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 .
a small aliquot was placed on coverglass and stored at 4c for 27 hr until its use on the 
 same day .
whole - cell current recording : whole - cell membrane current recordings were performed at room 
 temperature ( 2125c ) with standard patch - clamp techniques   with 47 
 m 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 ikatp 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 kv and bk channels [ 12 , 20 , 31 ] . under these conditions , the 
 k equilibrium potential ( ek )
ikv was recorded in cells that was bathed in ca - 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 cav1.2 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 ikv . when currents were recorded through ibk , 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 
 ( mitrpc ) 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 ca - 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 . ,
cromakalim and glibenclamide were dissolved in dimethyl sulfoxide 
 to form a stock solution and was stored at 20c .
the drug concentrations herein are expressed as the final 
 concentrations that were applied to the cells .
data analysis : the amplitude of ikatp that was induced by cromakalim was measured , 
 and the difference in the current level after the application of glibenclamide ( 10 m ) was 
 calculated . in order to determine the current - voltage ( i - v ) relationships of ikatp , 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 .
the amplitude of 
 ikv was measured by subtracting the leakage component from the outward current that was induced by 
 the depolarizing pulses .
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 ibk ( stoc ) and 
 caffeine - induced ibk were calculated as the difference from the holding current .
the amplitude of 
 mitrpc 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 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 
 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 ikatp , 
 ikv , stoc and mitrpc .
current through the katp 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 
 katp channel opener cromakalim ( 10 m ) induced an inward current , as has been 
 reported by nuttle & farley .
the current developed progressively 
 with time and reached a plateau about 13 min later ( fig .
1.characterization of cromakalim - induced katp channel currents ( ikatp ) 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 .
b shows the 
 current - voltage ( i - v ) relationship of cromakalim - induced ikatp .
( 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 m ) ( current b ) . 

( b ) : the i - v curve constructed by subtraction of the current b from the current a. ) .
further application of the katp channel inhibitor , glibenclamide ( 10 m ) , 
 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 ek 
 of 21 mv , thus indicating that this current was carried through k channels .
these results indicated 
 that the cromakalim - induced current was carried through katp channels .
characterization of cromakalim - induced katp channel currents ( ikatp ) 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 .
b shows the 
 current - voltage ( i - v ) relationship of cromakalim - induced ikatp .
( 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 m ) ( current b ) . 

( b ) : the i - v curve constructed by subtraction of the current b from the current a. skf reversibly suppressed the katp channel current ( ikatp ) : after the 
 cromakalim - induced ikatp reached a steady level , skf ( 10 m ) was applied in the 
 presence of cromakalim . as shown in fig .
ikatp was induced by 
 cromakalim ( 10 m ) in the cells under the same condition as those mentioned in fig .
1 . a shows a typical current trace when skf ( 10 
 m ) was extracellularly applied to the cell in the presence of cromakalim ( 10 
 m ) .
b shows a relationship between concentration of skf and its inhibitory effect on 
 ikatp .
( a ) : a typical example of the ikatp suppression induced by cumulative 
 application of skf ( 0.03 - 10 m ) .
( a ) : a typical example of ikatp 
 suppression induced by skf ( 1 m ) , where a voltage ramp pulse from 100 mv up to + 60 mv 
 over 350 msec was applied in the presence of cromakalim ( 10 m ) alone ( current a ) , 
 cromakalim + skf ( 1 m ) ( current b ) and those agents together with glibenclamide ( 10 
 m ) ( 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 ikatp suppression at each holding potential . 

, skf rapidly decreased the amplitude of ikatp and abolished the current within about 1 min . 

the time constant (  ) 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 ikatp .
the removal of skf from the bath 
 solution allowed the recovery of ikatp with a slower time course .
figure 2ba shows a typical example of ikatp suppression that was induced by cumulative 
 applications of skf with a series of ascending concentrations ( 0.0310 m ) .
the inhibitory 
 effects increased with increasing skf concentrations , and the maximal inhibition was attained at 10 
 m .
the mean ic50 value that was estimated by the curve fitting of the data from 
 each myocyte was 0.85  0.12 m ( n=6 ) ( fig .
ikatp was induced by 
 cromakalim ( 10 m ) in the cells under the same condition as those mentioned in fig .
1 . a shows a typical current trace when skf ( 10 
 m ) was extracellularly applied to the cell in the presence of cromakalim ( 10 
 m ) .
b shows a relationship between concentration of skf and its inhibitory effect on 
 ikatp .
( a ) : a typical example of the ikatp suppression induced by cumulative 
 application of skf ( 0.03 - 10 m ) .
( a ) : a typical example of ikatp 
 suppression induced by skf ( 1 m ) , where a voltage ramp pulse from 100 mv up to + 60 mv 
 over 350 msec was applied in the presence of cromakalim ( 10 m ) alone ( current a ) , 
 cromakalim + skf ( 1 m ) ( current b ) and those agents together with glibenclamide ( 10 
 m ) ( 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 ikatp suppression at each holding potential . 

the numbers of cells used are presented in parenthesis . in order to investigate the voltage dependency of the skf - induced ikatp suppression ,
the ramp pulses 
 were applied in the absence and presence of skf ( 1 m ) , and the i - v curves were obtained , as 
 shown in fig .
the i - v curves of ikatp 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 .
these results indicated that the skf - induced ikatp suppression was 
 voltage - independent .
comparison of the skf - induced mitrpc suppression with the ikatp 
 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 ( [ ca]i buffered to 100 nm ) . under these conditions , 

the application of cch ( 100 m ) induced an inward mitrpc , as previously reported by 
 sakamoto et al . .
skf ( 10 m ) , 
 which completely abolished ikatp , was applied to the bath solution in the presence of cch when the 
 mitrpc reached a steady level .
the amplitude of the mitrpc gradually decreased after the 
 application of skf and reached a plateau about 1 min later ( fig . 

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 m ) was extracellularly applied to the cell in 
 the presence of cch ( 100 m ) , respectively .
the numbers 
 of cells used are presented in parenthesis . * in ( b ) represents significantly smaller inhibition of 
 mitrpc induced by skf ( 10 m ) relative to the ikatp one 
 ( p<0.05 ) .
# in ( b ) represents significantly greater inhibition of mitrpc 
 induced by skf ( 30 m ) relative to the corresponding one induced by skf ( 10 
 m ) ( p<0.05 ) . ) .
however , the mean suppression of mitrpc was only 55.5  5.6% ( n=8 ) ( fig .
the time constant (  ) of the inhibitory effect was 38.7  8.3 sec ( n=7 ) , which 
 was significantly longer than the value in the ikatp suppression . when a higher concentration of skf 
 ( 30 m ) was applied , it almost completely abolished the mitrpc ( n=4 , fig .
the abolition had a longer time constant (  ) , 12.6  5.3 
 sec ( n=3 ) , than the value in ikatp suppression .
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 m ) was extracellularly applied to the cell in 
 the presence of cch ( 100 m ) , respectively .
the numbers 
 of cells used are presented in parenthesis . * in ( b ) represents significantly smaller inhibition of 
 mitrpc induced by skf ( 10 m ) relative to the ikatp one 
 ( p<0.05 ) .
# in ( b ) represents significantly greater inhibition of mitrpc 
 induced by skf ( 30 m ) relative to the corresponding one induced by skf ( 10 
 m ) ( p<0.05 ) .
skf reversibly suppressed ikv : the cells that were bathed in the 
 ca - 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 ikv .
applications of the depolarizing pulse evoked 
 an initial transient outward current ( ikvpeak ) with an additional sustained component 
 ( ikvsustained ) , as was previously described in mouse intestinal smooth muscles . as shown in fig .
cells bathed in the 
 ca - 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
aa : ikvpeak (  ) and ikvsustained (  ) 
 recorded in a cell when skf ( 1 m ) was extracellularly applied .
ab : time courses of the 
 change in ikvpeak (  ) and ikvsustained (  ) in the cell in a plotted against time 
 ( the beginning of depolarizing pulse applications was taken as zero ) when skf ( 1 m ) was 
 extracellularly applied .
points a - c in the graph correspond with actual ikv records ( a  c ) in 
 aa .
ac shows the mean percentages of ikatp and ikv suppressions induced by skf ( 1 
 m ) .
b ( a ) : time courses of the change in ikvpeak (  ) and ikvsustained (  ) 
 when skf ( 0.03 - 30 m ) was cumulatively applied .
bb : the averaged concentration - inhibition 
 curves of ikvpeak (  ) and ikvsustained (  ) .
each point in bb indicates the mean  
 s.e.m . of measurements in 35 cells .
# in ( ac ) represents significantly smaller inhibition of 
 ikv induced by skf ( 1 m ) relative to the ikatp one 
 ( p<0.05 )
. asterisks in ( ac ) and ( bb ) represent significantly greater inhibition of 
 the ikvsustained relative to the ikvpeak ( p<0.05 ) . , the amplitudes of both ikvpeak and ikvsustained reduced after the application of 
 skf ( 1 m ) .
the ikv suppressions developed progressively with time and reached a 
 plateau about 12 min later ( fig .
the suppression of the 
 ikvpeak had a longer time constant (  ) ( 66.8  23.1 sec , n=8 ) than the ikvsustained 
 suppression did ( 21.1  5.4 sec , n=8 ) , although the difference was not statistically significant .
the 
 mean suppressions of ikvpeak and ikvsustained by skf ( 1 m ) were 
 significantly smaller than ikatp suppression ( fig .
the removal of skf allowed for a partial recovery of ikvpeak and ikvsustained with a slow 
 time course ( fig .
the restored ikv was strongly 
 inhibited by the application of the k channel blocker tea ( 30 mm ) .
cells bathed in the 
 ca - 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
aa : ikvpeak (  ) and ikvsustained (  ) 
 recorded in a cell when skf ( 1 m ) was extracellularly applied .
ab : time courses of the 
 change in ikvpeak (  ) and ikvsustained (  ) in the cell in a plotted against time 
 ( the beginning of depolarizing pulse applications was taken as zero ) when skf ( 1 m ) was 
 extracellularly applied .
points a - c in the graph correspond with actual ikv records ( a  c ) in 
 aa .
ac shows the mean percentages of ikatp and ikv suppressions induced by skf ( 1 
 m ) .
b ( a ) : time courses of the change in ikvpeak (  ) and ikvsustained (  ) 
 when skf ( 0.03 - 30 m ) was cumulatively applied .
bb : the averaged concentration - inhibition 
 curves of ikvpeak (  ) and ikvsustained (  ) .
each point in bb indicates the mean  
 s.e.m . of measurements in 35 cells .
# in ( ac ) represents significantly smaller inhibition of 
 ikv induced by skf ( 1 m ) relative to the ikatp one 
 ( p<0.05 )
. asterisks in ( ac ) and ( bb ) represent significantly greater inhibition of 
 the ikvsustained relative to the ikvpeak ( p<0.05 ) .
the cumulative application of skf ( 0.0330 m ) at increasing concentrations inhibited both 
 ikvpeak and ikvsustained in a concentration - dependent manner .
4ba ) . however , the 
 ikvsustained suppressions were significantly larger than the ikvpeak suppressions at 3 , 
 10 and 30 m ( fig .
the mean ic50 
 value that was estimated by the curve fitting of the data from each myocyte was 3.3  0.24 m 
 in the ikvpeak ( n=5 ) , which was significantly higher than the value in the ikvsustained 
 ( 2.0  0.35 m , n=5 ) .
ibk was less sensitive to skf : it is well known that ca releases from 
 ca stores can induce ibk responses [ 34 , 40 ] .
thus , under the appropriate conditions ( see methods ) , ibk 
 that was evoked by the potent ca releaser caffeine was recorded in the presence of skf . as shown in 
 fig . 5afig .
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 ca release events were 
 detected as ibk .
a and b : typical current traces in the absence ( a ) and presence ( b ) of skf ( 1 
 m ) , respectively .
c shows the mean change rates of spontaneous transient outward 
 ibk ( stocs ) in the presence or absence of skf .
d shows summary of caffeine - induced 
 ibk in the presence or absence of skf .
the numbers of 
 cells used are presented in parentheses . * in ( c ) represents significantly larger reduction in stocs in 
 the presence of skf ( 10 m ) relative to the time - dependent rundown of stocs in the 
 absence of skf ( p<0.05 ) . , the stocs that were evoked by ca sparks   occurred 
 with various frequencies and amplitudes , as previously reported by sakamoto et al . .
when [ ca]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 ibk ( data not 
 shown , n=3 ) .
these findings strongly indicated that the stocs and caffeine - induced ibk were conducted 
 through bk channels .
skf ( 1 m ) 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 .
caffeine 
 ( 10 mm ) consistently evoked a brief ibk , even in the presence of skf ( 1 m ) ( fig .
the mean amplitude of the ibk that was induced by 
 caffeine did not differ significantly from the value without skf ( fig . 

the higher concentration of skf ( 10 m ) reduced the stocs , but not the 
 caffeine - induced ibk ( fig . 5c and 5d ) .
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 ca release events were 
 detected as ibk .
a and b : typical current traces in the absence ( a ) and presence ( b ) of skf ( 1 
 m ) , respectively .
c shows the mean change rates of spontaneous transient outward 
 ibk ( stocs ) in the presence or absence of skf .
d shows summary of caffeine - induced 
 ibk in the presence or absence of skf .
the numbers of 
 cells used are presented in parentheses . * in ( c ) represents significantly larger reduction in stocs in 
 the presence of skf ( 10 m ) relative to the time - dependent rundown of stocs in the 
 absence of skf ( p<0.05 ) .
activation of the muscarinic receptors induces the opening of cationic channels comprising trpc4 and 6 .
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 cav1 , 
 cav2 , cav3 [ 27 , 37 ] , nav1   and inwardly or outwardly rectifying 
 k channels . in this study , we investigated the effects 
 of skf on the activities of the katp , kv and bk channels that are expressed in 
 gastrointestinal smooth muscle cells .
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 
 .
the i - v curve of the cromakalim - induced current was almost 
 linear , which was consistent with the results obtained for rabbit esophageal   and swine tracheal   smooth muscle cells .
the reversal 
 potential of the current was similar to the estimated ek 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 
 katp channels .
the extracellular application of skf reversibly inhibited the cromakalim - induced 
 ikatp in a concentration - dependent manner .
although skf ( 10 m ) also inhibited the cch - induced 
 mitrpc , the mean percentage of suppression was only about 50% in the present study , which was 
 consistent with the ic50 ( 6.514.5 m ) that has been reported for skf - induced 
 mitrpc suppression .
the mean ic50 value 
 ( 0.85 m ) for the skf - induced ikatp suppression in the present study was obviously 
 lower than the reported ic50 value for the mitrpc suppression , which suggested that skf 
 suppressed the katp channels with a higher affinity than that required for inhibition of trpc 
 channels .
two kir6 genes ( kir6.1 and kir6.2 ) and two 
 sur genes ( sur1 and sur2 ) have been identified .
the pharmacological 
 properties of the katp channels , including the sensitivity to its channel opener and blocker , depend 
 on the channel subunits [ 16 , 17 , 
 49 ] .
katp channels , which have been reported in pancreatic  
 cells , cardiac muscles and vascular smooth muscles , consist of the kir6.2/sur1 [ 10 , 28 ] , kir6.2/sur2a 
 and kir6.1/sur2b [ 29 , 39 , 49 ] subunits , respectively .
quayle et al .   have reported that the 
 ic50 values of glibenclamide , tolbutamide , which is a selective katp channel inhibitor , 
 and tea , which is a nonselective k channel blocker , were 101 nm , 351 m 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 katp channels appears to be glibenclamide > skf > > tolbutamide 
 > > tea .
future investigations are needed on the sensitivity of skf to other types of katp 
 channels because the i - v curve of the ikatp that was obtained from the myocytes in the presence of skf 
 remained linear , the inhibitory effects of skf on ikatp seemed to be voltage - independent . 

the ikatp 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 katp in a 
 different manner from that of glibenclamide .
the short time course of the skf - induced ikatp 
 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 ikatp suppression .
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 .
have reported that the inhibition of 
 mitrpc 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 mitrpc may be somewhat different from those for the ikatp .
this idea is supported by 
 the fact that the time constant of the skf - induced mitrpc suppression was much longer than the 
 corresponding value of ikatp suppression .
the different characteristics of these suppressions can 
 provide useful information when skf is used as a blocker of trpc or katp .
kv 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 ikvpeak and 
 ikvsustained and that was strongly inhibited by tea .
skf ( 1 m ) , which was a 
 concentration that corresponded to the ic50 in the ikatp suppression , reversibly inhibited 
 the ikv .
however , the mean percentages of the skf - induced ikv suppressions were only ~30% , 
 and the ic50 values ( > 2 m ) , which were significantly higher than the value in 
 the ikatp suppression , were similar to the value reported for mitrpc suppression . a great 
 contribution of the kv4 and kv2 family to the ikvpeak and 
 ikvsustained , respectively , has been suggested [ 3 , 25 , 35 ] .
the heterogeneity of 
 ikv can make it difficult to determine the sensitivity of the kv family to skf due to 
 the lack of commercial blockers with high selectivity .
however , the ikvsustained suppression was 
 greater than the ikvpeak suppression with a lower ic50 value ( ikvsustained : 2.2 
 m vs. ikvpeak : 3.3 m ) .
furthermore , the time constant of the 
 ikvsustained suppression was shorter than the value for the ikvpeak 
 suppression .
these findings may reflect decreased sensitivity of the kv4 family for skf 
 compared with kv2 .
the short time course of the skf - induced ikvsustained suppression may 
 imply a direct bind of skf to the channel , as indicated in tea or 4-aminopyridine - induced kv channel 
 suppressions [ 38 , 42 ] as well as 
 the ikatp suppression .
in gastrointestinal smooth muscles , bk channels are expressed and supposed to involve in a negative feedback 
 mechanism to prevent cytosolic ca overload induced by the muscarinic ca mobilization 
 .
skf ( 1 m ) had no significant effects on stocs 
 and caffeine - induced ibk which reflected the ca release from the internal stores in the 
 myocytes .
taken together , skf can inhibit 
 katp , kv and bk channels in addition to trpc channel , and the order of affinity of skf 
 to those channels appears to be katp > kv  trpcs > bk .
skf ( 350 m ) 
 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
the order of affinity of skf to 
 those channels is katp > kv > bk .
 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 ca2 + activated k+ channels 
 ( ibk ) in the absence and presence of skf in single small intestinal myocytes of mice with 
 patch - clamp techniques . 
 skf ( 10 m ) reversibly abolished ikatp that was induced by 
 cromakalim ( 10 m ) , 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 m , which was obviously lower than that 
 reported for the muscarinic cationic current . 
 in addition , skf ( 1 m  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 m ) 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 . 
MATERIALS AND METHODS
RESULTS
DISCUSSION