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(+) (IKATP), voltage-gated K(+) (IKv) and Ca(2+) activated K(+) channels (IBK) in the absence and presence of SKF in single small intestinal myocytes of mice with patch-clamp techniques. SKF (10 µM) reversibly abolished IKATP 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 IKATP suppression) reversibly inhibited the IKv 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 IBK and caffeine-induced IBK. 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.

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.

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.

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).

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).

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).