the bladder was removed from adult guinea pigs of either sex weighing 250 to 600 gm .
the animals had been sacrificed by cervical dislocation or injection with pentobarbitone in accordance with schedule 1 united kingdom home office regulations .
cells were plated to the bottom of a recording bath and constantly perfused with hanks ' physiological salt solution at room temperature .
the cell of interest was continuously superfused by a drug delivery system consisting of a pipette with a tip diameter of 200 m placed approximately 300 m away .
the solution in the drug delivery system could be switched to one containing a drug with a dead space time of less than 10 seconds .
icc were identified by the presence of lateral branches as previously described and selected for patch clamp recordings using the whole cell or the amphotericin perforated patch technique .
patch pipette electrodes made from borosilicate glass ( resistance 2 to 4 m ) were connected via an analog to digital / digital to analog converter ( national instruments ) to an axopatch 1d patch clamp amplifier ( axon instruments , foster city , california ) and a personal computer running winwcp software ( http://spider.science.strath.ac.uk/sipbs/software_ses.htm ) .
in whole cell mode after gigaseals were obtained access to the cell interior was gained by sharp suction .
in perforated patch recordings the cell was given a test depolarization from 60 to 40 mv every 30 seconds until the outward current developed to its maximal amplitude .
series resistance and the capacitive surge were compensated using circuitry from the axopatch 1d amplifier .
hanks ' physiological salt solution was composed of 130 mm na , 5.8 mm k , 135 mm cl , 4.16 mm hco3 , 0.44 mm h2po4 , 0.34 mm hpo4 , 0.4 mm so4 , 1.8 mm ca , 0.9 mm mg , 10 mm hepes , 10 mm glucose and 2.9 mm sucrose ph buffered to 7.40 with naoh .
modified pipette solution to eliminate the contribution from bk and katp was composed of 130 mm k , 6.2 mm na , 110 mm gluconate , 21 mm cl , 0.5 mm mg , 3 mm atp ( na salt ) , 0.1 mm guanosine triphosphate , 5 mm hepes and 5 mm egta adjusted to ph 7.2 with koh .
statistical comparisons were made using student 's t test with p < 0.05 level considered significant .
the bladder was removed from adult guinea pigs of either sex weighing 250 to 600 gm .
the animals had been sacrificed by cervical dislocation or injection with pentobarbitone in accordance with schedule 1 united kingdom home office regulations .
cells were plated to the bottom of a recording bath and constantly perfused with hanks ' physiological salt solution at room temperature .
the cell of interest was continuously superfused by a drug delivery system consisting of a pipette with a tip diameter of 200 m placed approximately 300 m away .
the solution in the drug delivery system could be switched to one containing a drug with a dead space time of less than 10 seconds .
icc were identified by the presence of lateral branches as previously described and selected for patch clamp recordings using the whole cell or the amphotericin perforated patch technique .
patch pipette electrodes made from borosilicate glass ( resistance 2 to 4 m ) were connected via an analog to digital / digital to analog converter ( national instruments ) to an axopatch 1d patch clamp amplifier ( axon instruments , foster city , california ) and a personal computer running winwcp software ( http://spider.science.strath.ac.uk/sipbs/software_ses.htm ) .
in whole cell mode after gigaseals were obtained access to the cell interior was gained by sharp suction .
in perforated patch recordings the cell was given a test depolarization from 60 to 40 mv every 30 seconds until the outward current developed to its maximal amplitude .
series resistance and the capacitive surge were compensated using circuitry from the axopatch 1d amplifier .
hanks ' physiological salt solution was composed of 130 mm na , 5.8 mm k , 135 mm cl , 4.16 mm hco3 , 0.44 mm h2po4 , 0.34 mm hpo4 , 0.4 mm so4 , 1.8 mm ca , 0.9 mm mg , 10 mm hepes , 10 mm glucose and 2.9 mm sucrose ph buffered to 7.40 with naoh .
modified pipette solution to eliminate the contribution from bk and katp was composed of 130 mm k , 6.2 mm na , 110 mm gluconate , 21 mm cl , 0.5 mm mg , 3 mm atp ( na salt ) , 0.1 mm guanosine triphosphate , 5 mm hepes and 5 mm egta adjusted to ph 7.2 with koh .
statistical comparisons were made using student 's t test with p < 0.05 level considered significant .
mccloskey identified a bk and voltage dependent delayed rectifier k current in icc and described a noninactivating , voltage dependent unidentified component . the possibility that novel kcnq currents might contribute to the unidentified portion of icc
detrusor icc were voltage clamped using k filled pipettes containing 5 mm egta and 3 mm atp to inhibit bk and katp channels , respectively , and depolarized to evoke outward currents .
it avoids the use of channel blocking drugs , which may interfere with the conductance of interest .
mean icc capacitance was 59  1 pf in 80 cells . a depolarizing step from 60 to 40 mv elicited noninactivating currents ( fig . 1 ,  a ) .
xe991 ( 10 m ) significantly decreased the outward current from 498  44 to 432  39 pa and 30 m further decreased the current to 376  34 pa ( each p < 0.05 ) .
the current - voltage relationship was examined by stepping from 60 mv to a range of potentials up to 40 mv in 10 mv increments .
the resulting family of currents was decreased by 10 m xe991 across the voltage range ( fig . 1 , c ) .
mean current at each potential was plotted against the test potential in the absence and presence of drug ( fig . 1 , d )
. subtracting xe991 sensitive currents from control currents revealed that the xe991 sensitive kcnq current was a voltage dependent , outwardly rectifying current that activated positive to 60 mv ( fig . 1 , e ) .
figure 2 , a shows the concentration dependent decrease in current amplitude ( evoked by stepping from 60 to 40 mv ) by the lesser potent analogue of xe991 , linopirdine .
although it was less effective than xe991 , linopirdine ( 10 m ) decreased control current from 689  130 to 585  155 pa in 5 cells , while increasing the concentration to 30 m further decreased the current to 435  94 pa ( p > 0.05 ) .
chromanol ( 10 m ) significantly decreased the control current of 490  70 to 368  39 pa and 30 m further decreased the residual current to 292  29 pa in 11 cells each ( each p < 0.05 , fig .
the effect of the kcnq channel openers flupirtine and mfa were then tested on icc outward currents ( fig .
3 ) . flupirtine acts as an anticonvulsant and muscle relaxant but it was more recently shown to activate kcnq currents , resulting in decreased excitability in neurons .
likewise mfa , a well - known anti - inflammatory drug and a kcnq 2/3 k channel opener in neurons , also increased outward currents in 5 icc ( fig .
cells were patched with amphotericin b filled pipettes using the modified pipette solution and studied in current clamp mode , so that the effect of kcnq drugs on resting membrane potential could be assessed . mean resting membrane potential in 18 icc under these conditions was 38  3 mv . while most recordings were electrically quiescent , spontaneous oscillations in resting membrane potential
application of xe991 ( 10 m ) depolarized the membrane potential and mean depolarization in 4 cells was 18  5 mv ( p < 0.05 , fig .
4 , b ) . chromanol 293b also depolarized 4 icc by 5  2 mv ( p < 0.05 ) , although its effect was less marked than that of xe991 ( fig .
the kcnq opener flupirtine produced a mean membrane hyperpolarization of 6  1 mv in 6 cells ( p < 0.001 , fig .
figure 4 , g shows the overall effect of kcnq channel drugs on icc resting membrane potential .
mccloskey identified a bk and voltage dependent delayed rectifier k current in icc and described a noninactivating , voltage dependent unidentified component . the possibility that novel kcnq currents might contribute to the unidentified portion of icc
detrusor icc were voltage clamped using k filled pipettes containing 5 mm egta and 3 mm atp to inhibit bk and katp channels , respectively , and depolarized to evoke outward currents .
it avoids the use of channel blocking drugs , which may interfere with the conductance of interest .
mean icc capacitance was 59  1 pf in 80 cells . a depolarizing step from 60 to 40 mv elicited noninactivating currents ( fig . 1 ,  a ) .
xe991 ( 10 m ) significantly decreased the outward current from 498  44 to 432  39 pa and 30 m further decreased the current to 376  34 pa ( each p < 0.05 ) .
the current - voltage relationship was examined by stepping from 60 mv to a range of potentials up to 40 mv in 10 mv increments .
the resulting family of currents was decreased by 10 m xe991 across the voltage range ( fig . 1 , c ) .
mean current at each potential was plotted against the test potential in the absence and presence of drug ( fig . 1 , d )
. subtracting xe991 sensitive currents from control currents revealed that the xe991 sensitive kcnq current was a voltage dependent , outwardly rectifying current that activated positive to 60 mv ( fig . 1 , e ) .
figure 2 , a shows the concentration dependent decrease in current amplitude ( evoked by stepping from 60 to 40 mv ) by the lesser potent analogue of xe991 , linopirdine .
although it was less effective than xe991 , linopirdine ( 10 m ) decreased control current from 689  130 to 585  155 pa in 5 cells , while increasing the concentration to 30 m further decreased the current to 435  94 pa ( p > 0.05 ) .
chromanol ( 10 m ) significantly decreased the control current of 490  70 to 368  39 pa and 30 m further decreased the residual current to 292  29 pa in 11 cells each ( each p < 0.05 , fig .
the effect of the kcnq channel openers flupirtine and mfa were then tested on icc outward currents ( fig .
3 ) . flupirtine acts as an anticonvulsant and muscle relaxant but it was more recently shown to activate kcnq currents , resulting in decreased excitability in neurons .
likewise mfa , a well - known anti - inflammatory drug and a kcnq 2/3 k channel opener in neurons , also increased outward currents in 5 icc ( fig .
cells were patched with amphotericin b filled pipettes using the modified pipette solution and studied in current clamp mode , so that the effect of kcnq drugs on resting membrane potential could be assessed .
mean resting membrane potential in 18 icc under these conditions was 38  3 mv . while most recordings were electrically quiescent , spontaneous oscillations in resting membrane potential
application of xe991 ( 10 m ) depolarized the membrane potential and mean depolarization in 4 cells was 18  5 mv ( p < 0.05 , fig .
chromanol 293b also depolarized 4 icc by 5  2 mv ( p < 0.05 ) , although its effect was less marked than that of xe991 ( fig .
the kcnq opener flupirtine produced a mean membrane hyperpolarization of 6  1 mv in 6 cells ( p < 0.001 , fig .
figure 4 , g shows the overall effect of kcnq channel drugs on icc resting membrane potential .
more recently kcnq currents have been reported to be present in smooth muscle preparations , including the pulmonary artery , portal vein and mesenteric artery , and to have a role in smooth muscle excitability and vascular tone regulation .
the current study shows that , in addition to neuronal , cardiac and smooth muscle cells , icc from guinea pig detrusor also have kcnq currents .
we recently characterized similar currents in detrusor smooth muscle , in which kcnq currents appear to have a key role in the control of bladder spontaneous electrical and contractile activity ( anderson and mccloskey , unpublished data ) . in the current study kcnq currents from bladder icc showed many typical properties of kcnq channels , including voltage dependence , activation positive to 60 mv , outward rectification and little inactivation .
they were sensitive to inhibition by drugs commonly used to block kcnq channels , ie xe991 , linopirdine and chromanol 293b .
xe991 and linopirdine have been reported to be effective kcnq blockers , although the block was not found to be specific to a particular kcnq channel subtype .
xe991 was more potent for decreasing the residual outward current in icc compared with linopirdine in the current study , consistent with the findings of others .
a decrease in currents by xe991 and linopirdine similar to those in the current study were found in vascular smooth muscle cells from rat mesenteric arteries and the rat portal vein .
further support for the existence of kcnq currents in bladder icc comes from experiments with the kcnq channel openers flupirtine and mfa , which each increased outward currents , consistent with findings in rat mesenteric artery smooth muscle cells .
flupirtine and its more potent analogue retigabine increased kcnq / m current and decreased excitability in neurons .
moreover , mfa was found to augment kcnq currents by shifting the voltage activation curve to the left and slowing deactivation kinetics , leading to membrane hyperpolarization .
the potential physiological significance of kcnq currents in bladder icc was demonstrated in current clamp studies of cell resting membrane potential .
activation of kcnq currents in bladder icc appears to decrease excitability since application of the channel opener flupirtine hyperpolarized icc and inhibited the firing of spontaneous transient depolarizations .
this implies that kcnq channel opening could act as a brake , preventing electrical activity and depolarization of the cells .
consistent with this the blockade of kcnq channels depolarized the membrane potential sufficiently to trigger firing in otherwise quiescent cells .
however , several studies indicate that they act to modify or regulate the activity of neighboring smcs .
if this is indeed the case , blockade of kcnq channels on icc would depolarize the icc and subsequently affect smcs via gap junctions or evoke ca release from internal stores in icc and , therefore , activate cellular signaling mechanisms between icc and smcs . conversely kcnq openers would hyperpolarize icc , which would then hyperpolarize neighboring smcs .
therefore , icc may act to amplify the effect of kcnq drugs on smcs , representing a synergy of design .
for example , smc hyperpolarization by kcnq opening while neighboring icc remained depolarized would be unlikely to be maintained because the more positive icc would tend to depolarize the smcs .
thus , the presence of kcnq channels on icc as well as smcs might represent a fine tuning mechanism to control bladder contractility .
streng et al reported compelling evidence for the functional role of kcnq channels in the bladder in in vivo experiments in rats . in their study
intravesical administration of the kcnq opener retigabine decreased baseline bladder pressures in the rat detrusor , increased micturition volume and decreased capsaicin induced detrusor overactivity .
our study supports these findings and provides electrophysiological evidence for the existence of kcnq channels in bladder icc .
it seems feasible that the development of bladder specific kcnq openers could provide treatment for overactive bladder .
moreover , gene mutations of kcnq subunits , such as cardiovascular , nervous and auditory pathologies , may well underlie bladder disorders .
we investigated the hypothesis that a component of the outward k current in guinea pig bladder icc is mediated by kcnq channels .
icc outward currents were decreased by kcnq pharmacological blockers and enhanced by kcnq channel openers .
data suggest that kcnq currents have a role in the regulation of icc resting membrane potential and excitability .
these novel findings provide key information on the ion channels present in bladder icc and they may indicate relevant targets for the development of new therapy for bladder instability .
 purposethe presence of novel kcnq currents was investigated in guinea pig bladder interstitial cells of cajal and their contribution to the maintenance of the resting membrane potential was assessed.materials and methodsenzymatically dispersed interstitial cells of cajal were patch clamped with k+ filled pipettes in voltage clamp and current clamp modes . 
 pharmacological modulators of kcnq channels were tested on membrane currents and the resting membrane potential.resultscells were stepped from 60 to 40 mv to evoke voltage dependent currents using a modified k+ pipette solution containing ethylene glycol tetraacetic acid ( 5 mm ) and adenosine triphosphate ( 3 mm ) to eliminate large conductance ca activated k channel and kadenosine triphosphate currents . 
 application of the kcnq blockers xe991 , linopirdine ( tocris bioscience , ellisville , missouri ) and chromanol 293b ( sigma ) decreased the outward current in concentration dependent fashion . 
 the current - voltage relationship of xe991 sensitive current revealed a voltage dependent , outwardly rectifying current that activated positive to 60 mv and showed little inactivation . 
 the kcnq openers flupirtine and meclofenamic acid ( sigma ) increased outward currents across the voltage range . in current clamp mode xe991 or 
 chromanol 293b decreased interstitial cell of cajal resting membrane potential and elicited the firing of spontaneous transient depolarizations in otherwise quiescent cells . 
 flupirtine or meclofenamic acid hyperpolarized interstitial cells of cajal and inhibited any spontaneous electrical activity.conclusionsthis study provides electrophysiological evidence that bladder interstitial cells of cajal have kcnq currents with a role in the regulation of interstitial cell of cajal resting membrane potential and excitability . 
 these novel findings provide key information on the ion channels present in bladder interstitial cells of cajal and they may indicate relevant targets for the development of new therapies for bladder instability . 
Materials and Methods
Cell Preparation and Electrophysiological Recordings
Drugs and Solutions
Results
KCNQ Blockers and ICC Outward Currents
KCNQ Openers and ICC Outward Currents
KCNQ Drugs and ICC Resting Membrane Potential
Discussion
Conclusions