BACKGROUND
naringenin  is a major dietary flavonone, a type of flavonoid which is considered to have bioactive effects on human health.
several studies have indeed reported that it and other structurally related compounds could produce anxiolytic and antinociceptive actions  <cit> .
an earlier study also demonstrated its ability to exert anti-inflammatory properties in macrophages and ex vivo human whole-blood models  <cit> .
however, the ionic mechanism of their actions at the cellular level is largely unclear.figure  <dig>
chemical structure of ngen .
the kcnq <dig>  kcnq <dig>  and kcnq <dig> genes are known to encode the core subunits of kv <dig> , kv <dig>  and kv <dig>  channels.
the increased activity of kv <dig> ,  <dig>  and  <dig>  channels is known to generate the m-type k+ current ) which is a slowly activating and deactivating current suppressed by stimulation of muscarinic receptors  <cit> .
mutations of the kcnq <dig> gene are notably involved in peripheral nerve hyperexcitability, a syndrome which is characterized by spontaneous and continuous muscle overactivity  <cit> .
most of ik in neurons are made by heterologously expressed kv <dig> / <dig>  channels  <cit> .
targeting kv7/km channels is recognized to be valuable as an adjunctive regimen for the treatment of many neurological disorders  <cit> .
the large-conductance ca2+-activated k+ channels  have the largest single-channel conductance of all k+ selective channels.
they can be synergistically activated by membrane depolarization, elevation of intracellular ca2+, or both.
native bkca channels from mammalian tissues are composed of two structurally distinct subunits, α and β, arranged in a 1: <dig> stoichiometry.
these channels are a physiologically and structurally diverse group of k+ channels that are essential for neuronal excitability by participating in the repolarization and after-hyperpolarization of action potentials  <cit> .
ngen has been previously shown to bind to gabaa receptors and then to exert anxiolytic actions  <cit> .
earlier studies have demonstrated the ability of this agent to block herg-encoded currents  <cit> .
another work also reported that it could dilate endothelium-denuded aortic ring by the activation of bkca channels in vascular myocytes  <cit> .
naringin, another structurally similar compound, was recently reported to activate inwardly rectifying k+ channels  <cit> .
green tea flavonoids, such as epigallocatechin and epicatechin, were shown to reduce the activity of atp-sensitive k+ channels  <cit> .
quercetin, another natural flavonoid, was described to alter the amplitude of l-type ca2+ current in pituitary and motor neuron-like cells  <cit> .
a recent report also showed the ability of ngen to inhibit cl− secretion in isolated colonic epithelia  <cit> .
whether ngen and its structurally related compounds can produce any effects on other types of ion channels including kv channels remains incompletely understood.
the nsc- <dig> neuronal cell is a hybridoma cell line derived from the fusion of neuroblastoma cells with mice spinal cord cells.
these cells have attracted growing interest as a suitable model for evaluation of the effects of potential neuroprotective compounds against different insults including excitotoxins, mitochondrial toxins and oxidants  <cit> .
a recent study has reported the presence of na+-activated k+ channels functionally expressed in these cells  <cit> .
however, the biophysical or pharmacological properties in nsc- <dig> neuronal cells are incompletely characterized.
in the current study, we provide the first evidence that ngen can interact with km channels to increase the amplitude of macroscopic ik in motor neuron-like nsc- <dig> cells.
in hek293t cells transfected with α-hslo subunit, ngen was also noted to increase the activity of bkca channels.
therefore, similar to bms-204352-induced change in bkca and neuronal kcnq channels  <cit> , both stimulation of ik and ik caused by ngen can be a potential mechanisms through which it influences neuronal activity in central neurons, although these two compounds are structurally distinguishable.
furthermore, numerical simulation of ap bursting generated from a modeled pyramidal neurons  <cit>  clearly showed that as the conductances of gm and gkca channels were elevated by two-fold to mimic the stimulatory actions of ngen, the intraburst firing of aps was reduced accompanied by the increased after-hyperpolarization.
RESULTS
effect of ngen on ik in nsc- <dig> cells
in these experiments, nsc- <dig> cells were bathed in ca2+-free tyrode’s solution.
to ensure that the activity of kca or kna channels was not contaminated, iberiotoxin , apamin  and tetrodotoxin  were added to the bath medium.
iberiotoxin and apamin are, respectively, the blockers of bk channels and small-conductance ca2+-activated k+ channels, while tetrotodotxin is a potent blocker of voltage-gated na+ current.
under whole-cell configuration, the ik was elicited from a holding potential of -20 mv to different potentials which ranged from - <dig> to +10 mv with 10-mv increments at a rate of  <dig>  hz.
the voltage protocol used was previously described  <cit> .
as shown in figure  <dig>  addition of ngen at a concentration of 10 μm increased the amplitude of ik throughout the entire voltage-clamp steps examined.
for example, at the level of -10 mv, cell exposure to ngen  significantly elevated ik amplitude from  <dig>  ±  <dig> to  <dig>  ±  <dig> pa .
after washout of ngen, ik amplitude returned to 57 ±  <dig> pa .
similarly, flupirtine , an activator of ik  <cit> , also increased the ik amplitude found in nsc- <dig> cells.
a further application of linopirdine  reversed ik amplitude to  <dig>  ±  <dig> pa observed at the same level of holding potential.
linopirdine is a blocker of ik.
figure 2b illustrates averaged i-v relationships of ik obtained in the control and during exposure to ngen.
specifically, cell exposure to ngen significantly increased the slope of the linear fit of ik amplitudes to the voltages between - <dig> and -10 mv, namely, whole-cell conductance of ik, from  <dig>  ±  <dig>  to  <dig>  ±  <dig>  ns .
the data clearly indicate that as nsc- <dig> cells are exposed to ngen, the i-v relationship of ik in nsc- <dig> cells can be modified.figure  <dig>
effects of ngen on
i
k
recorded from motor neuron-like nsc- <dig> cells.
in , superimposed current traces obtained in the absence  and presence  of 10 μm ngen.
in these experiments, cells were bathed ca2+-tyrode’s solution which contained  <dig> nm iberiotoxin,  <dig> nm apamin and 1 μm tetrodotoxin.
the i
k was elicited from -20 mv to different potentials which ranged from - <dig> to +10 mv with 10-mv increments.
effect of ngen on the averaged i-v relations of i
k in nsc- <dig> cells .
■: control; □: 10 μm ngen.
current amplitude was measured at end of each voltage pulse.
voltage dependence of i
k conductance in the absence  and presence  of 10 μm ngen .
note that there is a leftward shift in the activation curve of i
k conductance during cell exposure to ngen, although the slope factor remains unchanged.
effect of ngen on the voltage dependence of ik activation in nsc- <dig> cells
figure 2c shows the activation curve of ik obtained with or without addition of ngen .
the plot of relative ik conductance as a function of membrane potential was constructed and fitted with a boltzmann function as described under methods.
in controls, v1/2 = - <dig>  ±  <dig>  mv, q =  <dig>  ±  <dig>  e , whereas in the presence of ngen , v1/2 = - <dig>  ±  <dig>  mv and q =  <dig>  ±  <dig>  e .
the data showed that the activation curve of ik was shifted along the voltage axis to more negative potentials by approximately 18 mv, as nsc- <dig> cells were exposed to ngen.
however, no significant change in the gating charge was clearly demonstrated in the presence of ngen.
the data thus indicate that in nsc- <dig> cells, ngen is capable of shifting the activation curve of ik to more negative potentials with no discernible change in the apparent gating charge.
the relationship between the ngen concentration and the percentage increase of ik is illustrated in figure  <dig>  to evoked ik obtained in controls and during exposure to different concentrations  of ngn, each cell was hyperpolarized from - <dig> to -50 mv.
the amplitude of ik measured at the end of hyperpolarizing pulse was compared with those obtained after subsequent application of linopirdine .
addition of ngen  was noted to increase the amplitude of linopirdine-sensitive current in a concentration-dependent manner.
the half-maximal concentration  required for stimulatory effect of ngen on ik was calculated to be  <dig>  ±  <dig>  μm, and at a concentration of 300 μm, it increased almost all of the ik amplitude in these cells.figure  <dig>
concentration-dependent stimulation of
i
k
by ngen in nsc- <dig> cells.
in these experiments, each cell was hyperpolarized from - <dig> to -50 mv with a duration of 2 sec.
original current traces obtained in the control and during exposure to ngen.
a: control; b: 3 μm ngen; c: 10 μm ngen; d: 30 μm ngen.
the upper part indicates the voltage protocol used.
concentration-response relationship for ngen-induced increase of i
k  measured at the end of hyperpolarizing pulses .
smooth blue line represents the best fit to the hill equation as described in methods.
effects of various related compounds on ik in nsc- <dig> cells
earlier reports have shown the ability of ngen or its structurally similar compounds to alter k+-channel activity  <cit> .
effects of ngen, flupirtine, ngen plus linopirdine and ngen plus blocker of ik on ik were further examined and compared.
as shown in figure  <dig>  similar to ngen, flupirtine at a concentration of 10 μm was effective in increasing the amplitude of ik.
flupirtine is recognized to be a specific activator of neuronal kcnq channels  <cit> .
however, neither iberiotoxin nor apamin produced any effects on ngen-stimulated ik, although subsequent application of linopirdine was able to reverse the increased ik caused by either ngen or flupirtine.
therefore, ngen-stimulated ik is subject to inhibition by linopirdine, but not linked to the activity of kca channels probably expressed in nsc- <dig> cells,figure  <dig>
effects of ngen and other k
+
current blockers on the amplitude of
i
k
in nsc- <dig> cells.
in these experiments, cells were bathed in ca2+-free tyrode's solution which contained  <dig> nm iberiotoxin,  <dig> nm apamin and 1 μm tetrodotoxin, and each cell was hyperpolarized from - <dig> to -50 mv with a duration of 2 sec.
current amplitude was measured at the end of voltage pulse.
each bar represents the mean ± sem .
flu: flupirtine; iber: iberiotoxin; aps: apamin; lino: linopirdine.
*significantly different from control.
**significantly different from ngen  alone group.
notably, further application of linopirdine reversed ngen-stimulated i
k, while neither iberiotoxin nor apamin produced any effects on it.
effect of ngen on km channels in nsc- <dig> neuronal cells
we next sought to investigate whether ngen can act by influencing the activity of km channels for changes in whole-cell ik amplitude in these cells.
in these experiments, cell-attached recordings were conducted in cells bathed in ca2+-free tyrode’s solution.
to ensure that the activity of kca or kna channels would not be contaminated, iberiotoxin , apamin  and tetrodotoxin  were added to the bathing solution.
the recording pipette was filled with k+-containing solution and the cell attached to the pipette was held at 0 mv relative to the bath.
as the cell was in a physiological external solution, the resting potential of the cells was about -71 mv.
the pipette solution had a high k+ concentration, which is the same as that of the cytoplasm.
therefore, a k+ channel in the membrane patch would be expected to have a reversal potential of 0 mv.
the membrane patch can be depolarized by applying negative command potentials to 0 mv.
figure 5a shows the current tracings of single-channel recordings obtained with or without addition of ngen .
when ngen was added to the bath, channel activity was greatly raised.
the probability of channel openings was significantly elevated from  <dig>  ±  <dig>  to  <dig>  ±  <dig>   in the presence of 10 μm ngen .
similarly, flupirtine  was effective in increasing channel activity.
a further application of 2 mm cacl <dig> or linopirdine  to the bath could effectively reverse ngen-stimulated activity of km channels.
however, single-channel amplitude between the absence and presence of ngen or flupirtine did not differ significantly.figure  <dig>
stimulatory effect of ngen on the activity of k
m
channels recorded from nsc- <dig> cells.
in , cells were bathed in ca2+-free tyrode’s solution which contained iberiotoxin , apamin  and tetrodotoxin .
cell-attached configuration was made as the cell attached was held at 0 mv relative to the bath.
channel activity was obtained in the control  and after addition of ngen  to the bath.
portion of tracing in the upper part of  is amplified in the lower part.
channel openings give a downward deflection in current.
summary of the data showing effects of ngen and flupirtine  on km-channel activity in nsc- <dig> cells .
*significantly different from control.
inability of ngen to block ik in nsc- <dig> neuronal cells
earlier work has demonstrated the ability of ngen to block cardiac herg currents  <cit> .
we further investigated the possible effects of ngen on ik found in nsc- <dig> cells.
as depicted in figure  <dig>  addition of ngen at a concentration of 10 μm did not cause any effect on peak ik amplitude elicited throughout the entire voltage-clamp steps examined.
the peak amplitude of ik elicited by membrane hyperpolarizations was not noted to differ significantly between the absence and presence of 10 μm ngen.
however, addition of azimilide, an blocker of ik  <cit> , reduced ik amplitude by 43%.
the results presented here are compatible with previous observations showing that the ic <dig> value for ngen-induced block of herg channels expressed in xenopus oocytes is about 100 μm  <cit> , which is much greater than that used to stimulate ik described here.figure  <dig>
no effects of ngen on
i
k
in nsc- <dig> cells.
in these experiments, cells were bathed in a high-k+, ca2+-free solution.
each cell was held at -10 mv and various voltage pulses ranging from - <dig> to 0 mv with 10-mv increments were applied as shown in the uppermost part of .
superimposed i
k obtained in the absence  and presence  of 10 μm ngen.
averaged i-v relationship of peak i
k obtained in control  and during exposure to 10 μm ngen  .
effects of ngen on spontaneous action currents recorded from nsc- <dig> cells
it was also examined whether ngen can produce any changes in spontaneous action currents emerging in these cells.
in these experiments, cells were bathed in normal tyrode's solution containing  <dig>  mm cacl <dig>  cell-attached voltage-clamp recordings were performed  <cit>  and patch pipettes were filled with a k+-containing solution.
the potential across the patch was set at the level of the resting membrane potential of the cells .
as illustrated in figure  <dig>  addition of ngen caused a significant reduction in the firing of spontaneous action currents.
the frequency of action currents in the presence of 10 μm ngen  was significantly smaller than that in control .
moreover, a subsequent application of linopirdine  reversed the firing frequency to  <dig>  ±  <dig>  hz , although iberiotoxin  had minimal effects on ngen-induced decrease of firing frequency.
in light of these data, we assumed that ngen-induced reduction of firing frequency is primarily linked to the simulation of km channels and unlikely to be mediated through the activation of bkca channels.figure  <dig>
effects of ngen and flupirtine on spontaneous action currents recorded from nsc- <dig> neuronal cells.
in , current traces were obtained in the absence  and presence  of 10 μm ngen.
the lower part shown at  and  indicates an expanded record from dashed box.
notably, the traces showing downward deflections indicate action currents.
summary of the data showing effects of ngen, flupirtine , ngen plus linopirdine  on the frequency of action currents in nsc- <dig> cells.
each point indicates the mean ± sem .
*significantly different from control.
stimulatory effect of ngen on ik in hek293t cells transfected with α-hslo
previous work has demonstrated the ability of ngen to stimulate the activity of bkca channels in vascular smooth myocytes  <cit> .
we also tested the hypothesis that this compound exerts any effects on ik in hek293t cells expressing α-hslo.
under our experimental conditions  <cit> , transfection with α-hslo into hek293t cells can result in the appearance of bkca channels, thereby elevating the amplitude of macroscopic ik.
in whole-cell configuration, as ngen was applied to the bath, the ik amplitude was significantly increased .
for example, ngen at a concentration of 30 μm increased ik amplitude at +50 mv from 403 ±  <dig> to 917 ±  <dig> pa .
subsequent application of verruculogen  or iberiotoxin  was noted to reverse ngen-stimulated ik significantly ; however, neither apamin  nor linopirdine  had any effects on it.figure  <dig>
effect of ngen on whole-cell
i
k
in α
-hslo
-expressing hek293t cells.
in these experiments, cells were bathed in normal tyrode’s solution containing  <dig>  mm cacl <dig>   current traces in response to membrane depolarization from  <dig> to +50 mv.
the upper part indicates the voltage protocol used.
a: control; b: 3 μm ngen; c: 10 μm ngen; and d: 30 μm ngen.
summary of the data showing effects of ngen, ngen plus verruculogen  and ngen plus iberiotoxin  on i
k amplitude in these cells .
*significantly different from control.
**significantly different from ngen  alone group.
stimulatory effect of ngen on the activity of bkca channels in hek293t cells expressing α-hslo
verruculogen and iberiotoxin, known inhibitors of bkca channels  <cit> , could clearly reverse ngen-stimulated ik in these cells.
the increased macroscopic ik caused by ngen could be due to either the elevated open probability, an increase in the number of active channels, or both.
the effect of ngen on bkca-channel activity was further investigated .
in this set of experiments, single-channel recordings with an inside-out configuration were performed in symmetrical k+ solution .
bath medium contained  <dig>  μm ca2+ and the potential was held at +60 mv.
when ngen  was applied to the intracellular leaflet of the detached patch, channel activity was significantly raised to  <dig>  ±  <dig>  from a control of  <dig>  ±  <dig>  .
a further application of verruculogen  decreased the probability of channel openings to  <dig>  ±  <dig>  , although linopirdine  applied to the bath had minimal effects on ngen-stimulated channel activity.
the single-channel conductance of bkca channels between the absence and presence of 30 μm ngen was not noted to differ significantly .
therefore, in agreement with previous observations made in vascular myocytes  <cit> , the ngen-induced increase of ik amplitude in hek293t cells which were transfected with α-hslo, tended to be associated with its increase in the probability of channel openings, rather than changes in the number of functional active channels.
ngen can thus interact with bkca channels to increase the amplitude of ik in these cells, even in the absence of bkca-channel β-subunits.figure  <dig>
stimulatory effect of ngen on bk
ca
-channel activity measured from α
-hslo
-expressing hek293t cells.
original current traces showing the activity of bkca channels before  and after application  of 30 μm ngen.
inside-out recordings were conducted with symmetrical k+ concentration .
the potential was constantly held at +60 mv, and bath medium contained  <dig>  μm ca2+.
channel openings give an upward deflection in current.
summary of the data showing effect of ngen and ngen plus verruculogen  on the probability of bkca-channel openings .
*significantly different from control.
**significantly different from ngen  alone group.
simulated firing and bursting pattern of aps in central modeled neuron with varying gm or gkca
we further explored how the dynamics of bursting firing elicited by continuous current injection can be altered by increasing the values of gm and gkca to mimic the stimulatory of ngen on ik and ik described above.
for studying this, a brief depolarizing current with  <dig>  ma was applied to the modeled central neuron  with the parameter values illustrated in table  <dig>  in an attempt to generate burst firing of neuronal aps.
the descriptions for this modeled neuron were detailed under methods.
initially, the gm value was arbitrarily elevated from  <dig>  to  <dig>  ms/cm <dig>  subsequently, to mimic the condition in which ngen  was added, the values of gm and gkca was arbitrarily raised by two-fold.
as illustrated in figure  <dig>  by elevating gm value alone, we are able to show an increase in the intra-burst interval in combination with the increased ik.
as both values of gm and gkca were elevated, intra-burst rate was reduced, along with the increase of after-hyperpolarizaton from - <dig> to -85 mv .
it also needs to be noted that in figure 10cb, the ik amplitude numerically generated was increased only by  <dig> -fold, although gm value was elevated by 2-fold as compared with the results in figure 10bb.
therefore, it is possible from these simulation results that when both gm and gkca are at work simultaneously, the outcome may not simply be due to the summation of individual element.table  <dig>
default parametric values used for the modeling of hippocampal ca <dig> pyramidal neurons
symbol
description
value
gna
gcat
gcal
gcan
gkdr
ga
gm
gkca
gkahp
gleak
iapp
vna
vk
vleakburst firing of aps  and the corresponding changes of
i
k
and
i
k
in modeled hippocampal neuron.
the detailed formulations were described under methods.
the firing was generated with the values of gm and gkca was set at  <dig>  and  <dig>  ms/cm2
,  <dig>  and  <dig>  ms/cm2
and  <dig>  and  <dig>  ms/cm2
, respectively.
ab, bb, and cb indicate the corresponding changes in i
k  and i
k , respectively.
in , the values of gm and gkca were arbitrarily elevated by two-fold to mimic the stimulatory effects of ngen .
note that in this simulation, the amplitudes of i
k are smaller than those of i
k.
fast-slow analysis of ap bursting in model neuron
finally, in order to develop a better quantitative understanding of km and kca channels, we further performed fast-slow analysis in a reduced comparable model.
such a maneuver can be allowed for separation of the variables into two subsystems, i.e., fast  and slow   <cit> .
one can analyze how the bursting from this modeled neuron is numerically generated owing to a delicate interaction of these two variables.
in other words, one is a slow autocatalytic variable n  and the other is a slow negative feedback variable o .
in this analysis, we treated slow variables n and o as the parameters to study the fast subsystem and subsequently to determine the regions of spiking and resting states, as the different values of gm and gkca are chosen.
as shown in figure  <dig>  the two-parameter phase plot clearly showed the ability of the burst trajectory to be projected onto the slow variables .
the solid and dashed lines shown in figure  <dig>  correspond to the voltage nullcline  for the fast system at the indicated values of n and o, where the saddle node bifurcation occurs.
it becomes clear from this plot that an interaction of n and o was able to generate slow oscillations that were allowed to move the fast subsystem in and out of the repetitive spiking regime.
the arrows shown in figure  <dig> indicate the direction of the trajectory movement.
the region above the nullcline corresponds to a region of repetitive firing, while the region below the nullcline is where the system remains silent.
the arbitrary increase of gm value from  <dig>  to  <dig>  ms/cm <dig> was noted to shift voltage nullcline in an upward direction with minimal changes in burst trajectory on n-o plane.
however, of interest, when the values of gm and gkca were simultaneously elevated by two-fold to mimic the stimulatory actions of ngen  described above, the nullcline was further shifted in an upward direction accompanied by the compression of burst trajectory in size on n-o plane.
such upward shift in the voltage nullcline indicates a reduction of repetitive spiking regime.
the results of these changes are therefore compatible with our experimental and computational data showing that the increased values of gm and gkca caused by ngen result in a reduction of intraburst firing accompanied by the prolongation of interburst period and the increase of the afterhyperpolarization of aps, if similar findings are observed in central neurons in vivo.figure  <dig>
fast-slow analysis of ap bursting generated from modeled neuron.
the phase plot of burst firing elicited by continuous current injection shows the projection of the burst trajectory onto n-o plane.
n and o denote the activation gates of cav <dig>  and kca channels, respectively.
arrows indicate the direction of trajectory movement.
the solid and dashed lines are the voltage nullcline  of the fast subsystem with n and o as parameters.
below the nullcline is the silent region.
notably, as the value of gm was increased from  <dig>  to  <dig>  ms/cm <dig>  the nullcline was shifted in an upward direction with minimal change in the burst trajectory.
when the values of gm and gkca were simultaneously elevated by two-fold to mimic the effect of ngeg , the burst trajectory was clearly compressed in combination with a further upward shift in the voltage nullcline.
discussion
in this study, we provide the direct evidence that ngen has a stimulatory action on ik in nsc motor neuron-like cells.
the half-maximal concentration  of ngen required for stimulation of ik was about  <dig>  μm, which is much smaller than that reported for the inhibition of herg channels  <cit> .
because of their lipophilicity, ngen was also reported to transverse the blood-brain barrier and subsequently to penetrate into different brain regions  <cit> , although the brain concentrations of ngen vary based on local extracellular milieu in and around membranes and synapses.
the importance of kv <dig> channels as regulators of neuronal excitability became apparent, when mutated neuronal kv <dig> channels were demonstrated to cause benign familial neonatal convulsions  <cit> .
it is thus anticipated that in motor neurons like nsc- <dig> cells, the kv7/km channels  are an important therapeutic target for the action of this agent or other structurally similar compounds within the cns.
addition of ngen could shift the activation curve of ik conductance to the hyperpolarized potential in this study.
inability of ngen to alter the gating charge of km channels observed in nsc- <dig> cells allows us to suggest that the stimulatory effect of this compound on km channels in nsc- <dig> cells is not mediated through a direct effect on the voltage sensor per se.
the binding site of ngen is most likely to lie outside of the transmembrane field around the channel.
nevertheless, as the neurons are exposed to ngen, the effects of this compound on ik amplitude could rely upon the ngen concentration used, the level of pre-existing membrane potential, or both.
the pharmacological and electrophysiological properties of km channels observed in nsc- <dig> cells are similar to those in other types of neurons described previously  <cit> .
in cell-attached configuration, addition of ngen  into the bath was noted to increase the probability of channel openings with no discernible change in single-channel amplitude.
the inability of ngen to modify single-channel conductance indicates that the increased responsiveness of the channel to this compound is not located at the central ion-conducting pore of the channel.
similarly, flupirtine at a concentration of 10 μm was capable of enhancing km-channel activity in these cells.
the stimulatory actions of ngen and flupirtine on the activity of km channels were not additive in nsc- <dig> neuronal cells .
our results suggest that the two compounds exert their major effects on the same component existing in km channels.
further work is also required to see whether this compound can interact with the kv7/km  channels to stimulate ik in other types of central neurons.
previous reports have demonstrated the ability of ngen to bind to gabaa receptors and to produce anxiolytic actions  <cit> .
a recent study also showed that ngen might inhibit cl− secretion in isolated colonic epithelia  <cit> .
in our study, macroscopic ik was measured in the recording pipette which was filled with a solution containing aspartic acid, rather than cl- ions.
moreover, gabazine , a blocker of gabaa receptors, had minimal effects on ngen-stimulated ik in nsc- <dig> cells.
it thus seems unlikely that the observed increase of ik caused by ngen is inherently associated with the increase of cl− current induced by its binding to gabaa receptors.
an earlier work has demonstrated that green tea flavonoids such as epigallocatechin and epicatechin were capable of reducing the activity of atp-sensitive k+ channels  <cit> .
under our experimental condition, the recording pipette used for measurements of whole-cell ik was filled with a solution which contained 3 mm atp.
moreover, ngen-stimulated ik observed in nsc- <dig> cells was not reversed by subsequent application of glibenclamide , but by linopirdine  .
therefore, in nsc- <dig> cells or in hek293t cells expressing α-hslo, the elevation of k+ currents induced by ngen is not necessarily related to any changes in atp-sensitive k+ channel conductance.
it is noteworthy that the observed increase in the whole-cell ik by ngen did not appear to match its increase in km-channel activity.
the reason for this discrepancy is currently unclear.
however, it is likely that the ngen effects are much dependent on some receptors and/or intracellular second messenger systems which are possibly washed out during our whole-cell recordings.
it remains to be further clarified whether any changes in receptors and/or transmembrane signal pathways are involved in the ngen effect on km channels.
in the present study, under a cell-attached recording, we found that addition of ngen decreased the firing frequency of spontaneous action currents in nsc- <dig> cells.
the reduction of firing frequency caused by ngen in these cells could be primarily explained by its activation of km channels, because linopirdine can significantly reverse ngen-mediated inhibition of spontaneous action currents.
it was also noted that when the recording pipette was filled with iberiotoxin  or apamin , ngen-induced reduction in the firing of action currents still existed .
moreover, because ik was relatively not subject to block by ngen at a concentration of 10 μm, lack of stimulatory effect of ngen on the spontaneous firing of action currents could be demonstrated in the presence of this compound.
activation of bkca channels induced by ngen in vascular myocytes reported previously  <cit>  was noted to share similar characteristics shown here in hek293t cells in which α-hslo channels are functionally expressed.
our results also suggest that ngen may bind to a site located in the cytoplasmic side of the α-subunit.
in addition, bms- <dig> and meclofenamic acid, known to be activators of bkca channels  <cit> , was shown to activate kv7/km channels  <cit> .
therefore, these two types of ion channels is most likely to share the unique motifs with which some small compounds such as ngen can interact.
another notable issue is the subunit composition of the bkca channels.
in this study, we used homomeric α-hslo channels and imply the consequences for neuronal firing.
however, in the central nervous system, α subunits are accompanied by accessory β <dig> proteins  <cit> .
β <dig> subunits were described to change channel pharmacology and current phenotype dramatically  <cit> , and even produce profound effect on the neuronal firing  <cit> .
therefore, it is important to investigate to what extent ngen produces any effects on α-hslo + β <dig> channels, although the results showing that homomeric α-hslo channels are activated by this compound are valuable from mechanistic standpoints.
there is evidence to show that ngen is a target for km channel.
in our study, we also found that this compound could be a direct stimulator of bkca channels.
with the aid of blastx program , we further examined the similarity of amino acid sequence between the α-subunit of bkca channel  and kcnq <dig> protein.
interestingly, a portion of bkca-channel α-subunit , to which the sequence of kcnq <dig>  shares the similarity , is located at 182- <dig>  this region are noted to correspond with the ion transport domain of the bkca-channel α-subunit.
therefore, it is tempting to speculate that ngen or other structurally similar compounds can interact at this region to influence the activity of bkca channels or/and kv7-encoded channels.
in the modeled hippocampal neurons presented herein, burst firing of aps in response to brief current injection was readily generated and an interaction of two slow variables could be derived.
in other words, as shown in figure  <dig>  a slow autocatalytic variable  correspond to the activation gate of t-type ca2+ channel, while a slow negative feedback variable  is the activation gate of kca channel.
in this system, the results from fast-slow analysis led us to suggest that the subtle interaction of activation gates between t-type ca2+ channel and kca channel is able to generate slow oscillations that move the fast subsystem from a stable non-oscillatory regime into a repetitive spiking regime and back again.
superimposition of the voltage nullcline  computed with the gates  designated as parameters is able to illustrate where this bifurcation emerges.
it is tempting to speculate that the presence of ngen potentially alters the firing behaviors of central neurons in vivo, assuming that those neurons functionally express the activity of both km and bkca channels.
CONCLUSIONS
the stimulatory effect of ngen on native ik expressed in central neurons could have an impact on the functioning of the neurons.
further work is required to see whether the activity of km channels stimulated by this compound is responsible for the antinociceptive or antioxidative actions of different compounds known to activate ik.
besides that, ngen-induced actions on the stimulation of km and bkca channels may combine to affect the functional activities if both channels are expressed in central neurons in vivo.
our findings raise the possibility that ngen-mediated changes in ion currents of neurons described above are closely linked to its possible neuroprotective actions  <cit> .

 naringenin is a major dietary flavonone, a type of flavonoid which is considered to have bioactive effects on human health. several studies have indeed reported that it and other structurally related compounds could produce anxiolytic and antinociceptive actions. an earlier study also demonstrated its ability to exert anti-inflammatory properties in macrophages and ex vivo human whole-blood models. however, the ionic mechanism of their actions at the cellular level is largely unclear.figure chemical structure of ngen. the kcnq kcnq and kcnq genes are known to encode the core subunits of kv, kv and kv channels. the increased activity of kv, and channels is known to generate the m-type k+ current ) which is a slowly activating and deactivating current suppressed by stimulation of muscarinic receptors. mutations of the kcnq gene are notably involved in peripheral nerve hyperexcitability, a syndrome which is characterized by spontaneous and continuous muscle overactivity. most of ik in neurons are made by heterologously expressed kv / channels. targeting kv7/km channels is recognized to be valuable as an adjunctive regimen for the treatment of many neurological disorders. the large-conductance ca2+-activated k+ channels have the largest single-channel conductance of all k+ selective channels. they can be synergistically activated by membrane depolarization, elevation of intracellular ca2+, or both. native bkca channels from mammalian tissues are composed of two structurally distinct subunits, α and β, arranged in a 1: stoichiometry. these channels are a physiologically and structurally diverse group of k+ channels that are essential for neuronal excitability by participating in the repolarization and after-hyperpolarization of action potentials. ngen has been previously shown to bind to gabaa receptors and then to exert anxiolytic actions. earlier studies have demonstrated the ability of this agent to block herg-encoded currents. another work also reported that it could dilate endothelium-denuded aortic ring by the activation of bkca channels in vascular myocytes. naringin, another structurally similar compound, was recently reported to activate inwardly rectifying k+ channels. green tea flavonoids, such as epigallocatechin and epicatechin, were shown to reduce the activity of atp-sensitive k+ channels. quercetin, another natural flavonoid, was described to alter the amplitude of l-type ca2+ current in pituitary and motor neuron-like cells. a recent report also showed the ability of ngen to inhibit cl− secretion in isolated colonic epithelia. whether ngen and its structurally related compounds can produce any effects on other types of ion channels including kv channels remains incompletely understood. the nsc- neuronal cell is a hybridoma cell line derived from the fusion of neuroblastoma cells with mice spinal cord cells. these cells have attracted growing interest as a suitable model for evaluation of the effects of potential neuroprotective compounds against different insults including excitotoxins, mitochondrial toxins and oxidants. a recent study has reported the presence of na+-activated k+ channels functionally expressed in these cells. however, the biophysical or pharmacological properties in nsc- neuronal cells are incompletely characterized. in the current study, we provide the first evidence that ngen can interact with km channels to increase the amplitude of macroscopic ik in motor neuron-like nsc- cells. in hek293t cells transfected with α-hslo subunit, ngen was also noted to increase the activity of bkca channels. therefore, similar to bms-204352-induced change in bkca and neuronal kcnq channels, both stimulation of ik and ik caused by ngen can be a potential mechanisms through which it influences neuronal activity in central neurons, although these two compounds are structurally distinguishable. furthermore, numerical simulation of ap bursting generated from a modeled pyramidal neurons clearly showed that as the conductances of gm and gkca channels were elevated by two-fold to mimic the stimulatory actions of ngen, the intraburst firing of aps was reduced accompanied by the increased after-hyperpolarization.  effect of ngen on ik in nsc- cells in these experiments, nsc- cells were bathed in ca2+-free tyrode’s solution. to ensure that the activity of kca or kna channels was not contaminated, iberiotoxin, apamin and tetrodotoxin were added to the bath medium. iberiotoxin and apamin are, respectively, the blockers of bk channels and small-conductance ca2+-activated k+ channels, while tetrotodotxin is a potent blocker of voltage-gated na+ current. under whole-cell configuration, the ik was elicited from a holding potential of -20 mv to different potentials which ranged from - to +10 mv with 10-mv increments at a rate of  hz. the voltage protocol used was previously described. as shown in figure  addition of ngen at a concentration of 10 μm increased the amplitude of ik throughout the entire voltage-clamp steps examined. for example, at the level of -10 mv, cell exposure to ngen significantly elevated ik amplitude from  ±  to  ±  pa. after washout of ngen, ik amplitude returned to 57 ±  pa. similarly, flupirtine, an activator of ik, also increased the ik amplitude found in nsc- cells. a further application of linopirdine reversed ik amplitude to  ±  pa observed at the same level of holding potential. linopirdine is a blocker of ik. figure 2b illustrates averaged i-v relationships of ik obtained in the control and during exposure to ngen. specifically, cell exposure to ngen significantly increased the slope of the linear fit of ik amplitudes to the voltages between - and -10 mv, namely, whole-cell conductance of ik, from  ±  to  ±  ns. the data clearly indicate that as nsc- cells are exposed to ngen, the i-v relationship of ik in nsc- cells can be modified.figure effects of ngen on i k recorded from motor neuron-like nsc- cells. in, superimposed current traces obtained in the absence and presence of 10 μm ngen. in these experiments, cells were bathed ca2+-tyrode’s solution which contained nm iberiotoxin, nm apamin and 1 μm tetrodotoxin. the i k was elicited from -20 mv to different potentials which ranged from - to +10 mv with 10-mv increments. effect of ngen on the averaged i-v relations of i k in nsc- cells. ■: control; □: 10 μm ngen. current amplitude was measured at end of each voltage pulse. voltage dependence of i k conductance in the absence and presence of 10 μm ngen. note that there is a leftward shift in the activation curve of i k conductance during cell exposure to ngen, although the slope factor remains unchanged. effect of ngen on the voltage dependence of ik activation in nsc- cells figure 2c shows the activation curve of ik obtained with or without addition of ngen. the plot of relative ik conductance as a function of membrane potential was constructed and fitted with a boltzmann function as described under methods. in controls, v1/2 = -  ±   mv, q =   ±  e, whereas in the presence of ngen, v1/2 = -  ±   mv and q =   ±  e. the data showed that the activation curve of ik was shifted along the voltage axis to more negative potentials by approximately 18 mv, as nsc- cells were exposed to ngen. however, no significant change in the gating charge was clearly demonstrated in the presence of ngen. the data thus indicate that in nsc- cells, ngen is capable of shifting the activation curve of ik to more negative potentials with no discernible change in the apparent gating charge. the relationship between the ngen concentration and the percentage increase of ik is illustrated in figure  to evoked ik obtained in controls and during exposure to different concentrations of ngn, each cell was hyperpolarized from - to -50 mv. the amplitude of ik measured at the end of hyperpolarizing pulse was compared with those obtained after subsequent application of linopirdine. addition of ngen was noted to increase the amplitude of linopirdine-sensitive current in a concentration-dependent manner. the half-maximal concentration required for stimulatory effect of ngen on ik was calculated to be  ±   μm, and at a concentration of 300 μm, it increased almost all of the ik amplitude in these cells.figure concentration-dependent stimulation of i k by ngen in nsc- cells. in these experiments, each cell was hyperpolarized from - to -50 mv with a duration of 2 sec. original current traces obtained in the control and during exposure to ngen. a: control; b: 3 μm ngen; c: 10 μm ngen; d: 30 μm ngen. the upper part indicates the voltage protocol used. concentration-response relationship for ngen-induced increase of i k measured at the end of hyperpolarizing pulses. smooth blue line represents the best fit to the hill equation as described in methods. effects of various related compounds on ik in nsc- cells earlier reports have shown the ability of ngen or its structurally similar compounds to alter k+-channel activity. effects of ngen, flupirtine, ngen plus linopirdine and ngen plus blocker of ik on ik were further examined and compared. as shown in figure  similar to ngen, flupirtine at a concentration of 10 μm was effective in increasing the amplitude of ik. flupirtine is recognized to be a specific activator of neuronal kcnq channels. however, neither iberiotoxin nor apamin produced any effects on ngen-stimulated ik, although subsequent application of linopirdine was able to reverse the increased ik caused by either ngen or flupirtine. therefore, ngen-stimulated ik is subject to inhibition by linopirdine, but not linked to the activity of kca channels probably expressed in nsc- cells,figure effects of ngen and other k + current blockers on the amplitude of i k in nsc- cells. in these experiments, cells were bathed in ca2+-free tyrode's solution which contained nm iberiotoxin, nm apamin and 1 μm tetrodotoxin, and each cell was hyperpolarized from - to -50 mv with a duration of 2 sec. current amplitude was measured at the end of voltage pulse. each bar represents the mean ± sem. flu: flupirtine; iber: iberiotoxin; aps: apamin; lino: linopirdine. *significantly different from control. **significantly different from ngen alone group. notably, further application of linopirdine reversed ngen-stimulated i k, while neither iberiotoxin nor apamin produced any effects on it. effect of ngen on km channels in nsc- neuronal cells we next sought to investigate whether ngen can act by influencing the activity of km channels for changes in whole-cell ik amplitude in these cells. in these experiments, cell-attached recordings were conducted in cells bathed in ca2+-free tyrode’s solution. to ensure that the activity of kca or kna channels would not be contaminated, iberiotoxin, apamin and tetrodotoxin were added to the bathing solution. the recording pipette was filled with k+-containing solution and the cell attached to the pipette was held at 0 mv relative to the bath. as the cell was in a physiological external solution, the resting potential of the cells was about -71 mv. the pipette solution had a high k+ concentration, which is the same as that of the cytoplasm. therefore, a k+ channel in the membrane patch would be expected to have a reversal potential of 0 mv. the membrane patch can be depolarized by applying negative command potentials to 0 mv. figure 5a shows the current tracings of single-channel recordings obtained with or without addition of ngen. when ngen was added to the bath, channel activity was greatly raised. the probability of channel openings was significantly elevated from  ±  to  ±  in the presence of 10 μm ngen. similarly, flupirtine was effective in increasing channel activity. a further application of 2 mm cacl or linopirdine to the bath could effectively reverse ngen-stimulated activity of km channels. however, single-channel amplitude between the absence and presence of ngen or flupirtine did not differ significantly.figure stimulatory effect of ngen on the activity of k m channels recorded from nsc- cells. in, cells were bathed in ca2+-free tyrode’s solution which contained iberiotoxin, apamin and tetrodotoxin. cell-attached configuration was made as the cell attached was held at 0 mv relative to the bath. channel activity was obtained in the control and after addition of ngen to the bath. portion of tracing in the upper part of is amplified in the lower part. channel openings give a downward deflection in current. summary of the data showing effects of ngen and flupirtine on km-channel activity in nsc- cells. *significantly different from control. inability of ngen to block ik in nsc- neuronal cells earlier work has demonstrated the ability of ngen to block cardiac herg currents. we further investigated the possible effects of ngen on ik found in nsc- cells. as depicted in figure  addition of ngen at a concentration of 10 μm did not cause any effect on peak ik amplitude elicited throughout the entire voltage-clamp steps examined. the peak amplitude of ik elicited by membrane hyperpolarizations was not noted to differ significantly between the absence and presence of 10 μm ngen. however, addition of azimilide, an blocker of ik, reduced ik amplitude by 43%. the results presented here are compatible with previous observations showing that the ic value for ngen-induced block of herg channels expressed in xenopus oocytes is about 100 μm, which is much greater than that used to stimulate ik described here.figure no effects of ngen on i k in nsc- cells. in these experiments, cells were bathed in a high-k+, ca2+-free solution. each cell was held at -10 mv and various voltage pulses ranging from - to 0 mv with 10-mv increments were applied as shown in the uppermost part of. superimposed i k obtained in the absence and presence of 10 μm ngen. averaged i-v relationship of peak i k obtained in control and during exposure to 10 μm ngen. effects of ngen on spontaneous action currents recorded from nsc- cells it was also examined whether ngen can produce any changes in spontaneous action currents emerging in these cells. in these experiments, cells were bathed in normal tyrode's solution containing  mm cacl cell-attached voltage-clamp recordings were performed and patch pipettes were filled with a k+-containing solution. the potential across the patch was set at the level of the resting membrane potential of the cells. as illustrated in figure  addition of ngen caused a significant reduction in the firing of spontaneous action currents. the frequency of action currents in the presence of 10 μm ngen was significantly smaller than that in control. moreover, a subsequent application of linopirdine reversed the firing frequency to  ±   hz, although iberiotoxin had minimal effects on ngen-induced decrease of firing frequency. in light of these data, we assumed that ngen-induced reduction of firing frequency is primarily linked to the simulation of km channels and unlikely to be mediated through the activation of bkca channels.figure effects of ngen and flupirtine on spontaneous action currents recorded from nsc- neuronal cells. in, current traces were obtained in the absence and presence of 10 μm ngen. the lower part shown at and indicates an expanded record from dashed box. notably, the traces showing downward deflections indicate action currents. summary of the data showing effects of ngen, flupirtine, ngen plus linopirdine on the frequency of action currents in nsc- cells. each point indicates the mean ± sem. *significantly different from control. stimulatory effect of ngen on ik in hek293t cells transfected with α-hslo previous work has demonstrated the ability of ngen to stimulate the activity of bkca channels in vascular smooth myocytes. we also tested the hypothesis that this compound exerts any effects on ik in hek293t cells expressing α-hslo. under our experimental conditions, transfection with α-hslo into hek293t cells can result in the appearance of bkca channels, thereby elevating the amplitude of macroscopic ik. in whole-cell configuration, as ngen was applied to the bath, the ik amplitude was significantly increased. for example, ngen at a concentration of 30 μm increased ik amplitude at +50 mv from 403 ±  to 917 ±  pa. subsequent application of verruculogen or iberiotoxin was noted to reverse ngen-stimulated ik significantly; however, neither apamin nor linopirdine had any effects on it.figure effect of ngen on whole-cell i k in α -hslo -expressing hek293t cells. in these experiments, cells were bathed in normal tyrode’s solution containing  mm cacl current traces in response to membrane depolarization from to +50 mv. the upper part indicates the voltage protocol used. a: control; b: 3 μm ngen; c: 10 μm ngen; and d: 30 μm ngen. summary of the data showing effects of ngen, ngen plus verruculogen and ngen plus iberiotoxin on i k amplitude in these cells. *significantly different from control. **significantly different from ngen alone group. stimulatory effect of ngen on the activity of bkca channels in hek293t cells expressing α-hslo verruculogen and iberiotoxin, known inhibitors of bkca channels, could clearly reverse ngen-stimulated ik in these cells. the increased macroscopic ik caused by ngen could be due to either the elevated open probability, an increase in the number of active channels, or both. the effect of ngen on bkca-channel activity was further investigated. in this set of experiments, single-channel recordings with an inside-out configuration were performed in symmetrical k+ solution. bath medium contained  μm ca2+ and the potential was held at +60 mv. when ngen was applied to the intracellular leaflet of the detached patch, channel activity was significantly raised to  ±  from a control of  ± . a further application of verruculogen decreased the probability of channel openings to  ± , although linopirdine applied to the bath had minimal effects on ngen-stimulated channel activity. the single-channel conductance of bkca channels between the absence and presence of 30 μm ngen was not noted to differ significantly. therefore, in agreement with previous observations made in vascular myocytes, the ngen-induced increase of ik amplitude in hek293t cells which were transfected with α-hslo, tended to be associated with its increase in the probability of channel openings, rather than changes in the number of functional active channels. ngen can thus interact with bkca channels to increase the amplitude of ik in these cells, even in the absence of bkca-channel β-subunits.figure stimulatory effect of ngen on bk ca -channel activity measured from α -hslo -expressing hek293t cells. original current traces showing the activity of bkca channels before and after application of 30 μm ngen. inside-out recordings were conducted with symmetrical k+ concentration. the potential was constantly held at +60 mv, and bath medium contained  μm ca2+. channel openings give an upward deflection in current. summary of the data showing effect of ngen and ngen plus verruculogen on the probability of bkca-channel openings. *significantly different from control. **significantly different from ngen alone group. simulated firing and bursting pattern of aps in central modeled neuron with varying gm or gkca we further explored how the dynamics of bursting firing elicited by continuous current injection can be altered by increasing the values of gm and gkca to mimic the stimulatory of ngen on ik and ik described above. for studying this, a brief depolarizing current with  ma was applied to the modeled central neuron with the parameter values illustrated in table  in an attempt to generate burst firing of neuronal aps. the descriptions for this modeled neuron were detailed under methods. initially, the gm value was arbitrarily elevated from to ms/cm subsequently, to mimic the condition in which ngen was added, the values of gm and gkca was arbitrarily raised by two-fold. as illustrated in figure  by elevating gm value alone, we are able to show an increase in the intra-burst interval in combination with the increased ik. as both values of gm and gkca were elevated, intra-burst rate was reduced, along with the increase of after-hyperpolarizaton from - to -85 mv. it also needs to be noted that in figure 10cb, the ik amplitude numerically generated was increased only by -fold, although gm value was elevated by 2-fold as compared with the results in figure 10bb. therefore, it is possible from these simulation results that when both gm and gkca are at work simultaneously, the outcome may not simply be due to the summation of individual element.table default parametric values used for the modeling of hippocampal ca pyramidal neurons symbol description value gna gcat gcal gcan gkdr ga gm gkca gkahp gleak iapp vna vk vleakburst firing of aps and the corresponding changes of i k and i k in modeled hippocampal neuron. the detailed formulations were described under methods. the firing was generated with the values of gm and gkca was set at and ms/cm2, and ms/cm2 and and ms/cm2, respectively. ab, bb, and cb indicate the corresponding changes in i k and i k, respectively. in, the values of gm and gkca were arbitrarily elevated by two-fold to mimic the stimulatory effects of ngen. note that in this simulation, the amplitudes of i k are smaller than those of i k. fast-slow analysis of ap bursting in model neuron finally, in order to develop a better quantitative understanding of km and kca channels, we further performed fast-slow analysis in a reduced comparable model. such a maneuver can be allowed for separation of the variables into two subsystems, i.e., fast and slow. one can analyze how the bursting from this modeled neuron is numerically generated owing to a delicate interaction of these two variables. in other words, one is a slow autocatalytic variable n and the other is a slow negative feedback variable o. in this analysis, we treated slow variables n and o as the parameters to study the fast subsystem and subsequently to determine the regions of spiking and resting states, as the different values of gm and gkca are chosen. as shown in figure the two-parameter phase plot clearly showed the ability of the burst trajectory to be projected onto the slow variables. the solid and dashed lines shown in figure  correspond to the voltage nullcline for the fast system at the indicated values of n and o, where the saddle node bifurcation occurs. it becomes clear from this plot that an interaction of n and o was able to generate slow oscillations that were allowed to move the fast subsystem in and out of the repetitive spiking regime. the arrows shown in figure  indicate the direction of the trajectory movement. the region above the nullcline corresponds to a region of repetitive firing, while the region below the nullcline is where the system remains silent. the arbitrary increase of gm value from to ms/cm was noted to shift voltage nullcline in an upward direction with minimal changes in burst trajectory on n-o plane. however, of interest, when the values of gm and gkca were simultaneously elevated by two-fold to mimic the stimulatory actions of ngen described above, the nullcline was further shifted in an upward direction accompanied by the compression of burst trajectory in size on n-o plane. such upward shift in the voltage nullcline indicates a reduction of repetitive spiking regime. the results of these changes are therefore compatible with our experimental and computational data showing that the increased values of gm and gkca caused by ngen result in a reduction of intraburst firing accompanied by the prolongation of interburst period and the increase of the afterhyperpolarization of aps, if similar findings are observed in central neurons in vivo.figure fast-slow analysis of ap bursting generated from modeled neuron. the phase plot of burst firing elicited by continuous current injection shows the projection of the burst trajectory onto n-o plane. n and o denote the activation gates of cav and kca channels, respectively. arrows indicate the direction of trajectory movement. the solid and dashed lines are the voltage nullcline of the fast subsystem with n and o as parameters. below the nullcline is the silent region. notably, as the value of gm was increased from to ms/cm the nullcline was shifted in an upward direction with minimal change in the burst trajectory. when the values of gm and gkca were simultaneously elevated by two-fold to mimic the effect of ngeg, the burst trajectory was clearly compressed in combination with a further upward shift in the voltage nullcline. discussion in this study, we provide the direct evidence that ngen has a stimulatory action on ik in nsc motor neuron-like cells. the half-maximal concentration of ngen required for stimulation of ik was about  μm, which is much smaller than that reported for the inhibition of herg channels. because of their lipophilicity, ngen was also reported to transverse the blood-brain barrier and subsequently to penetrate into different brain regions, although the brain concentrations of ngen vary based on local extracellular milieu in and around membranes and synapses. the importance of kv channels as regulators of neuronal excitability became apparent, when mutated neuronal kv channels were demonstrated to cause benign familial neonatal convulsions. it is thus anticipated that in motor neurons like nsc- cells, the kv7/km channels are an important therapeutic target for the action of this agent or other structurally similar compounds within the cns. addition of ngen could shift the activation curve of ik conductance to the hyperpolarized potential in this study. inability of ngen to alter the gating charge of km channels observed in nsc- cells allows us to suggest that the stimulatory effect of this compound on km channels in nsc- cells is not mediated through a direct effect on the voltage sensor per se. the binding site of ngen is most likely to lie outside of the transmembrane field around the channel. nevertheless, as the neurons are exposed to ngen, the effects of this compound on ik amplitude could rely upon the ngen concentration used, the level of pre-existing membrane potential, or both. the pharmacological and electrophysiological properties of km channels observed in nsc- cells are similar to those in other types of neurons described previously. in cell-attached configuration, addition of ngen into the bath was noted to increase the probability of channel openings with no discernible change in single-channel amplitude. the inability of ngen to modify single-channel conductance indicates that the increased responsiveness of the channel to this compound is not located at the central ion-conducting pore of the channel. similarly, flupirtine at a concentration of 10 μm was capable of enhancing km-channel activity in these cells. the stimulatory actions of ngen and flupirtine on the activity of km channels were not additive in nsc- neuronal cells. our results suggest that the two compounds exert their major effects on the same component existing in km channels. further work is also required to see whether this compound can interact with the kv7/km channels to stimulate ik in other types of central neurons. previous reports have demonstrated the ability of ngen to bind to gabaa receptors and to produce anxiolytic actions. a recent study also showed that ngen might inhibit cl− secretion in isolated colonic epithelia. in our study, macroscopic ik was measured in the recording pipette which was filled with a solution containing aspartic acid, rather than cl- ions. moreover, gabazine, a blocker of gabaa receptors, had minimal effects on ngen-stimulated ik in nsc- cells. it thus seems unlikely that the observed increase of ik caused by ngen is inherently associated with the increase of cl− current induced by its binding to gabaa receptors. an earlier work has demonstrated that green tea flavonoids such as epigallocatechin and epicatechin were capable of reducing the activity of atp-sensitive k+ channels. under our experimental condition, the recording pipette used for measurements of whole-cell ik was filled with a solution which contained 3 mm atp. moreover, ngen-stimulated ik observed in nsc- cells was not reversed by subsequent application of glibenclamide, but by linopirdine. therefore, in nsc- cells or in hek293t cells expressing α-hslo, the elevation of k+ currents induced by ngen is not necessarily related to any changes in atp-sensitive k+ channel conductance. it is noteworthy that the observed increase in the whole-cell ik by ngen did not appear to match its increase in km-channel activity. the reason for this discrepancy is currently unclear. however, it is likely that the ngen effects are much dependent on some receptors and/or intracellular second messenger systems which are possibly washed out during our whole-cell recordings. it remains to be further clarified whether any changes in receptors and/or transmembrane signal pathways are involved in the ngen effect on km channels. in the present study, under a cell-attached recording, we found that addition of ngen decreased the firing frequency of spontaneous action currents in nsc- cells. the reduction of firing frequency caused by ngen in these cells could be primarily explained by its activation of km channels, because linopirdine can significantly reverse ngen-mediated inhibition of spontaneous action currents. it was also noted that when the recording pipette was filled with iberiotoxin or apamin, ngen-induced reduction in the firing of action currents still existed. moreover, because ik was relatively not subject to block by ngen at a concentration of 10 μm, lack of stimulatory effect of ngen on the spontaneous firing of action currents could be demonstrated in the presence of this compound. activation of bkca channels induced by ngen in vascular myocytes reported previously was noted to share similar characteristics shown here in hek293t cells in which α-hslo channels are functionally expressed. our results also suggest that ngen may bind to a site located in the cytoplasmic side of the α-subunit. in addition, bms- and meclofenamic acid, known to be activators of bkca channels, was shown to activate kv7/km channels. therefore, these two types of ion channels is most likely to share the unique motifs with which some small compounds such as ngen can interact. another notable issue is the subunit composition of the bkca channels. in this study, we used homomeric α-hslo channels and imply the consequences for neuronal firing. however, in the central nervous system, α subunits are accompanied by accessory β proteins. β subunits were described to change channel pharmacology and current phenotype dramatically, and even produce profound effect on the neuronal firing. therefore, it is important to investigate to what extent ngen produces any effects on α-hslo + β channels, although the results showing that homomeric α-hslo channels are activated by this compound are valuable from mechanistic standpoints. there is evidence to show that ngen is a target for km channel. in our study, we also found that this compound could be a direct stimulator of bkca channels. with the aid of blastx program, we further examined the similarity of amino acid sequence between the α-subunit of bkca channel and kcnq protein. interestingly, a portion of bkca-channel α-subunit, to which the sequence of kcnq shares the similarity, is located at 182- this region are noted to correspond with the ion transport domain of the bkca-channel α-subunit. therefore, it is tempting to speculate that ngen or other structurally similar compounds can interact at this region to influence the activity of bkca channels or/and kv7-encoded channels. in the modeled hippocampal neurons presented herein, burst firing of aps in response to brief current injection was readily generated and an interaction of two slow variables could be derived. in other words, as shown in figure  a slow autocatalytic variable correspond to the activation gate of t-type ca2+ channel, while a slow negative feedback variable is the activation gate of kca channel. in this system, the results from fast-slow analysis led us to suggest that the subtle interaction of activation gates between t-type ca2+ channel and kca channel is able to generate slow oscillations that move the fast subsystem from a stable non-oscillatory regime into a repetitive spiking regime and back again. superimposition of the voltage nullcline computed with the gates designated as parameters is able to illustrate where this bifurcation emerges. it is tempting to speculate that the presence of ngen potentially alters the firing behaviors of central neurons in vivo, assuming that those neurons functionally express the activity of both km and bkca channels.  the stimulatory effect of ngen on native ik expressed in central neurons could have an impact on the functioning of the neurons. further work is required to see whether the activity of km channels stimulated by this compound is responsible for the antinociceptive or antioxidative actions of different compounds known to activate ik. besides that, ngen-induced actions on the stimulation of km and bkca channels may combine to affect the functional activities if both channels are expressed in central neurons in vivo. our findings raise the possibility that ngen-mediated changes in ion currents of neurons described above are closely linked to its possible neuroprotective actions. 
in this study, we attempted to evaluate the possible effects of ngen on k+ currents in nsc- neuronal cells and in hek293t cells expressing α-hslo. ngen had minimal effect on erg-mediated k+ currents. ngen shifted the activation curve of ik conductance to the more negative potentials. the present results demonstrate that activation of both km and bkca channels caused by ngen might combine to influence neuronal activity if similar channels were functionally co-expressed in central neurons in vivo. ngen increased m-type k+ current ) in a concentration-dependent manner with an ec value of  μm in nsc- cells. in cell-attached recordings, ngen or flupirtine enhanced the activity of m-type k+ channels with no changes in single-channel amplitude. naringeninm-type k+ channellarge-conductance ca2+-activated k+ channelmotor neuronissue-copyright-statement© the author 2014 under cell-attached voltage-clamp recordings, ngen decreased the frequency of spontaneous action currents and further application of linopirdine can reverse ngen-induced inhibition of firing. moreover, from the study of a modeled neuron, burst firing of simulated action potentials was reduced in the presence of the increased conductances of both km and kca channels. fast-slow analysis of ap bursting from this model also revealed that as the conductances of both km and bkca channels were increased by two-fold, the voltage nullcline was shifted in an upward direction accompanied by the compression of burst trajectory. under inside-out recordings, ngen applied to the intracellular side of the detached patch enhanced the activity of large-conductance ca2+-activated k+ channels. in hek293t cells expressing α-hslo, this compound increased the amplitude of ca2+-activated k+ current ). naringenin is a citrus bioflavonoid known to have beneficial health properties; however, the ionic mechanism of its actions remains largely unclear.
 naringenin is a citrus bioflavonoid known to have beneficial health properties; however, the ionic mechanism of its actions remains largely unclear. in this study, we attempted to evaluate the possible effects of ngen on k+ currents in nsc- neuronal cells and in hek293t cells expressing α-hslo.  ngen increased m-type k+ current ) in a concentration-dependent manner with an ec value of  μm in nsc- cells. ngen shifted the activation curve of ik conductance to the more negative potentials. in cell-attached recordings, ngen or flupirtine enhanced the activity of m-type k+ channels with no changes in single-channel amplitude. ngen had minimal effect on erg-mediated k+ currents. under cell-attached voltage-clamp recordings, ngen decreased the frequency of spontaneous action currents and further application of linopirdine can reverse ngen-induced inhibition of firing. in hek293t cells expressing α-hslo, this compound increased the amplitude of ca2+-activated k+ current ). under inside-out recordings, ngen applied to the intracellular side of the detached patch enhanced the activity of large-conductance ca2+-activated k+ channels. moreover, from the study of a modeled neuron, burst firing of simulated action potentials was reduced in the presence of the increased conductances of both km and kca channels. fast-slow analysis of ap bursting from this model also revealed that as the conductances of both km and bkca channels were increased by two-fold, the voltage nullcline was shifted in an upward direction accompanied by the compression of burst trajectory.  the present results demonstrate that activation of both km and bkca channels caused by ngen might combine to influence neuronal activity if similar channels were functionally co-expressed in central neurons in vivo. keywords naringeninm-type k+ channellarge-conductance ca2+-activated k+ channelmotor neuronissue-copyright-statement© the author 2014 