diphtheria toxin ( dt ) is a single , 535amino acid polypeptide secreted by corynebacterium diphtheriae and is responsible for the disease diphtheria .
1 ) : the nh2-terminal catalytic domain ( residues 1185 ) , the cooh - terminal receptor - binding domain ( residues 386535 ) , and the translocation , or t domain , lying between them ( residues 202378 ) . the catalytic domain is connected to the t domain by a protease - susceptible loop and by an easily reducible disulfide bridge ( for reviews see madshus and stenmark 1992 ; falnes and sandvig 2000 . ) cellular intoxication by dt is thought to proceed by the following mechanism
. pathogenic strains of corynebacterium diphtheriae infect a host and secrete the bacteriophage - encoded toxin as a monomer .
the receptor - binding domain targets it to the surface of cells harboring a heparin - binding epidermal growth factor  like precursor .
while the toxin is on the surface of the cell , a host protease nicks the loop connecting the catalytic domain to the t domain , leaving the two domains still connected by their disulfide bridge .
internalization occurs via receptor - mediated endocytosis , and the toxin now finds itself in an acidifying endosome .
the low ph of the endosome induces a conformational change in the toxin that inserts the t domain into the endosomal membrane and translocates the catalytic domain across the membrane into the reducing environment of the cytosol . here ,
subsequent adp - ribosylation of elongation factor 2 by this domain inhibits protein synthesis , thereby killing the cell .
the endosome function in this process is to provide a low ph environment ; experimentally it can be bypassed by exposing toxin - treated cells to a low ph , in which case the t domain translocates the catalytic domain directly across the plasma membrane ( draper and simon 1980 ; sandvig and olsnes 1980 ) .
the t domain alone , as well as whole toxin and a mutant lacking the r domain ( crm45 ) , form channels in planar bilayers when the ph of the cis side ( the solution to which t domain constructs are added ) is below 6 ( donovan et al .
associated with this channel formation , the entire catalytic domain along with 70 residues of the nh2 terminus of the t domain is translocated across the membrane ( oh et al .
thus the t domain contains all of the translocation machinery ; no cellular components , or even the toxin 's r domain , are required for translocation . in the open channel state ,
the topology of the t domain consists of only three transmembrane segments ( th5 , th8 , and th9 ; senzel et al .
2000 ) , and yet ions as large as glucosamine and nad can traverse the channel ( hoch et al .
how many subunits of dt are involved in making this channel and , thus , in translocating the catalytic domain ?
; steere and eisenberg 2000 ) , but it is not known whether these aggregates are functional ; in fact , purified dimers can not infect host cells ( carroll et al .
1992 ) , every cysteine mutant channel that reacted with thiol - specific methanethiosulfonate ( mts ) derivatives yielded only one transition in single - channel conductance ( huynh et al .
second , the conductance of the d352c channel after reacting with mts - ethylammonium is nearly identical to that of the d352k channel ( mindell et al .
this does not make sense for a multimeric channel , where the former has one positive charge ( ch2-s - s - ch2-ch2-nh3 ) from its one mts - ethylammonium reaction at d352c , and the latter would have n similar positively charged residues there ( ch2-ch2-ch2-ch2-nh3 ) , where n is the stoichiometry of the channel .
third , the conductance of the d352c channel is smaller than that of the wild - type channel , a consequence of replacing a negatively charged residue with a neutral one .
the subsequent reaction with mts - ethylsulfonate restores the channel conductance to that of wild type in a single step - change ( huynh et al .
again , it would be surprising , if this is a multimeric channel , that a mutant with only one negative charge at residue 352 had the same conductance as that of the wild - type channel with n negative charges there . the only observation suggesting that the t domain channel is composed of more than one subunit is that the rate of channel formation increases with about the second power of toxin concentration ( kagan et al .
however , it is possible that a cooperative process facilitates toxin entry into the membrane , thereby giving a nonlinear dependence of the rate of channel formation on concentration , whereas the channel that actually forms is a monomer .
all of this evidence , although not definitive , seems to argue against a channel composed of multiple subunits .
in fact , for most protein systems , one would probably accept the above as ample evidence of monomericity .
( how can three transmembrane segments alone create a pore large enough to conduct k and cl , let alone glucosamine and nad ? ) therefore , we have designed a set of experiments to test the hypothesis that the channel formed by the t domain of diphtheria toxin is monomeric .
details concerning plasmid construction of the t domain with an nh2-terminal hexahistidine tag ( h6 tag ) , as well as our methods of protein expression and purification for all the constructs used , were as previously reported ( zhan et al .
the nh2-terminal h6 construct was made by inserting wild - type t domain ( dt residues 202378 ) between the ndei and xhoi sites of the novagen pet-15b vector , which places an nh2-terminal h6 tag on the protein .
this h6 tag has a thrombin cleavage site so that it can be removed after purification of the expressed protein on a nickel column .
using stratagene 's quickchange site - directed mutagenesis kit , we mutated the arginine in this thrombin cleavage site to a glutamine to minimize unintended proteolysis of this h6 tag by trace proteases .
the cooh - terminal h6 tag construct was engineered by inserting the same t domain into novagen 's pet-22b plasmid . to do this ,
an xhoi site was introduced at the cooh - terminal end of the t domain in the pet-15b vector , using the site - directed mutagenesis kit .
the purified dna from that mutant was digested with the restriction endonucleases ndei and xhoi , gel - purified , and ligated into the pet-22b expression vector that had been cut with the same restriction enzymes and also gel - purified .
it was found that this protein was not an effective channel blocker . again using the site - directed mutagenesis kit
, we lengthened the h6 tag by inserting gggmgss between the cooh terminus of the t domain and the h6 tag ( primers used were cgtataatcgtcccctcgagggaggtggaatgggatcgtcgcaccaccaccaccaccac and its reverse compliment ; nucleotides inserted are shown in bold type ) .
we therefore inserted additional residues ssglvpr cooh - terminal of the h6 tag ( primers used were ccaccaccaccacagcagcggcctcgtccccaggtgagatccggctgc and its reverse compliment ) .
one can see that this is most of the nh2-terminal h6 tag oriented in the reverse direction .
purified plasmid containing the t domain with cooh - terminal h6 tag was digested with the restriction endonucleases ndei and bpu1102 , gel - purified , and cloned into the pet-15b expression vector .
this put the nh2-terminal h6 tag sequence mgssh6ssglvprgshm upstream of residue 202 in our cooh - terminal h6 construct .
thus , the final sequence of the t domain with both nh2- and cooh - terminal h6 tags is : mgssh6ssglvprgshm - i202  p378-legggmgssh6ssglvpr . after expression and purification ( zhan et al .
1995 ) on a novagen his - bind column , the proteins were dialyzed into 20 mm tris - cl , ph 8.0 , 1 mm edta and frozen at 80c . the t domain with the nh2-terminal h6 tag was at a concentration of 3 mg / ml ; that with the cooh - terminal h6 tag was at 1.5 mg / ml ; and that with both the nh2-terminal and cooh - terminal h6 tags was 3 mg / ml .
the appropriate ratio of nh2-terminal to cooh - terminal h6 tag t domain in the mixtures was determined as follows .
the t domains were first individually incubated at 37c for 2 h in 40% dmso ( sigma - aldrich ) to break up most of the preformed aggregates ( carroll et al .
this reliably disrupted > 90% of the dimeric t domain , as assayed by 15% native page ( fig .
they were then tested individually on bilayers to find dilutions that consistently gave from one to three single channels within a few minutes of being stirred into the cis solution .
this was usually achieved with a 1:300 to 1:500 dilution ( t domain construct:20 mm tris - cl , ph 8.0 ) .
the amount of each t domain in the individual dilutions was used to establish a suitable ratio in the mixtures of the original concentrated solutions .
after treatment with dmso , the mixtures were run on a superdex g-75 sizing column ( pharmacia ) in 50 mm nacl , 10 mm tris - cl , ph 8.0 , at a flow rate of 0.75 ml / min .
samples were collected in 0.5-ml fractions and concentrated 10-fold in a savant uvs 400 speed vac plus .
all mixtures used for bilayer experiments were taken from fractions located to the right of the monomer peak .
planar lipid ( asolectin ) bilayer membranes ( 70100 m in diameter ) were made by a modification of the folded film method as described by huynh et al .
the solutions on both sides of the membrane contained 1 m kcl , 2 mm cacl2 , and 1 mm edta ; in addition , the cis solution ( the solution to which t domain constructs were added ) contained 30 mm mes , ph 5.3 , and the opposite trans solution contained 50 mm hepes , ph 7.2 .
voltages are those of the cis solution with respect to the trans solution , whose potential was held at virtual ground .
current generated by single channels was measured by voltage clamping the cis side of the membrane to voltages between 60 and 80 mv .
1981 ) ; after a channel entered , the voltage was pulsed to negative values to determine the channel type : n - type h6 tag gating , c - type h6 tag gating , no gating at all , or both types of gating ( see fig .
if an entering channel left the membrane before application of this negative voltage pulse , it was not included in the dataset .
our method of counting channels was to take the maximum number of channels of each type seen coincidentally in the membrane .
for example , if one c - type channelappeared , stayed a while and then left , and another c - type channel entered the membrane later , this was counted as only one c - type channel .
this method of counting , by insuring our not counting the same channel multiple times , avoided biasing the data . if , however , two c - type channels were seen in the membrane at the same time , this and only this was counted as two c - type channels . to further clarify this point ,
if a membrane yielded a count of 3 n - type and 2 c - type channels , this meant that at some time a in the record there were 3 n - type channels coincidentally in the membrane , and at some time b ( not necessarily the same as a ) there were 2 c - type channels coincidentally in the membrane . due to the rapid flickering nature of the cooh - terminal h6 tag block ( see fig .
4 ) , we were unable to reliably count more than three of these channels in the membrane at one time , and thus usually stopped the experiment if three of these were seen coincidentally .
it was found that pulsing to large negative voltages ( 200 mv ) would close ( or drive out ) all of the channels in the membrane , so that multiple experiments could be done on the same membrane . in the end , though , for each membrane , only the maximum number of channels of each type seen in the membrane coincidentally were counted .
details concerning plasmid construction of the t domain with an nh2-terminal hexahistidine tag ( h6 tag ) , as well as our methods of protein expression and purification for all the constructs used , were as previously reported ( zhan et al .
the nh2-terminal h6 construct was made by inserting wild - type t domain ( dt residues 202378 ) between the ndei and xhoi sites of the novagen pet-15b vector , which places an nh2-terminal h6 tag on the protein .
this h6 tag has a thrombin cleavage site so that it can be removed after purification of the expressed protein on a nickel column .
using stratagene 's quickchange site - directed mutagenesis kit , we mutated the arginine in this thrombin cleavage site to a glutamine to minimize unintended proteolysis of this h6 tag by trace proteases .
the cooh - terminal h6 tag construct was engineered by inserting the same t domain into novagen 's pet-22b plasmid . to do this ,
an xhoi site was introduced at the cooh - terminal end of the t domain in the pet-15b vector , using the site - directed mutagenesis kit .
the purified dna from that mutant was digested with the restriction endonucleases ndei and xhoi , gel - purified , and ligated into the pet-22b expression vector that had been cut with the same restriction enzymes and also gel - purified .
it was found that this protein was not an effective channel blocker . again using the site - directed mutagenesis kit
, we lengthened the h6 tag by inserting gggmgss between the cooh terminus of the t domain and the h6 tag ( primers used were cgtataatcgtcccctcgagggaggtggaatgggatcgtcgcaccaccaccaccaccac and its reverse compliment ; nucleotides inserted are shown in bold type ) .
we therefore inserted additional residues ssglvpr cooh - terminal of the h6 tag ( primers used were ccaccaccaccacagcagcggcctcgtccccaggtgagatccggctgc and its reverse compliment ) .
one can see that this is most of the nh2-terminal h6 tag oriented in the reverse direction .
purified plasmid containing the t domain with cooh - terminal h6 tag was digested with the restriction endonucleases ndei and bpu1102 , gel - purified , and cloned into the pet-15b expression vector .
this put the nh2-terminal h6 tag sequence mgssh6ssglvprgshm upstream of residue 202 in our cooh - terminal h6 construct .
thus , the final sequence of the t domain with both nh2- and cooh - terminal h6 tags is : mgssh6ssglvprgshm - i202  p378-legggmgssh6ssglvpr . after expression and purification ( zhan et al .
1995 ) on a novagen his - bind column , the proteins were dialyzed into 20 mm tris - cl , ph 8.0 , 1 mm edta and frozen at 80c . the t domain with the nh2-terminal h6 tag was at a concentration of 3 mg / ml ; that with the cooh - terminal h6 tag was at 1.5 mg / ml ; and that with both the nh2-terminal and cooh - terminal h6 tags was 3 mg / ml .
the appropriate ratio of nh2-terminal to cooh - terminal h6 tag t domain in the mixtures was determined as follows .
the t domains were first individually incubated at 37c for 2 h in 40% dmso ( sigma - aldrich ) to break up most of the preformed aggregates ( carroll et al .
this reliably disrupted > 90% of the dimeric t domain , as assayed by 15% native page ( fig .
they were then tested individually on bilayers to find dilutions that consistently gave from one to three single channels within a few minutes of being stirred into the cis solution .
this was usually achieved with a 1:300 to 1:500 dilution ( t domain construct:20 mm tris - cl , ph 8.0 ) .
the amount of each t domain in the individual dilutions was used to establish a suitable ratio in the mixtures of the original concentrated solutions .
after treatment with dmso , the mixtures were run on a superdex g-75 sizing column ( pharmacia ) in 50 mm nacl , 10 mm tris - cl , ph 8.0 , at a flow rate of 0.75 ml / min .
samples were collected in 0.5-ml fractions and concentrated 10-fold in a savant uvs 400 speed vac plus .
all mixtures used for bilayer experiments were taken from fractions located to the right of the monomer peak .
planar lipid ( asolectin ) bilayer membranes ( 70100 m in diameter ) were made by a modification of the folded film method as described by huynh et al .
the solutions on both sides of the membrane contained 1 m kcl , 2 mm cacl2 , and 1 mm edta ; in addition , the cis solution ( the solution to which t domain constructs were added ) contained 30 mm mes , ph 5.3 , and the opposite trans solution contained 50 mm hepes , ph 7.2 .
voltages are those of the cis solution with respect to the trans solution , whose potential was held at virtual ground .
current generated by single channels was measured by voltage clamping the cis side of the membrane to voltages between 60 and 80 mv .
1981 ) ; after a channel entered , the voltage was pulsed to negative values to determine the channel type : n - type h6 tag gating , c - type h6 tag gating , no gating at all , or both types of gating ( see fig .
if an entering channel left the membrane before application of this negative voltage pulse , it was not included in the dataset .
our method of counting channels was to take the maximum number of channels of each type seen coincidentally in the membrane .
for example , if one c - type channelappeared , stayed a while and then left , and another c - type channel entered the membrane later , this was counted as only one c - type channel .
this method of counting , by insuring our not counting the same channel multiple times , avoided biasing the data . if , however , two c - type channels were seen in the membrane at the same time , this and only this was counted as two c - type channels . to further clarify this point ,
if a membrane yielded a count of 3 n - type and 2 c - type channels , this meant that at some time a in the record there were 3 n - type channels coincidentally in the membrane , and at some time b ( not necessarily the same as a ) there were 2 c - type channels coincidentally in the membrane . due to the rapid flickering nature of the cooh - terminal h6 tag block ( see fig .
4 ) , we were unable to reliably count more than three of these channels in the membrane at one time , and thus usually stopped the experiment if three of these were seen coincidentally .
it was found that pulsing to large negative voltages ( 200 mv ) would close ( or drive out ) all of the channels in the membrane , so that multiple experiments could be done on the same membrane . in the end , though , for each membrane , only the maximum number of channels of each type seen in the membrane coincidentally were counted .
the experiments to be described were performed in planar lipid bilayer membranes . their rationale came from our earlier work , which showed that a histidine tag ( h6 tag ) attached to the nh2 terminus of the t domain rapidly and completely blocked the channel at cis negative voltages , whereas when the h6 tag was attached to the cooh terminus , the channel was blocked ( a high frequency flickering block , occasionally entering a prolonged blocked state ) at cis positive voltages ( senzel et al .
these findings were used to demonstrate that the nh2 terminus of the protein ( and its h6 tag ) was on the trans side of the membrane and the cooh terminus ( and its h6 tag ) was on the cis side ( senzel et al .
, we verified that , as expected , when the t domain has both nh2- and cooh - terminal h6 tags , the channel is blocked at both negative and positive voltages .
we reasoned that if we mixed nh2-terminal and cooh - terminal h6-tagged t domains , and if the t domain channel is a multimer , then some fraction of channels should be blocked at both positive and negative voltages .
for example , if the channel is a dimer , and equal amounts of nh2-terminal and cooh - terminal h6-tagged t domains were mixed , then on average ( if the t domains have an equal preference to associate with one another ) one quarter of the channels should be blocked only at negative voltages ( both subunits have nh2-terminal h6 tags ) , one quarter blocked only at positive voltages ( both subunits have cooh - terminal h6 tags ) and one half blocked at both negative and positive voltages ( one subunit has an nh2-terminal h6 tag and the other has a cooh - terminal h6 tag ) . if , however , the channel is a monomer , one expects to see only channels that are blocked at either negative or positive voltages , but not at both .
one could argue that before mixing these constructs , there already exist preformed homo - multimers and that these are responsible for the channel - forming activity .
thus , when mixtures are made , one sees channels that only gate at either negative or positive voltages and falsely asserts that these channels are monomeric .
first , we dissociated dimers and higher order aggregates by incubating the mixture in 40% dmso ( carroll et al .
second , after this treatment , the mixture was applied to a size - exclusion column , and the monomeric band was purified away from any remaining dimer or higher aggregates .
the monomer came off in several fractions , and only fractions from the trailing half of the monomer peak , furthest from the multimers , were used in our experiments ( fig .
3 ) . after the above protocol , we performed experiments on two sets of purified mixtures of nh2- and cooh - terminal h6-tagged t domains .
we observed 75 single channels , none of which gated at both negative and positive voltages . from the first mixture ,
( a typical record from one bilayer is shown in fig . 5 . ) of these , 17 showed n - type and 14 showed c - type h6-tagged gating characteristics , whereas 4 did not gate at either positive or negative voltages ( see next paragraph ) . the failure to observe any channels that showed both n - type and c - type gating is , of course , which is consistent with a monomeric channel .
if , for the sake of argument , it is assumed that the channel is dimeric , the probability of our not seeing a single channel that showed both n - type and c - type h6 tag gating in 35 events is 4.5  10 ( see ) . from the second mixture , 9 individual bilayer experiments were performed giving a total of 40 observed single channels . of these ,
26 showed n - type and 9 showed c - type h6-tagged gating characteristics , and 5 did not gate at either positive or negative voltages ( see next paragraph ) .
if the channel is dimeric , the probability of our not having seen one channel that showed both n - type and c - type h6 tag gating in 40 events is 1.3  10 ( see ) . in the combined two sets of mixtures ,
the probability of our not having seen even one channel that showed both n- and c - type gating is 5.9  10 .
if more than two t domains are required to construct the channel , then the odds of our not having seen even one channel that was blocked at both negative and positive voltages become even more minuscule .
we attribute these  nulls  to a subpopulation of the nh2-terminal h6 tag proteins that had its nh2-terminal h6 tag cleaved off by trace amounts of protease in the solution .
( the region of the t domain immediately downstream of the nh2-terminal h6 tag is exposed in the water - soluble crystal structure [ bennett and eisenberg 1994 ] and could be a potential nicking site .
in fact , on sds - page we saw faint bands with molecular weights slightly smaller than the t domain . ) to test this hypothesis , we performed measurements on only the t domain with the nh2-terminal h6 tag .
in eight separate bilayer experiments yielding 36 channels , 31 were blocked at negative voltages and 5 were nulls , a ratio similar to those seen in our mixing experiments .
no nulls were seen in experiments with t domain having the cooh - terminal h6 tag .
we found that our extensive efforts to eliminate preformed dimers and aggregates were probably unnecessary . when we compared the activity of a sample that had a large preformed dimer population to its activity after receiving the 40% dmso treatment ( using the sample illustrated in fig .
2 ) , we saw no difference as assayed by the rate of channel entry into the membrane ( results not shown ) .
thus , it appears that the preformed dimers and higher aggregates are not contributing significantly to channel formation . with this in mind
, we can also include in our dataset the results from a mixture that was dmso treated , but was not run on the sizing column , and from a mixture that was neither dmso treated nor run on the sizing column . in the former case ,
21 single - channel events were recorded on six separate bilayers ; of these events , 11 showed n - type gating , 9 showed c - type gating , and 1 did not gate at all . in the latter case , of 19 single - channel events observed on five bilayers , 9 showed n - type gating and 10 showed c - type gating . combining these data with the results from the previous mixtures , we observed a total of 115 single channels in 28 bilayers and never saw a channel that showed both n - type and c - type gating .
associated with the translocation of diphtheria toxin 's catalytic domain across a planar lipid bilayer , the toxin 's t domain forms a channel . the t domain in this channel , which is permeable to ions as large as glucosamine and nad ( hoch et al .
therefore , it seems obvious that the channel must be a multimer , yet several independent pieces of evidence , reviewed in the introduction , suggest the contrary .
we mixed in a test tube t domain molecules having an nh2-terminal histidine tag ( h6 tag ) with those having a cooh - terminal h6 tag and observed the resulting channels formed by this mixture in planar lipid bilayers .
channels formed by t domain molecules with an nh2-terminal h6 tag show characteristic blocking at negative voltages , whereas those formed by t domain molecules with a cooh - terminal h6 tag block at positive voltages ( fig .
we reasoned that if the channel is a multimer , then some of the channels formed from the mixture should have both of these gating characteristics , just as do channels formed by t domain molecules that have both an nh2-terminal and a cooh - terminal h6 tag ( fig .
we recorded 115 channels in 28 bilayers and never saw one channel manifesting both n - type and c - type h6 tag gating !
therefore , we conclude , contrary to common sense , that the t domain channel contains only one t domain molecule .
it might be contended that the channels were generated from dimers or multimers that preexisted in the nh2-terminal and cooh - terminal h6-tagged t domain solutions , and so naturally we would not see any channels with both n - type and c - type h6 tag gating .
first , after incubating a t domain solution that had a relatively large dimer content for 2 h at 37c in 40% dmso , which converted almost all of the dimers and higher aggregates to monomers ( fig .
2 ) , we found the channel - forming ability of the solution unchanged , indicating that preformed dimers and higher multimers are not a significant source of channels .
second , in two sets of experiments , we went to the extreme of removing residual preformed dimers and multimers from mixtures of nh2-terminal and cooh - terminal h6-tagged t domains that had undergone the dmso treatment , by running the mixtures on a molecular sizing column and using , for the experiments , only fractions from the trailing half of the monomer peak ( fig .
3 ) . even though preexisting dimers and multimers are not the source of channel - forming activity
, this does not preclude that t domain monomers can come together on or within the membrane to form a multimeric channel .
for this to account for our failure to see even one channel out of 115 that manifested both n - type and c - type h6-tagged gating , however , one would have to assume that nh2-terminal h6-tagged t domains and cooh - terminal h6-tagged t domains associate exclusively with themselves ; i.e. , heteromultimer formation is precluded .
although this is logically possible , we can think of no justification for this assumption .
in fact , if one argues that somehow a t domain with a cooh - terminal h6 tag can not associate with one that has an nh2-terminal h6 tag , then why would t - domains containing both an nh2-terminal and cooh - terminal h6 tag associate in the postulated multimeric channel ? one would have to invoke a special attraction of nh2-terminal h6 tags and/or cooh - terminal h6 tags with themselves ; we can see no physical justification for this .
one might also argue that even though nh2-terminal h6-tagged t domains can form heteromultimers with cooh - terminal h6-tagged t domains , for some reason these do not form functional channels .
again , it is difficult to reconcile this position with the ability of t domains that have both an nh2-terminal and a cooh - terminal h6 tag to form channels .
thus , it seems to us that to believe that the t domain channel is a multimer , one must argue that heteromeric channels are formed by nh2-terminal and cooh - terminal h6-tagged t domains , but some of these exhibit only n - type gating , whereas others exhibit only c - type gating .
asymmetric structures can be envisioned that fulfill this condition , but this leads us into a fantasyland that is best avoided . throughout the analyses and discussions in this paper
, we have tacitly assumed that only a single h6 tag is required for channel blocking .
if this is not the case , one can imagine new scenarios in which the channel is multimeric , and yet no channels formed from our mixture exhibit both n - type and c - type gating .
for example , if the channels were a trimer and required at least two nh2- or cooh - terminal h6 tags to get nh2- or cooh - terminal gating , respectively , we would observe only n - type and c - type gating channels , never channels showing both types of gating .
in fact , in general , if the channel is formed by an odd number ( n ) of subunits and requires ( n + 1)/2 nh2- or cooh - terminal h6 tags to get n- or c - type gating , respectively , the above statement would hold .
( if n is even , the equivalent assumption that ( n/2 ) + 1 h6 tags are required for blocking predicts a significant number of channels showing neither n- nor c - type gating . )
we think that the requirement of multiple h6 tags to affect channel blocking is a priori unlikely ; we know of no precedent for this in the channel literature .
moreover , if more than one h6 tag were required to completely block the channel , we would anticipate substates ( partial block ) when less than one h6 tag entered the channel .
this paper presents what we feel are compelling arguments for the monomeric nature of the t domain channel formed in planar lipid bilayers .
the channels formed by whole toxin in planar bilayers are indistinguishable from these ( silverman et al . 1994 ) , and therefore it is not much of a stretch to assert that they too are monomeric .
the mechanism by which the catalytic domain of the toxin is translocated across planar bilayers in association with channel formation by its t domain ( oh et al .
1999 ) remains to be resolved , but whatever it is , we feel that it is now established that only one t domain molecule is involved in the process .
the structure of the t domain channel , having only three transmembrane segments ( senzel et al . 2000 ) , remains a mystery and probably involves the lipids in its architecture .
there is a feeling in the literature that in real life ( i.e. , the cell ) the translocation of the catalytic domain across the endosomal membrane into the cytosol is accomplished through an oligomer of dt ( steere and eisenberg 2000 ) .
the results in this paper do not , of course , directly address this issue .
all we can say in light of the present results and previous work ( oh et al .
1999 ) is that there is no necessity to invoke dt oligomers in cell intoxication , with the following caveat .
the rate of channel formation increases with about the second power of toxin concentration ( kagan et al .
thus , a cooperative interaction of two or more dt molecules may promote toxin entry into the membrane , even though the ultimate intoxicating unit is a monomer .
interestingly , proteins in a molten globule - like state promote the transmembrane insertion of the t domain ( ren et al .
dt itself partly unfolds into a molten globule - like state ( london 1992 ) , it may catalyze within the acidic endosome its own insertion into the endosomal membrane . in this sense
there are also data using liposomes that suggest that oligomerization is involved in dt pore formation .
for example , sharpe and london 1999 report that the size of liposome - entrapped molecules that are released is an increasing function of dt concentration ( at ph 4.5 ) .
they interpret this to mean that the pores formed by dt increase in size as the concentration of dt within the membrane increases .
furthermore , they observed that dt oligomerizes within the liposomal membrane under their experimental conditions .
thus , they conclude that the pore formed by dt is an oligomer and that the pore size is a function of the number of subunits in the oligomer .
we do not know how to relate our results to these , given the very different experimental conditions and techniques involved . in a previous paper ( senzel et al .
2000 ) , we discussed the difficulties involved in trying to reconcile planar lipid bilayer experiments with those on liposomes .
 in the presence of a low ph environment , the channel - forming t domain of diphtheria toxin undergoes a conformational change that allows for both its own insertion into planar lipid bilayers and the translocation of the toxin 's catalytic domain across them . 
 given that the t domain contributes only three transmembrane segments , and the channel is permeable to ions as large as glucosamine+ and nad , it would appear that the channel must be a multimer . 
 yet , there is substantial circumstantial evidence that the channel may be formed from a single subunit . to test the hypothesis that the channel formed by the t domain of diphtheria toxin is monomeric , we made mixtures of two t domain constructs whose 
 voltage - gating characteristics differ , and then observed the gating behavior of the mixture 's single channels in planar lipid bilayers . one of these constructs contained an nh2-terminal hexahistidine ( h6 ) tag that blocks the channel at negative voltages ; the other contained a cooh - terminal h6 tag that blocks the channel at positive voltages . 
 if the channel is constructed from multiple t domain subunits , one expects to see a population of single channels from this mixture that are blocked at both positive and negative voltages . 
 the observed single channels were blocked at either negative or positive voltages , but never both . 
 therefore , we conclude that the t domain channel is monomeric . 
INTRODUCTION
MATERIALS AND METHODS
Constructs
Ratios for Mixtures
Purification of Monomeric T Domain
Bilayer Experiments
RESULTS
DISCUSSION