Inhibitory effects of SKF96365 on the activities of K⁺ channels in mouse small intestinal smooth muscle cells

Abstract

In order to investigate the effects of SKF96365 (SKF), which is a non-selective cationic channel blocker, on K⁺ channel currents, we recorded currents through ATP sensitive K⁺ (I_KATP), voltage-gated K⁺ (I_Kv) and Ca²⁺ activated K⁺ channels (I_BK) in the absence and presence of SKF in single small intestinal myocytes of mice with patch-clamp techniques. SKF (10 µM) reversibly abolished I_KATP that was induced by cromakalim (10 µM), which is a selective ATP sensitive K⁺ channel opener. These inhibitory effects were induced in a concentration-dependent and voltage-independent manner. The 50% inhibitory concentration (IC₅₀) was 0.85 µM, which was obviously lower than that reported for the muscarinic cationic current. In addition, SKF (1 µM ≈ the IC₅₀ value in I_KATP suppression) reversibly inhibited the I_Kv that was induced by repetitive depolarizing pulses from −80 to 20 mV. However, the extent of the inhibitory effects was only ~30%. In contrast, SKF (1 µM) had no significant effects on spontaneous transient I_BK and caffeine-induced I_BK. These results indicated that SKF inhibited ATP sensitive K⁺ channels and voltage-gated K⁺ channels, with the ATP sensitive K⁺ channels being more sensitive than the voltage-gated K⁺ channels. These inhibitory effects on K⁺ channels should be considered when SKF is used as a cationic channel blocker.

In gastrointestinal smooth muscles, the stimulation of muscarinic receptors by cholinergic agonists, including acetylcholine, increases the intracellular concentration of Ca²⁺ ([Ca²⁺]_i), which results in a contractile response [45]. The increase in [Ca²⁺]_i is induced by the Ca²⁺ release from internal stores and Ca²⁺ entry into the cell through L-type voltage-gated Ca²⁺ channels (Ca_V1.2). The activation of Ca_V1.2 can be achieved by depolarization due to muscarinic stimulation of non-selective cationic channels [18, 23, 50]. Cationic channels are thought to consist of transient potential canonical channels 4 and 6 (TRPC4 and 6) [43], which are also permeable to Ca²⁺ and involved in Ca²⁺ entry [47]. In addition, muscarinic receptors directly or indirectly regulate the activities of K⁺ channels that are expressed in gastrointestinal smooth muscle cells, including ATP sensitive K⁺ (K_ATP), voltage-gated K⁺ (K_V) and large conductance Ca²⁺ activated K⁺ (BK) channels, through which the electrical activities of the muscles are modulated [5, 6, 12, 14, 20].

SKF96365 (SKF) is an imidazole compound that inhibits receptor-mediated Ca²⁺ entry [27]. SKF has been reported to inhibit the opening of TRPC channels in guinea-pig [52] and mouse ileal cells [9]. Although other inhibitors of TRPC channels, such as quinine, quinidine, La³⁺ and flufenamic acid, are well known [9, 15, 24], these chemicals are not specific for the TRPC channels. Therefore, in investigations of the physiological and pharmacological properties of muscarinic receptor-gated TRPC channels, SKF has been widely used as a channel inhibitor in gastrointestinal smooth muscle cells [9, 26, 52]. However, it has been reported that application of SKF evoked membrane depolarization in gastrointestinal smooth muscles [15, 51], indicating additional effects of SKF on ion channels other than TRPC channels. The depolarizing action can make it more difficult to analyze the physiological roles of TRPC channels. Schwarz et al. [36] have suggested that SKF blocks inwardly and outwardly rectifying K⁺ currents in endothelial cells (IC₅₀; ≥40 µM). Thus, it is possible that SKF can block K⁺ channels that are expressed in gastrointestinal smooth muscles together with TRPC channels, thus resulting in membrane depolarization. However, the effects of SKF on the activities of the K⁺ channels that are expressed in gastrointestinal smooth muscle cells remain to be elucidated. In the present study, we investigated the effects of SKF on the currents through the K_ATP (I_KATP), K_V (I_Kv) and BK (I_BK) channels in longitudinal smooth muscle cells that were isolated from mouse small intestine, which have often been used to investigate the activation mechanisms of TRPC channels.

MATERIALS AND METHODS

The nomenclature of the ion channels was in accordance with Alexander et al. [2]. All of the animal care and experimental procedures described below complied with the guidelines of the local animal ethics committee of the Faculty of Life Sciences, Kyoto Sangyo University.

Cell preparation: Mice (hybrid of 129S4 or 129S6 and CF1) of either sex who were above 2-month-old and who weighed (mean ± standard deviation) 25.1 ± 3.0 g were sacrificed by cervical dislocation. A 15-cm-long gut segment was then removed from the region over the jejunum and ileum, except for the 2.0-cm terminal portion, and placed in physiological salt solution (PSS; for composition, see below). The gut segment was cut into 1.0- to 1.5-cm pieces, and the longitudinal muscle layer was carefully peeled off from each of the pieces. Single smooth muscle cells were isolated enzymatically from the longitudinal muscle layers as described previously [40]. The cells were suspended in PSS containing 0.5 mM CaCl₂. A small aliquot was placed on coverglass and stored at 4°C for 2–7 hr until its use on the same day.

Whole-cell current recording: Whole-cell membrane current recordings were performed at room temperature (21–25°C) with standard patch-clamp techniques [11] with 4–7 MΩ patch pipettes. The current signals were amplified with a current amplifier (CEZ-2300 or 5100, Nihon Kohden Corp., Tokyo, Japan) filtered at 1 KHz and captured at a sampling rate of 4 KHz with an analog-digital converter (Digidata 1440A, Molecular Devices, LLC, Sunnyvale, CA, U.S.A.) that was interfaced with a computer (Think Centre A58 Small, Lenovo, Morrisville, NC, U.S.A.) that was running the pCLAMP program (version 10, Molecular Devices, LLC).

For the I_KATP recordings, the cells were bathed in a 60-mM K⁺ solution and intracellularly dialyzed with a K⁺-rich high BAPTA pipette solution (for compositions, see below) at a holding potential of −80 mV in order to increase the driving force of K⁺ and minimize the activities of the K_V and BK channels [12, 20, 31]. Under these conditions, the K⁺ equilibrium potential (E_K) was estimated at −21 mV, and the K⁺ currents were inwardly activated. I_Kv was recorded in cells that was bathed in Ca²⁺-free PSS (for composition, see below) and dialyzed with the K⁺-rich high-BAPTA pipette solution in order to minimize the currents through the BK and Ca_V1.2 channels. The whole-cell voltage clamp mode was held at −80 mV, and 2 sec step pulses to 20 mV that were generated by the pCLAMP program were repeatedly applied to the cell every 20 sec to elicit I_Kv. When currents were recorded through I_BK, a K⁺-rich low EGTA pipette solution (for composition, see below) was used. The cells were bathed in PSS and held at a holding potential of 0 mV. Recordings of the muscarinic cationic channel currents (mI_TRPC) were made from cells that were bathed in Cs⁺-rich external solution and intracellularly dialyzed with Cs⁺-rich pipette solution (for compositions, see below) at a holding potential of −60 mV in order to eliminate any K⁺ currents.

Solutions: The PSS that was used in the experiments had the following composition (mM): NaCl, 126; KCl, 6; CaCl₂, 2; MgCl₂, 1.2; glucose, 14; and HEPES, 10.5; and the pH was adjusted to 7.2 with NaOH. The 60-mM K⁺ solution was made by changing the concentrations of NaCl and KCl in the PSS to 72 mM and 60 mM, respectively. If necessary, the KCl in the 60-mM K⁺ solution was replaced with CsCl. The Ca²⁺-free PSS was prepared by omitting CaCl₂ from the PSS and adding 0.5 mM EGTA. The Cs⁺-rich external solution had the following composition (mM): CsCl, 120; glucose, 12; and HEPES, 10; and the pH was adjusted to pH 7.4 with CsOH. The compositions of each patch-pipette solution (mM) were as follows. The K⁺-rich low-EGTA pipette solution contained KCl, 134; Na₂GTP, 1.0; MgCl₂, 1.2; MgATP, 1.0; glucose, 14; EGTA, 0.05; and HEPES, 10.5; and the pH was adjusted to 7.2 with KOH. For the K⁺-rich high-BAPTA pipette solution, CaCl₂ was removed, and 20 mM BAPTA was added. In this case, in order to adjust the total concentration of K⁺ to 134 mM and the pH to 7.2, the concentration of KCl was reduced to ~67 mM, and KOH was added. The Cs⁺-rich pipette solution contained CsCl, 80; Na₂GTP, 1.0; creatine, 5; MgATP, 1.0; glucose, 20; HEPES, 10; BAPTA, 10; and CaCl₂, 4.6 (calculated free calcium=100 nM), with the pH adjusted to 7.4 with CsOH.

Drugs: The following drugs were used: 1-[2-(4-Methoxyphenyl)-2-[3-(4-methoxyphenyl)propoxy]ethyl-1H-imidazole hydrochloride (SKF), cromakalim (Tocris Bioscience, Bristol, U.K.), carbamylcholine chloride (CCh), glibenclamide (Sigma-Aldrich Co. LLC, St. Louis, MO, U.S.A.), tetraethylammonium Chloride (TEA; Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) and caffeine (Wako Pure Chemical Industries Ltd., Osaka, Japan). Cromakalim and glibenclamide were dissolved in dimethyl sulfoxide to form a stock solution and was stored at −20°C. The drug concentrations herein are expressed as the final concentrations that were applied to the cells.

Data analysis: The amplitude of I_KATP that was induced by cromakalim was measured, and the difference in the current level after the application of glibenclamide (10 µM) was calculated. In order to determine the current-voltage (I-V) relationships of I_KATP, a 350-msec ramp pulse that ranged from −100 mV to 60 mV was applied to the cells before and after SKF application, and the I-V curves were constructed by subtracting the current after the application of glibenclamide from the current in the presence of cromakalim alone or with SKF [31]. The amplitude of I_Kv was measured by subtracting the leakage component from the outward current that was induced by the depolarizing pulses [44]. The leakage component was estimated in each experiment as follows. A 10-mV hyperpolarizing pulse was applied to the cells at the holding potential (−40 mV), and the amplitude of the current was considered the leakage current for the 10-mV step. The leakage current that corresponded to the 100-mV depolarizing step pulse was subtracted from each of the outward currents that were evoked by the depolarizing pulse. The amplitudes of the spontaneous transient outward I_BK (STOC) and caffeine-induced I_BK were calculated as the difference from the holding current. The amplitude of mI_TRPC was calculated as the difference from the holding current before the application of CCh. The extent of the SKF-induced inhibition of each current was expressed as the percentage reduction from the control current level immediately before its application.

The values are presented herein as mean ± S.E.M. The statistical significance of the difference between two groups was tested with a Student’s unpaired or paired t-test. When multiple groups were compared, a one-way analysis of variance (ANOVA) that was followed by a posthoc Bonferroni-type multiple comparison test was used. The differences were considered significant when the P values were less than 0.05. The averaged curves that represented the relationship between the concentration of SKF and its inhibitory effects were constructed by curve fitting with GraphPad Prism 5.0 (GraphPad Software, Inc., La Jolla, CA, U.S.A.), and these provided the SKF concentration that was required to produce half maximal inhibition (IC₅₀). The cells that were obtained from male and female mice showed similar amplitudes for I_KATP, I_Kv, STOC and mI_TRPC.

RESULTS

Current through the K_ATP channels in mouse small intestinal smooth muscles: The cells were dialyzed with the K⁺-rich high-BAPTA pipette solution and bathed in the 60-mM K⁺ external solution at a holding potential of −80 mV. Under these conditions, application of the K_ATP channel opener cromakalim (10 µM) induced an inward current, as has been reported by Nuttle & Farley [31]. The current developed progressively with time and reached a plateau about 1–3 min later (Fig. 1A). Further application of the K_ATP channel inhibitor, glibenclamide (10 µM), almost completely abolished the current. The mean amplitude of the cromakalim-induced current was −895.2 ± 66.5 pA (n=39). When the extracellular K⁺ in the bath solution was completely replaced with Cs⁺, the cromakalim-induced inward current was immediately abolished at −80 mV (n=5; data not shown). The I-V relationships of the cromakalim-induced current were determined by applying a voltage ramp pulse that ranged from −100 mV up to +60 mV over 350 msec (Fig. 1Ba). As shown in Fig 1Bb, the I-V curve was linear across the voltages tested. The polarity of the current was reversed at −18.8 ± 2.0 mV (n=7), which corresponded to the estimated E_K of −21 mV, thus indicating that this current was carried through K⁺ channels. These results indicated that the cromakalim-induced current was carried through K_ATP channels.

Fig. 1.

Characterization of cromakalim-induced K_ATP channel currents (I_KATP) in single longitudinal smooth muscle cells isolated from mouse small intestine. Cells were dialyzed with the K⁺-rich high-BAPTA pipette solution and bathed in the 60-mM K⁺ external solution at a holding potential of −80 mV. Cromakalim (10 µM) was extracellularly applied to the cells. A: a typical example of the cromakalim-induced sustained inward I_KATP. B shows the current-voltage (I-V) relationship of cromakalim-induced I_KATP. (a): a typical current trace, where a voltage ramp pulse from −100 mV up to +60 mV over 350 msec was applied in the presence of cromakalim alone (current a) and in combination with glibenclamide (10 µM) (current b). (b): the I-V curve constructed by subtraction of the current b from the current a.

SKF reversibly suppressed the K_ATP channel current (I_KATP): After the cromakalim-induced I_KATP reached a steady level, SKF (10 µM) was applied in the presence of cromakalim. As shown in Fig. 2A, SKF rapidly decreased the amplitude of I_KATP and abolished the current within about 1 min. The time constant (τ) of the suppression (7.3 ± 1.6 sec, n=7) was significantly shorter than the value (35.6 ± 5.8 sec, n=10) in the glibenclamide-induced abolition of I_KATP. The removal of SKF from the bath solution allowed the recovery of I_KATP with a slower time course. Figure 2Ba shows a typical example of I_KATP suppression that was induced by cumulative applications of SKF with a series of ascending concentrations (0.03–10 µM). The inhibitory effects increased with increasing SKF concentrations, and the maximal inhibition was attained at 10 µM. The mean IC₅₀ value that was estimated by the curve fitting of the data from each myocyte was 0.85 ± 0.12 µM (n=6) (Fig. 2Bb).

Fig. 2.

Effects of SKF96365 (SKF) on cromakalim-induced I_KATP. I_KATP was induced by cromakalim (10 µM) in the cells under the same condition as those mentioned in Fig. 1. A shows a typical current trace when SKF (10 µM) was extracellularly applied to the cell in the presence of cromakalim (10 µM). B shows a relationship between concentration of SKF and its inhibitory effect on I_KATP. (a): a typical example of the I_KATP suppression induced by cumulative application of SKF (0.03-10 µM). (b): the averaged concentration-inhibition curve of I_KATP. Each point indicates the mean ± S.E.M. of measurements in 6 cells. C shows a voltage sensitivity of the SKF-induced I_KATP suppression. (a): a typical example of I_KATP suppression induced by SKF (1 µM), where a voltage ramp pulse from −100 mV up to +60 mV over 350 msec was applied in the presence of cromakalim (10 µM) alone (current a), cromakalim + SKF (1 µM) (current b) and those agents together with glibenclamide (10 µM) (current c). (b): the I-V curves constructed by subtraction of the current c from the current a or b. (c): the mean percentage of I_KATP suppression at each holding potential. Each column indicates mean + 1 S.E.M. The numbers of cells used are presented in parenthesis.

In order to investigate the voltage dependency of the SKF-induced I_KATP suppression, the ramp pulses were applied in the absence and presence of SKF (1 µM), and the I-V curves were obtained, as shown in Fig. 2Ca. The I-V curves of I_KATP remained linear, and the reversal potential was almost unchanged after the application of SKF (cromakalim alone, −18.5 ± 0.82 mV and cromakalim with SKF, −16.4 ± 0.97 mV; n=6) (Fig. 2Cb). I_KATP suppression at −80, −40 and 40 mV was comparable (Fig. 2Cc). These results indicated that the SKF-induced I_KATP suppression was voltage-independent.

Comparison of the SKF-induced mI_TRPC suppression with the I_KATP suppression: The cells that were bathed in the Cs⁺-rich external solution were clamped at a voltage of −60 mV with patch pipettes that were filled with the Cs⁺-rich pipette solution in order to block any K⁺ currents ([Ca²⁺]_i buffered to 100 nM). Under these conditions, the application of CCh (100 µM) induced an inward mI_TRPC, as previously reported by Sakamoto et al. [34]. SKF (10 µM), which completely abolished I_KATP, was applied to the bath solution in the presence of CCh when the mI_TRPC reached a steady level. The amplitude of the mI_TRPC gradually decreased after the application of SKF and reached a plateau about 1 min later (Fig. 3Aa). However, the mean suppression of mI_TRPC was only 55.5 ± 5.6% (n=8) (Fig. 3B). The time constant (τ) of the inhibitory effect was 38.7 ± 8.3 sec (n=7), which was significantly longer than the value in the I_KATP suppression. When a higher concentration of SKF (30 µM) was applied, it almost completely abolished the mI_TRPC (n=4, Fig. 3Ab and 3B). The abolition had a longer time constant (τ), 12.6 ± 5.3 sec (n=3), than the value in I_KATP suppression.

Fig. 3.

Effects of SKF96365 (SKF) on carbachol (CCh)-induced mI_TRPC. Cells bathed in the Cs⁺-rich external solution were held under voltage clamp at −60 mV using patch pipettes filled with the Cs⁺-rich pipette solution to block any K⁺ currents. Aa and Ab show a typical current trace when SKF (10 and 30 µM) was extracellularly applied to the cell in the presence of CCh (100 µM), respectively. B shows the mean percentages of I_KATP and mI_TRPC suppressions. Each column indicates mean + 1 S.E.M. The numbers of cells used are presented in parenthesis. * in (B) represents significantly smaller inhibition of mI_TRPC induced by SKF (10 µM) relative to the I_KATP one (P<0.05). # in (B) represents significantly greater inhibition of mI_TRPC induced by SKF (30 µM) relative to the corresponding one induced by SKF (10 µM) (P<0.05).

SKF reversibly suppressed I_Kv: The cells that were bathed in the Ca²⁺-free PSS were clamped at a voltage of −80 mV with patch pipettes that were filled with the K⁺-rich high-BAPTA pipette solution. Under these conditions, a 2 sec-step pulse to 20 mV was repeatedly applied every 20 sec in order to elicit I_Kv. Applications of the depolarizing pulse evoked an initial transient outward current (IK_Vpeak) with an additional sustained component (IK_Vsustained), as was previously described in mouse intestinal smooth muscles [25]. As shown in Fig. 4Aa, the amplitudes of both IK_Vpeak and IK_Vsustained reduced after the application of SKF (1 µM). The I_Kv suppressions developed progressively with time and reached a plateau about 1–2 min later (Fig. 4Ab). The suppression of the IK_Vpeak had a longer time constant (τ) (66.8 ± 23.1 sec, n=8) than the IK_Vsustained suppression did (21.1 ± 5.4 sec, n=8)_, although the difference was not statistically significant. The mean suppressions of IK_Vpeak and IK_Vsustained by SKF (1 µM) were significantly smaller than I_KATP suppression (Fig. 4Ac). The removal of SKF allowed for a partial recovery of IK_Vpeak and IK_Vsustained with a slow time course (Fig. 4Ab). The restored I_Kv was strongly inhibited by the application of the K⁺ channel blocker TEA (30 mM).

Fig. 4.

Effects of SKF on voltage-gated K⁺ channel currents (I_Kv). Cells bathed in the Ca²⁺-free PSS were held under voltage clamp at −80 mV using patch pipettes filled with the K⁺-rich high-BAPTA pipette solution. A 2 sec step pulse to 20 mV was repeatedly applied every 20 sec in order to elicit I_Kv. Aa: IK_Vpeak (●) and IK_Vsustained (Δ) recorded in a cell when SKF (1 µM) was extracellularly applied. Ab: time courses of the change in IK_Vpeak (●) and IK_Vsustained (Δ) in the cell in a plotted against time (the beginning of depolarizing pulse applications was taken as zero) when SKF (1 µM) was extracellularly applied. Points a-c in the graph correspond with actual I_Kv records (a–c) in Aa. Ac shows the mean percentages of I_KATP and I_Kv suppressions induced by SKF (1 µM). Each column indicates mean + 1 S.E.M. The numbers of cells used are presented in parenthesis. B (a): time courses of the change in IK_Vpeak (●) and IK_Vsustained (Δ) when SKF (0.03-30 µM) was cumulatively applied. Bb: the averaged concentration-inhibition curves of IK_Vpeak (●) and IK_Vsustained (Δ). Each point in Bb indicates the mean ± S.E.M. of measurements in 3–5 cells. # in (Ac) represents significantly smaller inhibition of I_Kv induced by SKF (1 µM) relative to the I_KATP one (P<0.05). Asterisks in (Ac) and (Bb) represent significantly greater inhibition of the IK_Vsustained relative to the IK_Vpeak (P<0.05).

The cumulative application of SKF (0.03–30 µM) at increasing concentrations inhibited both IK_Vpeak and IK_Vsustained in a concentration-dependent manner. The inhibitory effects reached a maximum at 30 µM (Fig. 4Ba). However, the IK_Vsustained suppressions were significantly larger than the IK_Vpeak suppressions at 3, 10 and 30 µM (Fig. 4Bb). The mean IC₅₀ value that was estimated by the curve fitting of the data from each myocyte was 3.3 ± 0.24 µM in the IK_Vpeak (n=5), which was significantly higher than the value in the IK_Vsustained (2.0 ± 0.35 µM, n=5). These IC₅₀ values were significantly higher than the value in the I_KATP suppression.

I_BK was less sensitive to SKF: It is well known that Ca²⁺ releases from Ca²⁺ stores can induce I_BK responses [34, 40]. Thus, under the appropriate conditions (see methods), I_BK that was evoked by the potent Ca²⁺ releaser caffeine was recorded in the presence of SKF. As shown in Fig. 5A, the STOCs that were evoked by Ca²⁺ sparks [7] occurred with various frequencies and amplitudes, as previously reported by Sakamoto et al. [33]. When [Ca²⁺]_i was strongly chelated, the STOCs were not observed at all (see Figs. 1 and 2). The K⁺ channel blocker TEA (5 mM) almost abolished the STOCs. In the presence of TEA, the application of CCh and the subsequent application of caffeine failed to evoke the I_BK (data not shown, n=3). These findings strongly indicated that the STOCs and caffeine-induced I_BK were conducted through BK channels. SKF (1 µM) had no obvious effects on the STOCs (Fig. 5A and 5B). The change rate of the STOC amplitude was similar to the time-dependent rundown that was observed in the cells without SKF (Fig. 5C). Caffeine (10 mM) consistently evoked a brief I_BK, even in the presence of SKF (1 µM) (Fig. 5B). The mean amplitude of the I_BK that was induced by caffeine did not differ significantly from the value without SKF (Fig. 5D). The higher concentration of SKF (10 µM) reduced the STOCs, but not the caffeine-induced I_BK (Fig. 5C and 5D).

Fig. 5.

Less sensitivity of SKF to Ca²⁺ activated K⁺ channel current (I_BK). Cells were bathed in PSS and held at a holding potential of 0 mV using the K⁺-rich low EGTA pipette solution. Spontaneous transient or caffeine (10 mM)-induced Ca²⁺ release events were detected as I_BK. A and B: typical current traces in the absence (A) and presence (B) of SKF (1 µM), respectively. C shows the mean change rates of spontaneous transient outward I_BK (STOCs) in the presence or absence of SKF. D shows summary of caffeine-induced I_BK in the presence or absence of SKF. Each column indicates mean + 1 S.E.M. The numbers of cells used are presented in parentheses. * in (C) represents significantly larger reduction in STOCs in the presence of SKF (10 µM) relative to the time-dependent rundown of STOCs in the absence of SKF (P<0.05).

DISCUSSION

Activation of the muscarinic receptors induces the opening of cationic channels comprising TRPC4 and 6 [43]. SKF has been used as an inhibitor of the TRPC channels [9, 26, 52]. However, it has been reported to inhibit other types of ion channels, including Ca_V1, Ca_V2, Ca_V3 [27, 37], Na_V1 [13] and inwardly or outwardly rectifying K⁺ channels [36]. In this study, we investigated the effects of SKF on the activities of the K_ATP, K_V and BK channels that are expressed in gastrointestinal smooth muscle cells.

K_ATP channels have been suggested to regulate electrical excitability in gastrointestinal smooth muscles [19,20,21]. Cromakalim induced a sustained inward current that was abolished by glibenclamide in small intestinal smooth muscles of the mouse, as has been reported by Nuttle & Farley [31]. The I-V curve of the cromakalim-induced current was almost linear, which was consistent with the results obtained for rabbit esophageal [12] and swine tracheal [31] smooth muscle cells. The reversal potential of the current was similar to the estimated E_K value, and the replacement of extracellular K⁺ with Cs⁺ abolished the current, which indicated that the charge carrier of this current was K⁺. These results indicated that the cromakalim-induced current was carried through the K_ATP channels. The extracellular application of SKF reversibly inhibited the cromakalim-induced I_KATP in a concentration-dependent manner. The I_KATP was abolished at a SKF concentration of 10 µM. Although SKF (10 µM) also inhibited the CCh-induced mI_TRPC, the mean percentage of suppression was only about 50% in the present study, which was consistent with the IC₅₀ (6.5–14.5 µM) that has been reported for SKF-induced mI_TRPC suppression [52]. The mean IC₅₀ value (0.85 µM) for the SKF-induced I_KATP suppression in the present study was obviously lower than the reported IC₅₀ value for the mI_TRPC suppression, which suggested that SKF suppressed the K_ATP channels with a higher affinity than that required for inhibition of TRPC channels.

The K_ATP channels are composed of Kir6 and SUR subunits. Two Kir6 genes (Kir6.1 and Kir6.2) and two SUR genes (SUR1 and SUR2) have been identified [1]. The pharmacological properties of the K_ATP channels, including the sensitivity to its channel opener and blocker, depend on the channel subunits [16, 17, 49]. K_ATP channels, which have been reported in pancreatic β cells, cardiac muscles and vascular smooth muscles, consist of the Kir6.2/SUR1 [10, 28], Kir6.2/SUR2A [4] and Kir6.1/SUR2B [29, 39, 49] subunits, respectively. Nakayama et al. [30] have reported the expression of Kir6.1 and SUR2B in mouse ileal smooth muscles. Quayle et al. [32] have reported that the IC₅₀ values of glibenclamide, tolbutamide, which is a selective K_ATP channel inhibitor, and TEA, which is a nonselective K⁺ channel blocker, were 101 nM, 351 µM and 6.2 mM, respectively, in the smooth muscles from the rabbit mesenteric artery. Taken together with our results, the order of affinity to the K_ATP channels appears to be glibenclamide >SKF >> tolbutamide >> TEA. Future investigations are needed on the sensitivity of SKF to other types of K_ATP channels

Because the I-V curve of the I_KATP that was obtained from the myocytes in the presence of SKF remained linear, the inhibitory effects of SKF on I_KATP seemed to be voltage-independent. Glibenclamide inhibits the K_ATP by occupying sulfonylurea receptors [8]. The I_KATP abolition that was induced by glibenclamide developed more slowly than that induced by SKF, as shown by the time constants of the abolitions. Thus, SKF may inhibit the K_ATP in a different manner from that of glibenclamide. The short time course of the SKF-induced I_KATP suppression implies the involvement of a simple mechanism, such as direct blockade of the channel pore, which is located in Kir6, as was suggested in caffeine-induced I_KATP suppression [41]. If so, the binding site might not be a voltage sensor of the channels, and its binding would be reversible. Future investigations with the single channel patch clamp technique are needed to elucidate the blockade mechanisms of SKF.

Zholos et al. [52] have reported that the inhibition of mI_TRPC by SKF was greater at positive membrane potentials than that at negative ones, which indicated the voltage-dependency of its inhibitory effects. Thus, the mechanisms of SKF-induced suppression of the mI_TRPC may be somewhat different from those for the I_KATP. This idea is supported by the fact that the time constant of the SKF-induced mI_TRPC suppression was much longer than the corresponding value of I_KATP suppression. The different characteristics of these suppressions can provide useful information when SKF is used as a blocker of TRPC or K_ATP.

K_V channels, which are widely expressed in gastrointestinal smooth muscles, are involved in the setting of the resting membrane potential and the determination of amplitude and rhythmicity of action potentials [22, 48]. Application of the depolarizing pulse evoked a biphasic outward current that consisted of IK_Vpeak and IK_Vsustained and that was strongly inhibited by TEA. SKF (1 µM), which was a concentration that corresponded to the IC₅₀ in the I_KATP suppression, reversibly inhibited the I_Kv. However, the mean percentages of the SKF-induced I_Kv suppressions were only ~30%, and the IC₅₀ values (>2 µM), which were significantly higher than the value in the I_KATP suppression, were similar to the value reported for mI_TRPC suppression. A great contribution of the K_V4 and K_V2 family to the IK_Vpeak and IK_Vsustained, respectively, has been suggested [3, 25, 35]. The heterogeneity of I_Kv can make it difficult to determine the sensitivity of the K_V family to SKF due to the lack of commercial blockers with high selectivity. However, the IK_Vsustained suppression was greater than the IK_Vpeak suppression with a lower IC₅₀ value (IK_Vsustained: 2.2 µM vs. IK_Vpeak: 3.3 µM). Furthermore, the time constant of the IK_Vsustained suppression was shorter than the value for the IK_Vpeak suppression_. These findings may reflect decreased sensitivity of the K_V4 family for SKF compared with K_V2. The short time course of the SKF-induced IK_Vsustained suppression may imply a direct bind of SKF to the channel, as indicated in TEA or 4-Aminopyridine-induced K_V channel suppressions [38, 42] as well as the I_KATP suppression.

In gastrointestinal smooth muscles, BK channels are expressed and supposed to involve in a negative feedback mechanism to prevent cytosolic Ca²⁺ overload induced by the muscarinic Ca²⁺ mobilization [46]. SKF (1 µM) had no significant effects on STOCs and caffeine-induced I_BK which reflected the Ca²⁺ release from the internal stores in the myocytes. These results indicated that BK channel is less sensitive to SKF. Taken together, SKF can inhibit K_ATP, K_V and BK channels in addition to TRPC channel, and the order of affinity of SKF to those channels appears to be K_ATP >K_V ≈ TRPCs >BK. SKF (3–50 µM) has been reported to evoke membrane depolarization in gastrointestinal muscles (15, 51). The depolarization can be explained by the inhibitory effects of SKF on those K⁺ channels. The depolarization can cause to underestimate a contribution of TRPC channel to mechanical and electrical responses. Thus, the inhibitory effects of those K⁺ channels should be considered when SKF is used as a TRPC channel blocker in gastrointestinal smooth muscles. Characterizing the SKF-induced suppression, such as IC₅₀ and voltage-dependency, and additional tests with other TRPC blockers may be necessary to assess an involvement of TRPC channel in a response.

In conclusion, SKF can suppress K_ATP, K_V and BK channels. The order of affinity of SKF to those channels is K_ATP > K_V > BK.