel function in rat dorsal vagal neurones. Journal of Physiology 527, 283-290 [Abstract/Full Text]

PÉREZ, G. J., BONEV, A. D. & NELSON, M. T. (2001). Micromolar Ca²⁺ from sparks activates Ca²⁺-sensitive K⁺ channels in rat cerebral artery smooth muscle. American Journal of Physiology - Cell Physiology 281, C1769-1775 [Abstract/Full Text]

PÉREZ, G. J., BONEV, A. D., PATLAK, J. B. & NELSON, M. T. (1999). Functional coupling of ryanodine receptors to K_Ca channels in smooth muscle cells from rat cerebral arteries. Journal of General Physiology 113, 229-237 [Abstract/Full Text]

PETKOV, G. V., BONEV, A. D., HEPPNER, T. J., BRENNER, R., ALDRICH, R. W. & NELSON, M. T. (2001). $beta$ 1-subunit of the Ca²⁺-activated K⁺ channel regulates contractile activity of mouse urinary bladder smooth muscle. Journal of Physiology 537, 443-452 [Abstract/Full Text]

ROBERTSON, B. E. & NELSON, M. T. (1994). Aminopyridine inhibition and voltage dependence of K⁺ currents in smooth muscle cells from cerebral arteries. American Journal of Physiology 267, C1589-1597 [Medline]

ROUSSEAU, E., SMITH, J. S. & MEISSNER, G. (1987). Ryanodine modifies conductance and gating behavior of single Ca²⁺ release channel. American Journal of Physiology 253, C364-368 [Medline]

RUBART, M., PATLAK, J. & NELSON, M. (1996). Ca²⁺ currents in cerebral artery smooth muscle cells of rat at physiological Ca²⁺ concentrations. Journal of General Physiology 107, 459-472 [Abstract]

SCHUMACHER, M. A., RIVARD, A., BÄCHINGER, H. P. & ADELMAN, J. P. (2001). Structure of the gating domain of a Ca²⁺-activated K⁺ channel complexed with Ca²⁺/calmodulin. Nature 410, 1120-1124 [Medline]

SHAH, M. & HAYLETT, D. G. (2000). The pharmacology of hSK1 Ca²⁺-activated K⁺ channels expressed in mammalian cell lines. British Journal of Pharmacology 129, 627-630 [Abstract/Full Text]

STOCKER, M., KRAUSE, M. & PEDARZANI, P. (1999). An apamin-sensitive Ca²⁺-activated K⁺ current in hippocampal pyramidal neurons. Proceedings of the National Academy of Sciences of the USA 96, 4662-4667 [Abstract/Full Text]

STRØBÆK, D., JØRGENSEN, T. D., CHRISTOPHERSEN, P., AHRING, P. K. & OLESEN, S.-P. (2000). Pharmacological characterization of small-conductance Ca²⁺-activated K⁺ channels stably expressed in HEK 293 cells. British Journal of Pharmacology 129, 991-999 [Abstract/Full Text]

VERGARA, C., LATORRE, R., MARRION, N. V. & ADELMAN, J. P. (1998). Calcium activated potassium channels. Current Opinion in Neurobiology 8, 321-329 [Medline]

VOGALIS, F. & GOYAL, R. K. (1997). Activation of small conductance Ca²⁺-dependent K⁺ channels by purinergic agonists in smooth muscle cells of the mouse ileum. Journal of Physiology 502, 497-508 [Abstract]

VOLK, K. A., MATSUDA, J. J. & SHIBATA, E. F. (1991). A voltage-dependent potassium current in rabbit coronary artery smooth muscle cells. Journal of Physiology 439, 751-768 [Abstract]

WOLFART, J., NEUHOFF, H., FRANZ, O. & ROEPER, J. (2001). Differential expression of the small-conductance, calcium-activated potassium channel SK3 is critical for pacemaker control in dopaminergic midbrain neurons. Journal of Neuroscience 21, 3443-3456 [Abstract/Full Text]

XIA, X.-M., FAKLER, B., RIVARD, A., WAYMAN, G., JOHNSON-PAIS, T., KEEN, J. E., ISHII, T., HIRSCHBERG, B., BOND, C. T., LUTSENKO, S., MAYLIE, J. & ADELMAN, J. P. (1998). Mechanism of calcium gating in small-conductance calcium-activated potassium channels. Nature 395, 503-507 [Medline]

ZHUGE, R., TUFT, R. A., FOGARTY, K. E., BELLVE, K., FAY, F. S. & WALSH, J. V. JR (1999). The influence of sarcoplasmic reticulum Ca²⁺ concentration on Ca²⁺ sparks and spontaneous transient outward currents in single smooth muscle cells. Journal of General Physiology 113, 215-228 [Abstract/Full Text]

ZOGRAFOS, P., LI, J. H. & KAU, S. T. (1992). Comparison of the in vitro effects of K⁺ channel modulators on detrusor and portal vein strips from guinea pigs. Pharmacology 45, 216-230 [Medline]

Acknowledgements

The authors would like to thank Drs David Hill-Eubanks, Georgi Petkov and L. Fernando Santana for helpful discussions and comments relating to this manuscript. This work was supported by National Institute of Health Grant DK-53832 to M.T.N. and by a Training Grant from the National Institute of Health T32 HL/AR 07944. During portions of this study G.M.H. was a National Science Foundation Minority Graduate Research Fellow.

This article has been cited by other articles:

J. Layne, M. Werner, D. Hill-Eubanks, and M. Nelson
NFATc3 regulates BK channel function in murine urinary bladder smooth muscle
Am J Physiol Cell Physiol, September 1, 2008; 295(3): C611 - C623.
[Abstract] [Full Text] [PDF]

K. S. Thorneloe, A. M. Knorn, P. E. Doetsch, E. S. R. Lashinger, A. X. Liu, C. T. Bond, J. P. Adelman, and M. T. Nelson
Small-conductance, Ca2+-activated K+ channel 2 is the key functional component of SK channels in mouse urinary bladder
Am J Physiol Regulatory Integrative Comp Physiol, May 1, 2008; 294(5): R1737 - R1743.
[Abstract] [Full Text] [PDF]

Y.-K. Ng, W. C. de Groat, and H.-Y. Wu
Smooth muscle and neural mechanisms contributing to the downregulation of neonatal rat spontaneous bladder contractions during postnatal development
Am J Physiol Regulatory Integrative Comp Physiol, May 1, 2007; 292(5): R2100 - R2112.
[Abstract] [Full Text] [PDF]

E. T. Barfod, A. L. Moore, M. W. Roe, and S. D. Lidofsky
Ca2+-activated IK1 Channels Associate with Lipid Rafts upon Cell Swelling and Mediate Volume Recovery
J. Biol. Chem., March 23, 2007; 282(12): 8984 - 8993.
[Abstract] [Full Text] [PDF]

A. Nolting, T. Ferraro, D. D'hoedt, and M. Stocker
An Amino Acid Outside the Pore Region Influences Apamin Sensitivity in Small Conductance Ca2+-activated K+ Channels
J. Biol. Chem., February 9, 2007; 282(6): 3478 - 3486.
[Abstract] [Full Text] [PDF]

A. Brown, T. Cornwell, I. Korniyenko, V. Solodushko, C. T. Bond, J. P. Adelman, and M. S. Taylor
Myometrial expression of small conductance Ca2+-activated K+ channels depresses phasic uterine contraction
Am J Physiol Cell Physiol, February 1, 2007; 292(2): C832 - C840.
[Abstract] [Full Text] [PDF]

M. E. Werner, A.-M. Knorn, A. L. Meredith, R. W. Aldrich, and M. T. Nelson
Frequency encoding of cholinergic- and purinergic-mediated signaling to mouse urinary bladder smooth muscle: modulation by BK channels
Am J Physiol Regulatory Integrative Comp Physiol, January 1, 2007; 292(1): R616 - R624.
[Abstract] [Full Text] [PDF]

G. Ji, M. Feldman, R. Doran, W. Zipfel, and M. I. Kotlikoff
Ca2+-Induced Ca2+ Release through Localized Ca2+ Uncaging in Smooth Muscle
J. Gen. Physiol., February 27, 2006; 127(3): 225 - 235.
[Abstract] [Full Text] [PDF]

A. F. Brading
Spontaneous activity of lower urinary tract smooth muscles: correlation between ion channels and tissue function
J. Physiol., January 1, 2006; 570(1): 13 - 22.
[Abstract] [Full Text] [PDF]

M. E. Werner, P. Zvara, A. L. Meredith, R. W. Aldrich, and M. T. Nelson
Erectile dysfunction in mice lacking the large-conductance calcium-activated potassium (BK) channel
J. Physiol., September 1, 2005; 567(2): 545 - 556.
[Abstract] [Full Text] [PDF]

K. S. Thorneloe, A. L. Meredith, A. M. Knorn, R. W. Aldrich, and M. T. Nelson
Urodynamic properties and neurotransmitter dependence of urinary bladder contractility in the BK channel deletion model of overactive bladder
Am J Physiol Renal Physiol, September 1, 2005; 289(3): F604 - F610.
[Abstract] [Full Text] [PDF]

G. M. Herrera, B. Etherton, B. Nausch, and M. T. Nelson
Negative feedback regulation of nerve-mediated contractions by KCa channels in mouse urinary bladder smooth muscle
Am J Physiol Regulatory Integrative Comp Physiol, August 1, 2005; 289(2): R402 - R409.
[Abstract] [Full Text] [PDF]

G. V. Petkov and M. T. Nelson
Differential regulation of Ca2+-activated K+ channels by {beta}-adrenoceptors in guinea pig urinary bladder smooth muscle
Am J Physiol Cell Physiol, June 1, 2005; 288(6): C1255 - C1263.
[Abstract] [Full Text] [PDF]

T. J Heppner, A. D Bonev, and M. T Nelson
Elementary purinergic Ca2+ transients evoked by nerve stimulation in rat urinary bladder smooth muscle
J. Physiol., April 1, 2005; 564(1): 201 - 212.
[Abstract] [Full Text] [PDF]

BARRETT, J. N., MAGLEBY, K. L. & PALLOTTA, B. S. (1982). Properties of single calcium-activated potassium channels in cultured rat muscle. Journal of Physiology 331, 211-230	[Medline]
BOND, C. T., MAYLIE, J. & ADELMAN, J. P. (1999). Small-conductance calcium-activated potassium channels. Annals of the New York Academy of Sciences 868, 370-378	[Medline]
BRADING, A. F. (1992). Ion channels and control of contractile activity in urinary bladder smooth muscle. Japanese Journal of Pharmacology 58, 120-127P
CANNELL, M. B., CHENG, H. & LEDERER, W. J. (1995). The control of calcium release in heart muscle. Science 268, 1045-1049
COLLIER, M. L., JI, G., WANG, Y.-X. & KOTLIKOFF, M. I. (2000). Calcium-induced calcium release in smooth muscle: loose coupling between the action potential and calcium release. Journal of General Physiology 115, 653-662	[Abstract/Full Text]
COX, D. H., CUI, J. & ALDRICH, R. W. (1997). Allosteric gating of a large conductance Ca-activated K⁺ channel. Journal of General Physiology 110, 257-281	[Abstract/Full Text]
CREED, K. E., ISHIKAWA, S. & ITO, Y. (1983). Electrical and mechanical activity recorded from rabbit urinary bladder in response to nerve stimulation. Journal of Physiology 338, 149-164	[Abstract]
FUJII, K., FOSTER, C. D., BRADING, A. F. & PAREKH, A. B. (1990). Potassium channel blockers and the effects of cromakalim on the smooth muscle of the guinea-pig bladder. British Journal of Pharmacology 99, 779-785	[Abstract]
GALVEZ, A., GIMENEZ-GALLEGO, G., REUBEN, J. P., ROY-CONTACIN, L., FEIGENBAUM, P., KACZOROWSKI, G. J. & GARCIA, M. L. (1990). Purification and characterization of a unique, potent, peptidyl probe for the high conductance calcium-activated potassium channel from venom of the scorpion Buthus tamulus. Journal of Biological Chemistry 265, 11083-11090	[Abstract]
GANITKEVICH, V. Y. & ISENBERG, G. (1991). Depolarization-mediated intracellular calcium transients in isolated smooth muscle cells of guinea-pig urinary bladder. Journal of Physiology 435, 187-205	[Abstract]
GUIA, A., WAN, X., COURTEMANCHE, M. & LEBLANC, N. (1999). Local Ca²⁺ entry through L-type Ca²⁺ channels activates Ca²⁺-dependent K⁺ channels in rabbit coronary myocytes. Circulation Research 84, 1032-1042	[Abstract/Full Text]
HABERMANN, E. (1984). Apamin. Pharmacological Therapeutics 25, 255-270
HEPPNER, T. J., BONEV, A. D. & NELSON, M. T. (1997). Ca²⁺-activated K⁺ channels regulate action potential repolarization in urinary bladder smooth muscle. American Journal of Physiology 273, C110-117	[Medline]
HERRERA, G. M., HEPPNER, T. J. & NELSON, M. T. (2000). Regulation of urinary bladder smooth muscle contractions by ryanodine receptors and BK and SK channels. American Journal of Physiology - Regulatory, Integrative and Comparative Physiology 279, R60-68	[Medline]
HERRERA, G. M., HEPPNER, T. J. & NELSON, M. T. (2001). Voltage dependence of the coupling of Ca²⁺ sparks to BK_Ca channels in urinary bladder smooth muscle. American Journal of Physiology - Cell Physiology 280, C481-490	[Medline]
HIRSCHBERG, B., MAYLIE, J., ADELMAN, J. P. & MARRION, N. V. (1998). Gating of recombinant small-conductance Ca-activated K⁺ channels by calcium. Journal of General Physiology 111, 565-581	[Abstract/Full Text]
HIRSCHBERG, B., MAYLIE, J., ADELMAN, J. P. & MARRION, N. V. (1999). Gating properties of single SK channels in hippocampal CA1 pyramidal neurons. Biophysical Journal 77, 1905-1913	[Abstract/Full Text]
HORN, R. & MARTY, A. (1988). Muscarinic activation of ionic currents measured by a new whole cell recording method. Journal of General Physiology 92, 145-159	[Abstract]
ISHII, T. M., MAYLIE, J. & ADELMAN, J. P. (1997). Determinants of apamin and d-tubocurarine block in SK potassium channels. Journal of Biological Chemistry 272, 23195-23200	[Abstract/Full Text]
JAGGAR, J. H., MAWE, G. M. & NELSON, M. T. (1998a). Voltage-dependent K⁺ currents in smooth muscle cells from mouse gallbladder. American Journal of Physiology 274, G687-693	[Medline]
JAGGAR, J. H., PORTER, V. A., LEDERER, W. J. & NELSON, M. T. (2000). Calcium sparks in smooth muscle. American Journal of Physiology - Cell Physiology 278, C235-256
JAGGAR, J. H., STEVENSON, A. S. & NELSON, M. T. (1998b). Voltage-dependence of Ca²⁺ sparks in intact cerebral arteries. American Journal of Physiology 274, C1755-1761	[Medline]
KEEN, J. E., KHAWALED, R., FARRENS, D. L., NEELANDS, T., RIVARD, A., BOND, C. T., JANOWSKY, A., FAKLER, B., ADELMAN, J. P. & MAYLIE, J. (1999). Domains responsible for constitutive and Ca²⁺-dependent interactions between calmodulin and small conductance Ca²⁺-activated potassium channels. Journal of Neuroscience 19, 8830-8838	[Abstract/Full Text]
KLÖCKNER, U. & ISENBERG, G. (1985a). Action potentials and net membrane currents of isolated smooth muscle cells (urinary bladder of the guinea-pig). Pflügers Archiv 405, 329-339	[Medline]
KLÖCKNER, U. & ISENBERG, G. (1985b). Calcium currents of cesium loaded isolated smooth muscle cells (urinary bladder of the guinea pig). Pflügers Archiv 405, 340-348	[Medline]
KOH, S. D., DICK, G. M. & SANDERS, K. M. (1997). Small-conductance Ca²⁺-dependent K⁺ channels activated by ATP in murine colonic smooth muscle. American Journal of Physiology 273, C2010-2021	[Medline]
KÖHLER, M., HIRSCHBERG, B., BOND, C. T., KINZIE, J. M., MARRION, N. V., MAYLIE, J. & ADELMAN, J. P. (1996). Small-conductance, calcium-activated potassium channels from mammalian brain. Science 273, 1709-1714
LANGTON, P. D., NELSON, M .T., HUANG, Y. & STANDEN, N. B. (1991). Block of calcium-activated potassium channels in mammalian arterial myocytes by tetraethylammonium ions. American Journal of Physiology 260, H927-934	[Medline]
LOPEZ-LOPEZ, J. R., SHACKLOCK, P. S., BALKE, C. W. & WIER, W. G. (1995). Local calcium transients triggered by single L-type calcium channel currents in cardiac cells. Science 268, 1042-1045
MARRION, N. V. & TAVALIN, S. J. (1998). Selective activation of Ca²⁺-activated K⁺ channels by colocalized Ca²⁺ channels in hippocampal neurons. Nature 395, 900-905	[Medline]
PEDARZANI, P., KULIK, A., MULLER, M., BALLANYI, K. & STOCKER, M. (2000). Molecular determinants of Ca²⁺-dependent K⁺ channel function in rat dorsal vagal neurones. Journal of Physiology 527, 283-290	[Abstract/Full Text]
PÉREZ, G. J., BONEV, A. D. & NELSON, M. T. (2001). Micromolar Ca²⁺ from sparks activates Ca²⁺-sensitive K⁺ channels in rat cerebral artery smooth muscle. American Journal of Physiology - Cell Physiology 281, C1769-1775	[Abstract/Full Text]
PÉREZ, G. J., BONEV, A. D., PATLAK, J. B. & NELSON, M. T. (1999). Functional coupling of ryanodine receptors to K_Ca channels in smooth muscle cells from rat cerebral arteries. Journal of General Physiology 113, 229-237	[Abstract/Full Text]
PETKOV, G. V., BONEV, A. D., HEPPNER, T. J., BRENNER, R., ALDRICH, R. W. & NELSON, M. T. (2001). $beta$ 1-subunit of the Ca²⁺-activated K⁺ channel regulates contractile activity of mouse urinary bladder smooth muscle. Journal of Physiology 537, 443-452	[Abstract/Full Text]
ROBERTSON, B. E. & NELSON, M. T. (1994). Aminopyridine inhibition and voltage dependence of K⁺ currents in smooth muscle cells from cerebral arteries. American Journal of Physiology 267, C1589-1597	[Medline]
ROUSSEAU, E., SMITH, J. S. & MEISSNER, G. (1987). Ryanodine modifies conductance and gating behavior of single Ca²⁺ release channel. American Journal of Physiology 253, C364-368	[Medline]
RUBART, M., PATLAK, J. & NELSON, M. (1996). Ca²⁺ currents in cerebral artery smooth muscle cells of rat at physiological Ca²⁺ concentrations. Journal of General Physiology 107, 459-472	[Abstract]
SCHUMACHER, M. A., RIVARD, A., BÄCHINGER, H. P. & ADELMAN, J. P. (2001). Structure of the gating domain of a Ca²⁺-activated K⁺ channel complexed with Ca²⁺/calmodulin. Nature 410, 1120-1124	[Medline]
SHAH, M. & HAYLETT, D. G. (2000). The pharmacology of hSK1 Ca²⁺-activated K⁺ channels expressed in mammalian cell lines. British Journal of Pharmacology 129, 627-630	[Abstract/Full Text]
STOCKER, M., KRAUSE, M. & PEDARZANI, P. (1999). An apamin-sensitive Ca²⁺-activated K⁺ current in hippocampal pyramidal neurons. Proceedings of the National Academy of Sciences of the USA 96, 4662-4667	[Abstract/Full Text]
STRØBÆK, D., JØRGENSEN, T. D., CHRISTOPHERSEN, P., AHRING, P. K. & OLESEN, S.-P. (2000). Pharmacological characterization of small-conductance Ca²⁺-activated K⁺ channels stably expressed in HEK 293 cells. British Journal of Pharmacology 129, 991-999	[Abstract/Full Text]
VERGARA, C., LATORRE, R., MARRION, N. V. & ADELMAN, J. P. (1998). Calcium activated potassium channels. Current Opinion in Neurobiology 8, 321-329	[Medline]
VOGALIS, F. & GOYAL, R. K. (1997). Activation of small conductance Ca²⁺-dependent K⁺ channels by purinergic agonists in smooth muscle cells of the mouse ileum. Journal of Physiology 502, 497-508	[Abstract]
VOLK, K. A., MATSUDA, J. J. & SHIBATA, E. F. (1991). A voltage-dependent potassium current in rabbit coronary artery smooth muscle cells. Journal of Physiology 439, 751-768	[Abstract]
WOLFART, J., NEUHOFF, H., FRANZ, O. & ROEPER, J. (2001). Differential expression of the small-conductance, calcium-activated potassium channel SK3 is critical for pacemaker control in dopaminergic midbrain neurons. Journal of Neuroscience 21, 3443-3456	[Abstract/Full Text]
XIA, X.-M., FAKLER, B., RIVARD, A., WAYMAN, G., JOHNSON-PAIS, T., KEEN, J. E., ISHII, T., HIRSCHBERG, B., BOND, C. T., LUTSENKO, S., MAYLIE, J. & ADELMAN, J. P. (1998). Mechanism of calcium gating in small-conductance calcium-activated potassium channels. Nature 395, 503-507	[Medline]
ZHUGE, R., TUFT, R. A., FOGARTY, K. E., BELLVE, K., FAY, F. S. & WALSH, J. V. JR (1999). The influence of sarcoplasmic reticulum Ca²⁺ concentration on Ca²⁺ sparks and spontaneous transient outward currents in single smooth muscle cells. Journal of General Physiology 113, 215-228	[Abstract/Full Text]
ZOGRAFOS, P., LI, J. H. & KAU, S. T. (1992). Comparison of the in vitro effects of K⁺ channel modulators on detrusor and portal vein strips from guinea pigs. Pharmacology 45, 216-230	[Medline]

				K. S. Thorneloe, A. M. Knorn, P. E. Doetsch, E. S. R. Lashinger, A. X. Liu, C. T. Bond, J. P. Adelman, and M. T. Nelson Small-conductance, Ca2+-activated K+ channel 2 is the key functional component of SK channels in mouse urinary bladder Am J Physiol Regulatory Integrative Comp Physiol, May 1, 2008; 294(5): R1737 - R1743. [Abstract] [Full Text] [PDF]

				Y.-K. Ng, W. C. de Groat, and H.-Y. Wu Smooth muscle and neural mechanisms contributing to the downregulation of neonatal rat spontaneous bladder contractions during postnatal development Am J Physiol Regulatory Integrative Comp Physiol, May 1, 2007; 292(5): R2100 - R2112. [Abstract] [Full Text] [PDF]

				E. T. Barfod, A. L. Moore, M. W. Roe, and S. D. Lidofsky Ca2+-activated IK1 Channels Associate with Lipid Rafts upon Cell Swelling and Mediate Volume Recovery J. Biol. Chem., March 23, 2007; 282(12): 8984 - 8993. [Abstract] [Full Text] [PDF]

				A. Nolting, T. Ferraro, D. D'hoedt, and M. Stocker An Amino Acid Outside the Pore Region Influences Apamin Sensitivity in Small Conductance Ca2+-activated K+ Channels J. Biol. Chem., February 9, 2007; 282(6): 3478 - 3486. [Abstract] [Full Text] [PDF]

				A. Brown, T. Cornwell, I. Korniyenko, V. Solodushko, C. T. Bond, J. P. Adelman, and M. S. Taylor Myometrial expression of small conductance Ca2+-activated K+ channels depresses phasic uterine contraction Am J Physiol Cell Physiol, February 1, 2007; 292(2): C832 - C840. [Abstract] [Full Text] [PDF]

				M. E. Werner, A.-M. Knorn, A. L. Meredith, R. W. Aldrich, and M. T. Nelson Frequency encoding of cholinergic- and purinergic-mediated signaling to mouse urinary bladder smooth muscle: modulation by BK channels Am J Physiol Regulatory Integrative Comp Physiol, January 1, 2007; 292(1): R616 - R624. [Abstract] [Full Text] [PDF]

				G. Ji, M. Feldman, R. Doran, W. Zipfel, and M. I. Kotlikoff Ca2+-Induced Ca2+ Release through Localized Ca2+ Uncaging in Smooth Muscle J. Gen. Physiol., February 27, 2006; 127(3): 225 - 235. [Abstract] [Full Text] [PDF]

				A. F. Brading Spontaneous activity of lower urinary tract smooth muscles: correlation between ion channels and tissue function J. Physiol., January 1, 2006; 570(1): 13 - 22. [Abstract] [Full Text] [PDF]

				M. E. Werner, P. Zvara, A. L. Meredith, R. W. Aldrich, and M. T. Nelson Erectile dysfunction in mice lacking the large-conductance calcium-activated potassium (BK) channel J. Physiol., September 1, 2005; 567(2): 545 - 556. [Abstract] [Full Text] [PDF]

				K. S. Thorneloe, A. L. Meredith, A. M. Knorn, R. W. Aldrich, and M. T. Nelson Urodynamic properties and neurotransmitter dependence of urinary bladder contractility in the BK channel deletion model of overactive bladder Am J Physiol Renal Physiol, September 1, 2005; 289(3): F604 - F610. [Abstract] [Full Text] [PDF]

				G. M. Herrera, B. Etherton, B. Nausch, and M. T. Nelson Negative feedback regulation of nerve-mediated contractions by KCa channels in mouse urinary bladder smooth muscle Am J Physiol Regulatory Integrative Comp Physiol, August 1, 2005; 289(2): R402 - R409. [Abstract] [Full Text] [PDF]

				G. V. Petkov and M. T. Nelson Differential regulation of Ca2+-activated K+ channels by {beta}-adrenoceptors in guinea pig urinary bladder smooth muscle Am J Physiol Cell Physiol, June 1, 2005; 288(6): C1255 - C1263. [Abstract] [Full Text] [PDF]

				T. J Heppner, A. D Bonev, and M. T Nelson Elementary purinergic Ca2+ transients evoked by nerve stimulation in rat urinary bladder smooth muscle J. Physiol., April 1, 2005; 564(1): 201 - 212. [Abstract] [Full Text] [PDF]