voltage - dependent ion channels are major contributors to decide physiological functions and the generation of pathophysiological conditions in mammalian excitable cells , such as neurons and cardiac myocytes . therefore , agonists and antagonists of ion channels are widely used for biological and therapeutical approaches in researches and clinic applications [ 1 - 5 ] . in nature , a number of toxins acquired from insects , snakes , and fish play roles to block and modulate the permeability of ion channels expressed in cellular membranes . for example , na channels are blocked by tetrodotoxin from pufferfish and mu - conotoxin from conusstriatus , while they are opened by versutoxin from spiders [ 6 - 8 ] . because the pharmacological modification of gating properties of ion channels is most simple but powerful for testing their functions in neurons , the discovery of new toxins also leads biological researchers to find unknown physiological functions of cells that are correlated with specific ion channels in mammalian systems . in addition , most ion channels have several subunits exhibiting distinctive kinetics and permeability , indicating that the classification of specific toxins for each ion channel subunit is also important to define their delicate functions [ 9 - 12 ] . as they can determine cardiac rhythm and neuronal transmission , voltage - dependent k channels ( kv channels ) are most critical channels working in both cardiac and nervous systems [ 13 - 16 ] . in electrophysiological experiments , major k outward currents through kv channels can be recorded by using voltage - clamp methods and classified into two patterns , transient or rapidly inactivating currents - known as a - type ( ia ) - and sustained or not inactivating currents . in hippocampal ca1 neurons of mammalian brains , kv4.2 and kv4.1 subunits of kv channels previous papers have reported that these k subunits are sensitive to several natural toxins and chemicals [ 16,18 - 21 ] for example , hanatoxins have been reported to block kv4.2 as well as kv2.1 channel subunits expressed in xenopus laevis oocytes , indicating that these toxins are not specific for one of several subunits . recently , heteropodatoxins ( hptxs ) isolated from a spider , heteropoda venatoria , are known as more specific blockers of ia by blocking kv4 families but not kv1.4 subunits . in particular , hptx2 has dose- and voltage - dependent blockade effects on ia via binding the s3-s4 linker region of kv4.2 channels . these previous results are not sufficient to define and specify the effects of hptx2 on ia channels , because the blocking effects were tested only on channels expressed experimentally in xenopus oocytes or cardiac cells . in addition , subunits of voltage - dependent k channels in native mammalian neurons have homologue structures with six membrane spanning portions and share auxiliary subunits , insisting that the effects of hptx2 on k currents must be demonstrated in neurons . it is also possible that , because of the hydrophilic property of the toxin and the location of binding sites , hptx2 might have effects on other k subunits according to application protocols . in the present study , we attempted to test effects of hptx2 on both transient and sustained k outward currents in ca1 neurons of cultured organotypic hippocampal slices . in nucleated - patch experiments , however , when hptx2 was added to internal pipette solution , sustained but not transient currents showed a significant reduction . although we did not discover the binding mechanism in this study , these results clearly indicate that hptx2 effects to modulate or block k outward currents may be dependent on the location of binding sites of each subunit . organotypic hippocampal slices ( 350 m thick ) were prepared from postnatal day 7~8 sprague - dawley rats . after preparation of hippocampal slices in a cutting solution containing ( in mm ) : 10 mgcl2 , 25 glucose , 20 hepes in gey 's balanced salt solution ( sigma ) , slices were transferred to an incubator gassed with 95% o2 and 5% co2 at 35 , and cultured for 5~6 days before performance of electrophysiological measurements . the animal care and use committee of jeju national university approved all animal protocols . for patch - clamp recordings from organotypic slices , slices were transferred to a submerged recording chamber with continuous flow of acsf containing ( in mm ) : 125 nacl , 2.5 kcl , 25 nahco3 , 1.25 nah2po4 , 25 glucose , 2 cacl2 , 1 mgcl2 . 5 m 2-chloroadenosine , and 5 m bicuculine were added in some recordings . ttx ( 1 m ) was added for recording voltage - dependent k currents immediately after pulling nucleated - patches . normal patch electrodes ( 4~6 m ) were filled with ( in mm ) : 20 kcl , 125 k - gluconate , 10 hepes , 4 nacl , 0.5 egta , 4 atp , 0.3 trisgtp , and 10 phosphocreatin . in some experiments , patch electrodes were filled with cs - gluconate internal solution , including ( in mm ) : 100 cs - gluconate , 5 mgcl2 , 0.6 egta , 8 nacl , 40 hepes , 2 naatp , and 0.3 trisgtp . ph and osmolarity were adjusted to 7.2~7.3 and 280~300 mosm , respectively , in all experiments . k outward current recordings using a nucleated - patch were performed at 31~32 and low - pass filtered at 5 khz and digitized at 10 khz by an heka 9/2 , which was controlled by software pulse ( heka ) . command pulse generation and data acquisition were performed using pulse , and igor pro ( wavemetrics ) and excel ( microsoft ) software were used for further data and statistical analysis . unpaired t - tests were used in performance of statistical analysis , and significance was set to p<0.05 or 0.01 . recording voltage - gated k currents was made in voltage - clamp mode of nucleated - patches ( 18.350.5 pf , n=37 ) . hptx2 recombinant ( sigma - aldrich , usa ) was added to either external acsf or internal pipette solution to test blocking effects on k outward currents , and 4-ap was added to internal pipette solution . in this study , to clarify the effects of drugs added intracellularly , all nucleated - patches were pulled within 2 min after making a whole - cell . leakage and capacitive currents were subtracted digitally using either a p/6 protocol or null traces . the transient current was isolated from the sustained current using 200 ms pre - pulse step to -20 mv for inactivation of transient channels . more detailed protocols for isolation of each pure current can be found in our previous paper . organotypic hippocampal slices ( 350 m thick ) were prepared from postnatal day 7~8 sprague - dawley rats . after preparation of hippocampal slices in a cutting solution containing ( in mm ) : 10 mgcl2 , 25 glucose , 20 hepes in gey 's balanced salt solution ( sigma ) , slices were transferred to an incubator gassed with 95% o2 and 5% co2 at 35 , and cultured for 5~6 days before performance of electrophysiological measurements . for patch - clamp recordings from organotypic slices , slices were transferred to a submerged recording chamber with continuous flow of acsf containing ( in mm ) : 125 nacl , 2.5 kcl , 25 nahco3 , 1.25 nah2po4 , 25 glucose , 2 cacl2 , 1 mgcl2 . ttx ( 1 m ) was added for recording voltage - dependent k currents immediately after pulling nucleated - patches . normal patch electrodes ( 4~6 m ) were filled with ( in mm ) : 20 kcl , 125 k - gluconate , 10 hepes , 4 nacl , 0.5 egta , 4 atp , 0.3 trisgtp , and 10 phosphocreatin . in some experiments , patch electrodes were filled with cs - gluconate internal solution , including ( in mm ) : 100 cs - gluconate , 5 mgcl2 , 0.6 egta , 8 nacl , 40 hepes , 2 naatp , and 0.3 trisgtp . ph and osmolarity were adjusted to 7.2~7.3 and 280~300 mosm , respectively , in all experiments . k outward current recordings using a nucleated - patch were performed at 31~32 and low - pass filtered at 5 khz and digitized at 10 khz by an heka 9/2 , which was controlled by software pulse ( heka ) . command pulse generation and data acquisition were performed using pulse , and igor pro ( wavemetrics ) and excel ( microsoft ) software were used for further data and statistical analysis . unpaired t - tests were used in performance of statistical analysis , and significance was set to p<0.05 or 0.01 . recording voltage - gated k currents was made in voltage - clamp mode of nucleated - patches ( 18.350.5 pf , n=37 ) . hptx2 recombinant ( sigma - aldrich , usa ) was added to either external acsf or internal pipette solution to test blocking effects on k outward currents , and 4-ap was added to internal pipette solution . in this study , to clarify the effects of drugs added intracellularly , all nucleated - patches were pulled within 2 min after making a whole - cell . leakage and capacitive currents were subtracted digitally using either a p/6 protocol or null traces . the transient current was isolated from the sustained current using 200 ms pre - pulse step to -20 mv for inactivation of transient channels . more detailed protocols for isolation of each pure current can be found in our previous paper . kv4.2 subunit , a major contributor to transient k outflow , is targeted by activity - dependent synaptic modulations and its functions are considerably mediated with the activities of synaptic glutamate receptors in ca1 neurons of rat hippocampi . these previous papers indicate that trafficking properties of kv4.2 channels are rapidly driven by cellular signalings activated by synaptic activities . because the rapid internalization of kv4.2 channels by synaptic activities or plasticities resulted in the reduction of transient currents , the blockade of synaptic activities in ca1 neurons during recording was necessary for testing effects of hptx2 on k outward currents . for this , we used a nucleated - patch technique for isolating soma membrane of a ca1 neuron ( fig . this methodological approach allowed us to test the intracellular or extracellular effects of hptx2 without influences of synaptic activities . in all experiments performed in this study , nucleated - patches were completely pulled within 2 min after making whole - cells , and neurons were rejected in cases of over 2 min . in the previous paper , the extracellular application of 100~200 nm hptx2 blocked only ia currents through kv4 subunits in rat ventricular myocytes . therefore , we first tested 200 nm hptx2 , which was added to external acsf , to determine whether it could block transient currents in ca1 neurons . total k outward currents were recorded at + 60 mv holding potential in a voltage - clamp mode , and sustained and transient currents were isolated by using a pre - pulse protocol ( fig . consistent with previous reports , the prominent effect of hptx2 was observed only on transient currents . ca1 neurons showed a small but significant reduction of transient current amplitude in the presence of 200 nm hptx2 , compared with neurons tested in normal recording solution ( fig . 1b and c , transient , control=1.570.45 , n=5 ; hptx2=1.320.71 na , n=6 ; p<0.05 ) . however , sustained currents were not affected by extracellular hptx2 ( fig . 1b and c , sustained , control=0.190.01 ; hptx2=0.180.01 na ) . these results indicate that existence of hptx2 in extracellular spaces can modulate the gating properties of kv4.2 subunit in ca1 hippocampal neurons . 1 ) were also tested for confirming the concentration - dependent effects on ca1 neurons . all concentration of hptx2 significantly reduced transient currents of ca1 neurons , but no differences of reduction rates according to each concentration were observed . this indicates a restricted modulation of gating kinetics of ia channels by hptx2 in ca1 neurons . also , we confirmed that long exposure to hptx2 ( 2 m , 24 hours ) exhibited the similar rate of currents reduction , compared with short exposure effects . it is possible that blocking or modulating effects of toxins on gating properties of channels are variable regarding binding affinities between toxins and channels . in fig . 2 , the next experiments were performed in order to determine if intracellular application of hptx2 also has effects to block only transient currents in neurons . using internal pipette solution including either 100 or 200 nm hptx2 , unexpectedly , both 100 and 200 nm of hptx2 were effective on sustained but not transient currents . approximately 30% of the peak amplitude of sustained currents was reduced by intracellular hptx2 five minutes after making a whole cell ( fig . 2 , 200 nm hptx2 ; sustained , 0 min=0.190.08 , 5 min=0.140.04 , p<0.01 ; transient , 0 min=1.590.11 , 5 min=1.520.12 na ; n=11 ) . the reduction of sustained currents by hptx2 seemed not to be dose - dependent , showing a similar decrement between 100 ( n=5 ) and 200 nm treatments ( fig . altering intracellular components is critical for determining channel permeability and neuronal responses , and cytosolic space of soma is significantly restricted under a nucleated - patch condition . therefore , addition of hptx2 to an internal solution might possibly affect channel conductances without pharmacological actions . for clarifying this issue , we analyzed changes of current density , which was measured by whole - cell patch capacitances ( fig . 2c ) . all neurons recorded with intracellular hptx2 ( n=16 ) did not show significant changes of cell capacitances between 0 min ( before recording , 19.83.2 pf ) and 15 min ( after recording , 21.12.2 pf ) after pulling nucleated - patches , so it is clear that the densities of transient and sustained currents reflected the changes of peak amplitudes ( fig . 2c , transient : 0 min=81.693.79 , 15 min=74.814.02 , p=0.248 ; sustained : 0 min=9.620.35 , 15 min=6.540.29 pa / pf , p<0.01 ) . in addition , we tested other agents known as blockers of k channels to determine if they also have intracellular effects on either transient or sustained currents . 4-ap is widely used for suppressing k outward currents in dendrites and soma , and was previously confirmed to be effective on transient currents when it was added to internal pipette solution . in this study , 3 mm of 4-ap was added to internal solution , and nucleated - patches were then made ( fig . 4-ap application resulted in a rapid and significant reduction of the peak amplitude in transient currents but not in sustained currents ( fig . 3a , normalized amplitude at 10 min compared with 0 min ; transient=0.350.04 , p<0.05 ; sustained=0.860.02 , n=5 ) . we also tested cs - gluconate internal solution ( fig . both transient and sustained currents showed a gradual and significant reduction 10 min after pulling nucleated - patches ( fig . 3b , normalized amplitude at 10 min ; transient=0.670.04 , p<0.05 ; sustained=0.710.03 , p<0.05 , n=5 ) . therefore , the results shown in fig . 2 and 3 suggest that blocking effects of agents tested in this study may possibly be variable according to the location of channel effective sites . in fig . 4 , the reduction rates of transient and sustained currents by intracellular drug treatments , which were calculated by the ratio between 0 min and 15 min peak after pulling nucleated - patches , were compared . the reduction rate of hptx2 in sustained currents was significantly larger than that in transient currents , while 4-ap reduced only transient currents ( fig . 4 , hptx2 , transient : sustained=6.432.55 : 27.683.22% ; 4-ap , transient : sustained=64.153.51:18.026.08% ) . these results suggest the possibility that k outward flows in ca1 neurons can be selectively blocked by chemicals or toxins according to their application methods . the opening properties and current flows through k channels are critical factors in regulation of neuronal excitabilities . therefore , studying agents to block specific subunits of k channels is valuable for understanding neuronal functions . major findings in this study are that hptx2 is not a specific agent to block or modulate transient currents , and that the intracellular application of hptx2 can reduce sustained k outward currents in ca1 hippocampal neurons . although these results are not consistent with previous reports demonstrating hptx2 effects to be specific on kv4 families , it should be considered that sustained and transient currents were sensitive to intracellular and extracellular hptx2 , respectively . because testing hptxs effects has previously and dominantly been performed in xenopus oocytes or cardiac myocytes , the effective reduction in both sustained and transient currents in ca1 neurons of hippocampal slices indicates that hptx2 is also valuable for studying the physiological functions of k channels working in neuronal excitabilities of mammalian cns . transient currents through kv4.2 channels in ca1 neurons play crucial roles in modulation of ap back - propagation , dendritic integration of excitatory postsynaptic potentials , synaptic composition of glutamate receptors and intrinsic excitabilities . in particular , synaptic activities of hippocampal neurons can directly regulate kv4.2 trafficking in dendrites and soma . therefore , most studies investigating modulatory functions of k channels in neuronal excitability , primarily focus on either kv4.2 or kv1.4 subunit . however , pharmacological approaches to block kv4 and kv1 families have difficulties in the absence of specific chemical agents , even though some chemicals were useful for blocking the various k outward currents in cardiac and neuronal cells . in the last decade , pharmacological studies of some toxins from insects and snakes have suggested a possibility for the manipulation of specific kv subunits , and provided credible results that hptxs and hanatoxins are valuable to determine neuronal functions of kv channels [ 12,18 - 20 ] . of them , hptx2 is known as a gating regulator specific to kv4 subunits via binding to the s3-s4 linker region of kv4.2 . however , because of insufficient evidence in mammalian cns , it is not suitable for determining pharmacological effects of hptx2 on native channels in neurons . in addition , rapid changes in trafficking and gating properties of kv4 subunits are induced by synaptic activities . in this study , extracellular application of 200 nm hptx2 resulted in a small but significant reduction of transient but not sustained k outward currents in nucleated - patched ca1 neurons . this indicates that hptx2 can act as a gating modulator of kv4.2 subunit natively expressed in ca1 neurons , consistent with effects on channels expressed experimentally in xenopus oocytes . however , intracellular hptx2 significantly reduced sustained currents , showing no changes in transient currents . compared with intracellular 4ap that reduced only transient currents , it is possible that the blocking effect of cytosolic hptx2 on sustained currents may be due to pharmacological interactions with kv2.1 subunit showing homologue structures with other kv families . because hydrophilic or hydrophobic property of chemicals is correlated with their membrane permeability , different actions between intracellular and extracellular hptx2 can be explained by a hypothesis that hydrophilic hptx2 and the location of effective sites in channels may determine the toxin effects on kv channels . kv2.1 channels , as well as kv4 families , are major determinants of neuronal excitabilities , so they can regulate neuronal responses based on synaptic inputs and dendritic integrations . in addition , several reports have provided evidence that sustained k currents can continuously compensate for activity - dependent changes of transient currents and membrane excitabilities via regulating intracellular k concentrations [ 28 - 31 ] . therefore , distinctive actions of hptx2 inside and outside of neurons are very effective for the selective reduction of specific k outward currents , compared with effects of cs - gluconate internal solution acting in both transient and sustained currents . in the present study , we did not confirm the interaction mechanism between intracellular hptx2 and channels contributing to sustained currents . the precise actions of cytosolic hptx2 to correlate with kv subunits should be addressed in a future study .
blocking or regulating k+ channels is important for investigating neuronal functions in mammalian brains , because voltage - dependent k+ channels ( kv channels ) play roles to regulate membrane excitabilities for synaptic and somatic processings in neurons . 
 although a number of toxins and chemicals are useful to change gating properties of kv channels , specific effects of each toxin on a particular kv subunit have not been sufficiently demonstrated in neurons yet . in this study , we tested electrophysiologically if heteropodatoxin2 ( hptx2 ) , known as one of kv4-specific toxins , might be effective on various k+ outward currents in ca1 neurons of organotypic hippocampal slices of rats . using a nucleated - patch technique and a pre - pulse protocol in voltage - clamp mode 
 , total k+ outward currents recorded in the soma of ca1 neurons were separated into two components , transient and sustained currents . 
 the extracellular application of hptx2 weakly but significantly reduced transient currents . however , 
 when hptx2 was added to internal solution , the significant reduction of amplitudes were observed in sustained currents but not in transient currents . 
 this indicates the non - specificity of hptx2 effects on kv4 family . 
 compared with the effect of cytosolic 4-ap to block transient currents , it is possible that cytosolic hptx2 is pharmacologically specific to sustained currents in ca1 neurons . 
 these results suggest that distinctive actions of hptx2 inside and outside of neurons are very efficient to selectively reduce specific k+ outward currents .