inward rectifiers , a subset of k channels , play many important biological roles by controlling and regulating the resting membrane potential ( hille 1992 ) . these channels conduct much larger inward than outward currents even when k concentrations on both sides of the membrane are equal ( katz 1949 ; noble 1965 ; hille 1992 ) . this unusual conduction feature , now commonly referred to as inward rectification , enables these channels to control and regulate the resting membrane potential without impeding the generation of action potentials ( hille 1992 ) . inward - rectifier k channels are inhibited by intracellular mg and polyamines ( horie et al . channel inhibition by these intracellular cations depends strongly on membrane voltage , which causes the channel to conduct in an inwardly rectifying manner . inward rectification in some weak rectifiers , such as romk1 , results from simple voltage - dependent block of their ion conduction pore by the intracellular cations , as in the case of block of the voltage - activated k channels in squid by intracellular tea ( armstrong and binstock 1965 ) . initially , the strong rectification in the absence of intracellular mg was thought to reflect intrinsic channel gating ( e.g. , ishihara et al . 1989 ; silver and decoursey 1990 ) . this apparently intrinsic gating was subsequently interpreted as a manifestation of channel block by intracellular polyamines ( ficker et al . however , a cloned strong rectifier ( irk1 ) still exhibits modest inward rectification even after the excised membrane patch is exhaustively perfused with a solution nominally devoid of mg and polyamines ( e.g. , aleksandrov et al . in other words , removing mg and polyamines diminishes , but does not eliminate , inward rectification . these observations caused a reconsideration of intrinsic ( voltage - dependent ) channel gating , rather than pore block , as the mechanism of inward rectification , and led to the proposal that inward rectification results primarily from enhancement of voltage - dependent channel gating by the action of intracellular mg and/or polyamines ( aleksandrov et al . we notice that the extent of the residual inward rectification in the absence of mg and polyamines varies among laboratories . for example , it is considerably smaller in our previously published report than in those of others ( e.g. , compare guo and lu 2000a , with aleksandrov et al . however , incomplete removal of high - affinity polyamines such as spermidine or spermine alone can not explain the remaining , variable rectification , since we found its voltage dependence to be much weaker than that associated with channel inhibition by those polyamines ( guo and lu 2000a ) . irk1 cdna was cloned into the pcdna1/amp plasmid ( invitrogen ) ( kubo et al . 1993 ) . the cdna encoding the d172n mutant channel was produced using the polymerase chain reaction primed with a mutagenic oligonucleotide . a sequenced fragment containing the mutation ( between sali and bstbi sites ) was subcloned into a wild - type recipient version of the irk1 cdna . oocytes harvested from xenopus laevis ( xenopus one ) were incubated in a solution containing ( mm ) : 82.5 nacl , 2.5 kcl , 1.0 mgcl2 , 5 hepes , ph 7.6 , and 24 mg / ml collagenase . the oocyte preparation was agitated using a platform shaker ( 80 rpm ) for 6090 min . it was then rinsed thoroughly and stored in a solution containing ( mm ) : 96 nacl , 2.5 kcl , 1.8 cacl2 , 1.0 mgcl2 , 5 hepes , ph 7.6 , and 50 g / ml gentamicin . defolliculated oocytes were selected and injected with rna at least 2 and 16 h , respectively , after collagenase treatment . macroscopic irk1 currents were recorded in the inside - out configuration from xenopus oocytes ( injected with irk1 crna ) with an axopatch 200b amplifier ( axon instruments , inc . ) , filtered at 5 khz , and sampled at 25 khz using an analogue - to - digital converter ( digidata 1200 ; axon instruments , inc . ) interfaced with a personal computer . pclamp6 software ( axon instruments , inc . ) was used to control the amplifier and acquire the data . during current recording , the voltage across the membrane patch was first hyperpolarized from the 0 mv holding potential to 100 mv for 25 ms , and then stepped to a test voltage between 100 and + 100 mv for a period of 100 ms ; the increment between consecutive test voltages was 10 mv . background leak current correction was carried out as previously described ( lu and mackinnon 1994 ; spassova and lu 1998 ) . during the recording , the tip of the patch pipette was immersed in a stream of intracellular solution exiting 1 of 10 glass capillaries ( 0.2 mm i.d . ) all recording solutions contained 100 mm k contributed by : kcl , k2edta , k2hpo4 , kh2po4 , k2b4o7 , and koh that was used to adjust ph . the hepes - containing pipette ( extracellular ) solution contained ( mm ) : 100 k ( cl + oh ) , 0.3 cacl2 , 1.0 mgcl2 , and 10 hepes , ph 7.6 ( adjusted with koh ) . in the mops- , phosphate- , and borate - buffered pipette solutions , ph 7.6 , hepes was replaced by an equal concentration of mops ( ph adjusted with koh ) ,  k2hpo4 + kh2po4  and  k2b4o7 + h3b03 ,  respectively . the hepes - containing bath ( intracellular ) solution contained ( mm ) : 90 k ( cl + oh ) , 5 k2edta [ or 98 k ( cl + oh ) and 1 k2edta , when specified ] , and 10 hepes , ph 7.6 . in the mops- , phosphate- , and borate - buffered bath solutions , ph 7.6 , hepes was replaced by an equal concentration of mops ( adjusted with koh ) ,  k2hpo4 + kh2po4  and  k2b4o7 + h3b03 ,  respectively . when its concentration dependence was examined , hepes ( free acid ) at the specified concentration was included in the phosphate - containing bath solution ( the final ph of the solution was adjusted to 7.6 with koh ) . ( a ) , sigma - aldrich ( b ) , calbiochem ( c ) , icn biomedicals ( d ) , or fisher scientific ( e1 and e2 ) . irk1 cdna was cloned into the pcdna1/amp plasmid ( invitrogen ) ( kubo et al . 1993 ) . the cdna encoding the d172n mutant channel was produced using the polymerase chain reaction primed with a mutagenic oligonucleotide . a sequenced fragment containing the mutation ( between sali and bstbi sites ) was subcloned into a wild - type recipient version of the irk1 cdna . oocytes harvested from xenopus laevis ( xenopus one ) were incubated in a solution containing ( mm ) : 82.5 nacl , 2.5 kcl , 1.0 mgcl2 , 5 hepes , ph 7.6 , and 24 mg / ml collagenase . the oocyte preparation was agitated using a platform shaker ( 80 rpm ) for 6090 min . it was then rinsed thoroughly and stored in a solution containing ( mm ) : 96 nacl , 2.5 kcl , 1.8 cacl2 , 1.0 mgcl2 , 5 hepes , ph 7.6 , and 50 g / ml gentamicin . defolliculated oocytes were selected and injected with rna at least 2 and 16 h , respectively , after collagenase treatment . macroscopic irk1 currents were recorded in the inside - out configuration from xenopus oocytes ( injected with irk1 crna ) with an axopatch 200b amplifier ( axon instruments , inc . ) , filtered at 5 khz , and sampled at 25 khz using an analogue - to - digital converter ( digidata 1200 ; axon instruments , inc . ) interfaced with a personal computer . pclamp6 software ( axon instruments , inc . ) was used to control the amplifier and acquire the data . during current recording , the voltage across the membrane patch was first hyperpolarized from the 0 mv holding potential to 100 mv for 25 ms , and then stepped to a test voltage between 100 and + 100 mv for a period of 100 ms ; the increment between consecutive test voltages was 10 mv . background leak current correction was carried out as previously described ( lu and mackinnon 1994 ; spassova and lu 1998 ) . during the recording , the tip of the patch pipette was immersed in a stream of intracellular solution exiting 1 of 10 glass capillaries ( 0.2 mm i.d . ) all recording solutions contained 100 mm k contributed by : kcl , k2edta , k2hpo4 , kh2po4 , k2b4o7 , and koh that was used to adjust ph . the hepes - containing pipette ( extracellular ) solution contained ( mm ) : 100 k ( cl + oh ) , 0.3 cacl2 , 1.0 mgcl2 , and 10 hepes , ph 7.6 ( adjusted with koh ) . in the mops- , phosphate- , and borate - buffered pipette solutions , ph 7.6 , hepes was replaced by an equal concentration of mops ( ph adjusted with koh ) ,  k2hpo4 + kh2po4  and  k2b4o7 + h3b03 ,  respectively . the hepes - containing bath ( intracellular ) solution contained ( mm ) : 90 k ( cl + oh ) , 5 k2edta [ or 98 k ( cl + oh ) and 1 k2edta , when specified ] , and 10 hepes , ph 7.6 . in the mops- , phosphate- , and borate - buffered bath solutions , ph 7.6 , hepes was replaced by an equal concentration of mops ( adjusted with koh ) , when its concentration dependence was examined , hepes ( free acid ) at the specified concentration was included in the phosphate - containing bath solution ( the final ph of the solution was adjusted to 7.6 with koh ) . all chemicals were purchased from fluka chemical corp . , except hepes , which was purchased from either fluka chemical corp . ( a ) , sigma - aldrich ( b ) , calbiochem ( c ) , icn biomedicals ( d ) , or fisher scientific ( e1 and e2 ) . 1 a shows a series of irk1 current traces at membrane voltages between 100 and + 100 mv in 10-mv increments , with 100 mm k on both sides of the membrane ( ph 7.6 , buffered with hepes ) . 1 c. as previously shown , the outward irk1 current after a step to positive voltages exhibited significant relaxation and , consequently , the corresponding steady state i - v curve ( determined at the end of the voltage steps ) exhibited inward rectification even in a patch exhaustively perfused with the artificial intracellular solution ( fig . furthermore , a slight curvature was also present in the negative portion of the steady state i - v curve . interestingly , when phosphate replaced hepes as the ph buffer in the intracellular ( and extracellular ) solutions , relaxation of the outward current at positive voltages vanished , except at + 100 mv , where it is barely discernible ( fig . 1 b ) . consequently , the steady state i - v curve in phosphate is practically linear ( fig . a similarly linear i - v curve was also observed when borate replaced hepes ( fig in contrast , the i - v curve remained inwardly rectifying as hepes was replaced by another organic zwitterionic buffer , mops ( fig . 1 e ,  ) . we also found that the channel exhibited some slight , but clearly noticeable , inward rectification when we lowered the concentration of intracellular edta from 5 to 1 mm ( fig . 1 e ,  ) . based on this finding and the fact that the channel has an extremely high affinity for intracellular cations , we surmise that the barely discernible residual current relaxation at + 100 mv results from block of the channel by trace amounts of endogenous and/or exogenous cationic blockers , such as metal ions or amines , that we could not completely eliminate . if this is the case , the minimal remaining current relaxation should be further reduced or eliminated altogether by a mutation in the channel pore , d172n , that reduces the affinity of the channel for intracellular cations ( ficker et al . 1994 ; lopatin et al . 1994 ; lu and mackinnon 1994 ; stanfield et al . 1994 ; wible et al . 1994 ; fakler et al . 1995 ; yang et al . 2 shows the behavior of the d172n channel in the presence of the various ph buffers . as expected , the i - v curve of this mutant channel with reduced affinity for intracellular cations exhibited somewhat reduced inward rectification when hepes , or mops , was used as the ph buffer ( compare fig . 2 c with 1 e ) , but became completely linear when phosphate or borate , was used ( fig . 2 c ) . note the absence of current relaxation even at + 100 mv when phosphate replaced hepes as the ph buffer ( compare fig . all remaining data were obtained with the wild - type irk1 channel . unless specified otherwise , they were collected with phosphate in both the intracellular and extracellular solutions , regardless of whether hepes was also present in the intracellular solution . 3 shows several series of irk1 current traces in the presence of various concentrations of intracellular hepes . the current relaxed more strongly with increasing hepes concentration , which suggests that the relaxation results primarily from channel block by hepes and/or some accompanying impurity . the i - v curves without and with various concentrations of hepes are plotted in fig . 4 a. adding increasing amounts of hepes to the phosphate - containing intracellular solution caused an increasingly pronounced downward deflection in the i - v curve at positive voltages . in fig . 4 b , the fraction of unblocked current in the presence of various concentrations of hepes is plotted against membrane voltage . the curves superimposed on the data are fits of the woodhull equation ( woodhull 1973 ) . the fit yields an apparent kd ( at 0 mv ) equivalent to 1 m hepes with an apparent valence ( z ) of 1 . hepes from different commercial sources or even from different lots of the same source affects the channel differently . 5 shows several series of current traces recorded from the same patch in the presence of hepes from various common commercial sources or , for the data labeled e1 and e2 , from different lots of the same source . since both the extent and the rate of current relaxation vary significantly among preparations , the observed channel block must at least in part be caused by a contaminant(s ) . the i - v curves with either phosphate or hepes from the various sources tested are plotted in fig . 6 a , while ratios of the i - v curves with hepes relative to that with phosphate are plotted in fig . regardless of the source of the hepes used , the apparent valence of channel block varies little , whereas the blocking potency varies considerably , most likely reflecting different amounts of contaminant(s ) present . previous studies have shown that intracellular polyamines block the irk1 channel in a complex manner ( e.g. , guo and lu 2000a ) . since we performed the experiments in the quoted study with hepes as the ph buffer , we wondered whether hepes and/or its impurity had significantly affected the general blocking behavior of polyamines . c , shows i - v curves of the channels in the absence and presence of various concentrations of intracellular putrescine , spermidine , or spermine obtained with phosphate , rather than hepes , as the ph buffer . the fractions of unblocked current for each of the three polyamines are plotted in fig . the blocking curve for putrescine tends to a nonzero level at positive membrane voltages , which is lower for higher putrescine concentrations ; qualitatively similar features were seen with spermidine and spermine . nevertheless , only the blocking curves for spermidine and spermine , but not for putrescine , exhibit a significant hump . these peculiar characteristics of polyamine block essentially mirror those previously observed when hepes was used as the ph buffer ( guo and lu 2000a ) . however , the blocking curves shown in fig . 7 differ from the earlier ones by the absence of apparent voltage - independent channel inhibition by polyamines ( compare fig . 7 , d  f , at negative voltages with guo and lu 2000a ) . since , in the present study , we limited patch exposure to polyamine - containing solutions to < 2 min , the apparent voltage - independent inhibition we previously observed may reflect a decrease in the level of membrane - associated pip2 , which is required for channel activity ( huang et al . under commonly employed experimental conditions , the outward irk1 current at positive voltages exhibits significant relaxation , even in excised membrane patches exhaustively perfused with an artificial intracellular solution to remove endogenous blocking ions . as a result , these phenomena led to the hypothesis that inward rectification results mainly from intrinsic ( voltage - dependent ) channel gating , which is enhanced when intracellular cations bind to the gating machinery , located at the intracellular side of the channel ( e.g. , aleksandrov et al . , we found that the current relaxation and the resulting nonlinearity of the steady state i - v curve are primarily related to the use of hepes ( or a similar organic zwitterionic ph buffer , mops ) , because the current relaxation essentially vanished and the i  v curve became practically linear when phosphate ( or borate ) , instead of hepes ( or mops ) , was used as a ph buffer ( fig . 1 ) . the barely discernible current relaxation with phosphate at + 100 mv most likely results from channel block by residual endogenous and/or contaminating exogenous blocking ions ( fig . 1 b ) . this interpretation is supported by the fact that no current relaxation even at + 100 mv was observed in the d172n mutant channel ( fig . 2 b ) , which has a reduced affinity for intracellular cations ( ficker et al . 1994 ; lopatin et al . 1994 ; lu and mackinnon 1994 ; stanfield et al . 1994 ; wible et al . 1994 ; fakler et al . 1995 ; yang et al . 1995 ) . 4 ) . the extent of channel block varied significantly when hepes from different sources or even different lots of the same source was used ( fig . 5 and fig . 6 ) . therefore , some impurity in hepes , such as amines used or produced in its synthesis , must block the channel , although channel block by zwitterionic hepes itself would not surprise . it is noteworthy that the negative portion of the i - v curve also exhibits a slight curvature when hepes is used ( fig . 1 ) . however , when we replaced hepes with phosphate in the extracellular solution , the i - v curve became practically linear . therefore , the nonlinearity in the negative portion of the i - v curve results also from channel block by hepes and/or some impurity . we showed previously , in experiments using hepes buffer , that the irk1 current in the presence of intracellular putrescine decreased with increasing membrane voltage , but tended to a nonzero level at very positive voltages ( guo and lu 2000a ) ; the nonzero current level was lower for higher putrescine concentrations . in the presence of intracellular spermidine or spermine , the current also tends to a nonzero level as voltage is increased , but with a noticeable intervening hump . we accounted for the blocking behavior of putrescine by a model in which putrescine acts as a permeant blocker , whereas that of spermidine and spermine required a model in which each blocker acts in two conformations with differing affinity and probability of traversing the pore ; the different conformations reflect different protonation states of the polyamines ( guo and lu 2000a , guo and lu 2000b ) . in light of the present findings , our previous data were , in reality , collected in the presence of ( an)other blocker(s ) ( hepes and/or its impurity ) at constant concentration . to account for the then observed voltage - dependent current reduction ( relaxation ) , we added a nonconducting state to our model whose cause was unknown : possibly intrinsic gating or channel block by a contaminating blocker ( guo and lu 2000a ) . as discussed there , this additional state , now found unnecessary , should not fundamentally impact our analyses , but only slightly modify the fitted values of the equilibrium dissociation constants for polyamine binding and associated valence factors . indeed , as shown in the present study , which avoids the unknown contaminant blocker(s ) , the voltage - dependent blocking curves for each of the di- and polyamines retain the same characteristics and are still well fitted by the same model , with parameter values quite comparable with those from our previous study .
the irk1 channel is inhibited by intracellular cations such as mg2 + and polyamines in a voltage - dependent manner , which renders its i - v curve strongly inwardly rectifying . however , even in excised patches exhaustively perfused with a commonly used artificial intracellular solution nominally free of mg2 + and polyamines , the macroscopic i - v curve of the channels displays modest rectification . 
 this observation forms the basis of a hypothesis , alternative to the pore - blocking hypothesis , that inward rectification reflects the enhancement of intrinsic channel gating by intracellular cations . 
 we find , however , that residual rectification is caused primarily by the commonly used ph buffer hepes and/or some accompanying impurity . 
 therefore , inward rectification in the strong rectifier irk1 , as in the weak rectifier romk1 , can be accounted for by voltage - dependent block of its ion conduction pore by intracellular cations .