ion channels gated by cyclic nucleotides are widely distributed throughout eukaryotes ( kaupp and seifert , 2002 ; robinson and siegelbaum , 2003 ) . as the molecular hardware linking changes of intracellular cyclic nucleotide levels occurring in sensory transduction to cellular electrical responses , these proteins are especially important in the coherent functioning of electrically excitable tissues . they have been most widely studied in sensory neurons , where they carry out essential tasks in the detection of photons or odorants , in cardiac myocytes , where they underlie pacemaking activity , and in cerebellar neurons , where they contribute to fine tuning in motor - coordination circuits .
all such channels belong to the s4 superfamily ( yellen , 2002 ; yu and catterall , 2003 ) , which also includes voltage - dependent ca , na , and k channels ; these proteins are tetramers of identical or similar subunits , or of honorary subunits inhabiting the same polypeptide in tandem , with each subunit having six transmembrane sequences and a pore - forming sequence positioned between the fifth and sixth of these .
the cyclic nucleotide - modulated channels additionally carry a cyclic nucleotide - binding domain ( cnbd ) in the cytoplasmic cooh - terminal region .
these channels fall operationally into two broad subclasses , denoted cng ( cyclic nucleotide gated ) and hcn ( hyperpolarization and cyclic nucleotide activated ) .
channels of the first class depend absolutely on cyclic nucleotides for opening , gate in a voltage - independent manner , and permit conduction only by monovalent cations , with little ionic selectivity among the group ia cations .
in contrast , hcn channels are merely modulated by cyclic nucleotides in their primary mode of voltage - dependent gating , and , while also conductive to monovalent cations , favor k over na by a factor of approximately four . at the molecular level ,
both cng and hcn channels show the arginine - decorated fourth transmembrane sequence characteristic of the s4 family , and both carry similar cnbd sequences . in keeping with their k selectivity ,
pore - forming selectivity sequence unique and common to k channels ( heginbotham et al . , 1992 ) , while cng channels lack this sequence .
the recent high - resolution structures of several k channels ( doyle et al . , 1998 ; jiang et al . , 2002 , 2003 )
provide an outline of molecular architecture in this protein family but leave many questions specific to the cyclic nucleotide - binding subfamily unaddressed .
are these , like k channels , built as fourfold symmetric structures , as in the structure of an isolated cnbd of an hcn channel ( zagotta et al . , 2003 ) , or as dimers - of - dimers , as suggested by functional experiments on cng and hcn channels ( liu et al .
, 1998 ; ulens and siegelbaum , 2003 ) ? what accounts for the hyperpolarization - dependent opening of hcn channels , a voltage dependence with polarity opposite to that of conventional kv - type k channels ( mannikko et al . , 2002
the 80-residue sequence connecting the cnbd to the sixth transmembrane helix , mediate between cyclic nucleotide binding and channel opening ( gordon and zagotta , 1995 ) ?
so much less k selective than other k channels , which typically show 1001,000-fold selectivity for k over na ?
the only productive avenue toward high - resolution ion channel structure is currently provided by bacterial homologues , which in some cases allow overexpression in escherichia coli of properly assembled proteins amenable to purification and crystallization . for this reason , we searched prokaryotic databases for s4-type ion channels containing cyclic nucleotide - binding domains .
we now describe the overexpression , purification , and functional reconstitution of an s4-superfamily homologue from mesorhizobium loti , with a k selectivity sequence and a cnbd .
the channel 's chromatographic behavior in detergent micelles and its ionic flux properties in liposomes respond to micromolar levels of cyclic nucleotides , and its selectivity for k is higher than that of either cng or hcn subtypes .
a putative bacterial channel protein homologous to eukaryotic cyclic nucleotide - gated channels was identified by a homology search of the tigr bacterial database .
the coding sequence was pcr cloned from m. loti genomic dna and inserted into an e. coli expression vector ( pask - iba2 ; sigma - genosys ) with a cooh - terminal hexahistidine tag .
e. coli jm83 were transformed with this construct , and after plating on lb - amp , cells were harvested and grown in terrific broth .
expression of protein was induced for 90 min with 0.2 mg / l anhydrotetracycline at a cell density ( a550 ) of 1.0 .
cells were pelleted , resuspended in 100 mm kcl , 50 mm tris - hcl , ph 7.5 ( 50 ml for 13 liter culture ) , and lysed by sonication on ice in the presence of protease inhibitors ( complete edta - free , roche , and pmsf 0.17 mg / ml ) .
membrane protein was extracted 2 h by adding 50 mm n - decylmaltoside ( dm ; anatrace ) , and the extract , clarified by centrifugation , was applied to a ni affinity column ( qiagen ) .
after washing off nonspecifically bound proteins with running buffer ( 100 mm kcl , 20 mm tris - hcl , 5 mm dm , ph 7.6 ) supplemented with 40 mm imidazole , the protein was eluted by raising imidazole to 400 mm , concentrated to 815 mg / ml in amicon ultracentrifugal concentrators , and chromatographed on a superdex 200 size exclusion column ( amersham biosciences ) in running buffer . when needed , cyclic adenosine monophosphate or cyclic guanosine monophosphate ( camp , cgmp , fluka ) , 50200 m unless otherwise specified , was added to the bacterial suspension before sonication and maintained during all purification and reconstitution steps . in experiments
in which cyclic nucleotide concentration was varied , protein was always prepared in high cyclic nucleotide , and the desired final concentration was introduced in the reconstitution step .
eluted protein was analyzed by sds - page gel electrophoresis and the proteins visualized by coomassie staining .
the purified protein was immediately reconstituted into liposomes at a concentration of 0.150 g protein / mg lipid in 20 mg / ml e. coli polar lipids ( avanti polar lipids , inc . ) in the presence or absence of camp or cgmp .
detergent was removed either by overnight dialysis against 1,000 volumes of 400 mm kcl , 10 mm hepes , 5 mm n - methyl glucamine ( nmg ) , ph 7.6 , or by chromatography on sephadex g-50 fine , equilibrated in this same solution .
the rb flux assay has been described in detail ( heginbotham et al . , 1998 ) .
in brief , liposomes were thawed , sonicated , and passed over g-50 spin - columns ( 100 l liposome , 1.5 ml column ) equilibrated in uptake buffer ( 400 mm sorbitol , 50 m kcl , 10 mm hepes , 5 mm nmg , and the desired concentration of camp or cgmp ) to establish a k gradient across the liposome membrane .
uptake of rb was initiated by mixing the liposomes with two to six volumes of uptake buffer containing trace amounts of rb ( 0.1 ci / ml ) , as depicted in the inset of fig .
, extraliposomal rb was removed by passing a 100 l aliquot over a 1.5-ml cation - exchange dowex column ( 50wx4 - 100 ; sigma - aldrich ) converted to the nmg form , and radioactivity was measured in a liquid scintillation counter .
valinomycin ( 1 g / ml ) was added at the end of each experiment to assay maximum rb uptake into all liposomes present . to follow proton uptake into electrically polarized liposomes , protein - free or
reconstituted vesicles were thawed and supplemented with 50 mm kpi , ph 7.3 , and were then sonicated 510 s to trap the buffer in the internal space . for each assay , 100 l of vesicles
was applied to a 1.5-ml spin column equilibrated with proton uptake solution ( 350 mm nacl , 10 mm kcl , 2 mm kpi , ph 7.3 ) to exchange the external solution and establish a k gradient .
the liposomes ( 100 l ) were immediately diluted into a stirred vial containing 1.9 ml of proton uptake solution , in which a ph electrode was positioned .
the ph of the solution was recorded on chart paper on a high - sensitivity scale ( 0.06 ph units full scale ) .
fccp and valinomycin were used to permeabilize the liposomes to h and k , respectively .
a putative bacterial channel protein homologous to eukaryotic cyclic nucleotide - gated channels was identified by a homology search of the tigr bacterial database .
the coding sequence was pcr cloned from m. loti genomic dna and inserted into an e. coli expression vector ( pask - iba2 ; sigma - genosys ) with a cooh - terminal hexahistidine tag .
e. coli jm83 were transformed with this construct , and after plating on lb - amp , cells were harvested and grown in terrific broth .
expression of protein was induced for 90 min with 0.2 mg / l anhydrotetracycline at a cell density ( a550 ) of 1.0 .
cells were pelleted , resuspended in 100 mm kcl , 50 mm tris - hcl , ph 7.5 ( 50 ml for 13 liter culture ) , and lysed by sonication on ice in the presence of protease inhibitors ( complete edta - free , roche , and pmsf 0.17 mg / ml ) .
membrane protein was extracted 2 h by adding 50 mm n - decylmaltoside ( dm ; anatrace ) , and the extract , clarified by centrifugation , was applied to a ni affinity column ( qiagen ) .
after washing off nonspecifically bound proteins with running buffer ( 100 mm kcl , 20 mm tris - hcl , 5 mm dm , ph 7.6 ) supplemented with 40 mm imidazole , the protein was eluted by raising imidazole to 400 mm , concentrated to 815 mg / ml in amicon ultracentrifugal concentrators , and chromatographed on a superdex 200 size exclusion column ( amersham biosciences ) in running buffer . when needed , cyclic adenosine monophosphate or cyclic guanosine monophosphate ( camp , cgmp , fluka ) , 50200 m unless otherwise specified , was added to the bacterial suspension before sonication and maintained during all purification and reconstitution steps . in experiments
in which cyclic nucleotide concentration was varied , protein was always prepared in high cyclic nucleotide , and the desired final concentration was introduced in the reconstitution step .
eluted protein was analyzed by sds - page gel electrophoresis and the proteins visualized by coomassie staining .
the purified protein was immediately reconstituted into liposomes at a concentration of 0.150 g protein / mg lipid in 20 mg / ml e. coli polar lipids ( avanti polar lipids , inc . ) in the presence or absence of camp or cgmp .
detergent was removed either by overnight dialysis against 1,000 volumes of 400 mm kcl , 10 mm hepes , 5 mm n - methyl glucamine ( nmg ) , ph 7.6 , or by chromatography on sephadex g-50 fine , equilibrated in this same solution .
the rb flux assay has been described in detail ( heginbotham et al . , 1998 ) .
in brief , liposomes were thawed , sonicated , and passed over g-50 spin - columns ( 100 l liposome , 1.5 ml column ) equilibrated in uptake buffer ( 400 mm sorbitol , 50 m kcl , 10 mm hepes , 5 mm nmg , and the desired concentration of camp or cgmp ) to establish a k gradient across the liposome membrane .
uptake of rb was initiated by mixing the liposomes with two to six volumes of uptake buffer containing trace amounts of rb ( 0.1 ci / ml ) , as depicted in the inset of fig .
, extraliposomal rb was removed by passing a 100 l aliquot over a 1.5-ml cation - exchange dowex column ( 50wx4 - 100 ; sigma - aldrich ) converted to the nmg form , and radioactivity was measured in a liquid scintillation counter .
valinomycin ( 1 g / ml ) was added at the end of each experiment to assay maximum rb uptake into all liposomes present . to follow proton uptake into electrically polarized liposomes , protein - free or reconstituted vesicles were thawed and supplemented with 50 mm kpi , ph 7.3 , and were then sonicated 510 s to trap the buffer in the internal space . for each assay , 100 l of vesicles
was applied to a 1.5-ml spin column equilibrated with proton uptake solution ( 350 mm nacl , 10 mm kcl , 2 mm kpi , ph 7.3 ) to exchange the external solution and establish a k gradient .
the liposomes ( 100 l ) were immediately diluted into a stirred vial containing 1.9 ml of proton uptake solution , in which a ph electrode was positioned .
the ph of the solution was recorded on chart paper on a high - sensitivity scale ( 0.06 ph units full scale ) .
fccp and valinomycin were used to permeabilize the liposomes to h and k , respectively .
in a blast search for s4-type channels containing cnbds , we noticed , among four other hits , a putative k channel homologue in the plant symbiont m. loti , an organism commonly used for genetic studies of rhizobial symbiosis and nitrogen fixation .
np104392 ) encodes a 355-residue polypeptide of 38 kd with six putative transmembrane sequences ( s1s6 ) , where s4 bears three basic residues ( two arginines , one lysine ) , a k selectivity sequence ( ttgygd ) between s5 and s6 , and a cooh - terminal cnbd ( fig .
the pore sequence for this protein , which we call mlok1 , is homologous to the corresponding region of other k channels such as kcsa ( 60% identity ) and shaker ( 50% ) , and its cnbd displays convincing similarity ( 30% ) to those of eukaryotic cng and hcn subtypes . unlike its eukaryotic counterparts
, mlok1 does not contain the four - helix c - linker domain , which connects the end of s6 to the cnbd and is thought to transmit the cyclic nucleotide binding signal to the channel 's gate ( gordon and zagotta , 1995 ;
; johnson and zagotta , 2001 ; wang et al . , 2001 ) . in mlok1 ,
mlok1 in the s4 ion channel superfamily . predicted transmembrane segments shown as cylinders , cnbd in yellow , pore region in cyan .
the completely conserved arg is shown in green and the adjacent conserved hydroxy amino acid in red .
asterisk marks the position of acidic residues in cng channels ( varnum et al . , 1995 )
channel abbreviations , with ncbi accession nos . , are as follows : hcn1 , mouse hcn ( o88704 ) , hcn2 , human hcn ( q9ul51 ) , cnga1 , bovine cng ( q00194 ) , cnga2 , bovine cng ( q03041 ) , spih , sea urchin hcn ( np999729 ) . the channel protein , engineered with a cooh - terminal his6 tag and placed behind a tetracycline promoter , is efficiently expressed in e. coli ( fig .
2 ) , with an induction - dependent band of 35 kd visible on sds - page even in the whole - cell lysate . after extraction of the cells in detergent ,
high purity is achieved in a single pass over a ni - chelate column with a yield of 3 mg / l culture .
the chromatographic behavior of the affinity - purified protein on gel filtration is cyclic nucleotide dependent ( fig .
much of the protein elutes as an aggregate in the void volume , with some tetrameric material ( 150 kd ) following , along with several other peaks .
however , with 50 m camp present , most of the material runs as a single tetrameric peak , as though the protein population is monodisperse under this condition .
although gel filtration chromatography does not rigorously determine particle size , we are reasonably confident of our measurement here , since the column was calibrated with six membrane proteins of known size , run under identical detergent conditions .
we note that these experiments were performed on two separate preparations processed in parallel in which the protein was extracted , purified , and chromatographed in the presence or absence of camp .
this result implies that occupancy of the cnbd by ligand promotes native folding and perhaps stabilizes the channel during the purification procedure ; we have found ( unpublished data ) that the tetrameric peak purified without camp and maintained in its absence remains tetrameric for at least 7 d at 4c .
protein samples were run on a 10% polyacrylamide sds gel stained with coomassie blue as follows : lane 1 , protein ladder with molecular weights indicated ; lanes 2 and 3 , pre- and post - induction whole e. coli lysates ; lane 4 , clarified membrane extract of induced cells ; lane 5 , ni column flow - through of membrane extract ; lane 6 , nonspecifically bound protein wash of ni column ; lane 7 , ni column 
eluted mlok1 ( 7 g ) ; lane 8 , gel filtration column - purified mlok1 ( 7 g ) .
preparations of ni column  purified ( solid curve ) or gel filtration  purified ( dashed curve in b ) mlok1 were applied to a superdex gel filtration column monitored at 280 nm in the absence ( a ) or presence ( b ) of 50 m camp
. dashed lines mark the tetrameric mlok1 peak and arrows indicate the void volume and molecular weights of two calibrated membrane proteins run under identical conditions : mthk k channel tetramer ( 250 kd ) and clc - ecl cl transporter dimer ( 92 kd ) . according to a calibration using five membrane proteins of known sizes run under identical conditions , the elution volume of mlok1 corresponds to 150 kd .
scale bar for absorbance at 280 nm represents 20 mau for a and 100 mau for b. does mlok1 function as a cyclic nucleotide - gated ion channel ?
we reconstituted the protein into liposomes and examined its ability to catalyze transmembrane movement of rb , a convenient radioactive k analogue .
influx was followed under conditions wherein liposomes loaded with high kcl are suspended in low kcl solution containing radioactive tracer , a circumstance that leads to concentrative accumulation of radioactivity . under these conditions ,
significant tracer uptake can occur only in liposomes that are permeable to k but not to cl ( heginbotham et al . , 1998 ) .
4 a illustrates a typical influx time course , in which rb becomes trapped within the liposomes with approximately exponential time dependence characterized by a half - time of 30 min .
however , even on the long , 2-h time scale of this experiment , the influx does not reach completion , and so the equilibrium level of trapped rb remains uncertain .
we measure the maximum capacity of rb uptake by adding valinomycin , an ionophore which instantly permeabilizes all liposomes to k and rb .
thus , in this experiment , mlok1 catalyzes rb uptake into 35% of the liposome space in 2 h , but because of the slowness of the time course , we can not claim that this represents the fraction of liposomes carrying functional mlok1 ; it does place a lower limit on this fraction , however . henceforth we normalize influx time courses to the maximum uptake measured by valinomycin to compare results obtained from different reconstitution conditions , as in fig . 4 b. here , the effect of camp on influx is shown . over the 1-h time course
reconstitution of mlok1 increases the initial rate of uptake , and a maximal concentration of camp ( 200 m ) approximately doubles this .
these results show that mlok1-mediated ion transport is not absolutely dependent on camp , but that the ligand enhances the protein 's intrinsic activity .
( a ) representative time course of accumulation of rb into liposomes reconstituted with mlok1 ( 10 g / mg lipid , 200 m camp ) .
each point represents radioactivity of a single 100-l sample removed from the reaction mix at the time point indicated .
after the 2-h sample was collected , valinomycin was added ( arrow ) and a sample was taken 2 min later .
( b ) valinomycin - normalized rb uptake in liposomes reconstituted with mlok1 ( 10 g / mg lipid ) in the presence of camp ( 200 m , black circles ) or in its absence ( gray circles ) .
inset , cartoon of rb uptake assay , with liposomes loaded with high k ( 400 mm ) immersed in low k solution ( 50 m ) containing tracer rb ; this leads to a high negative intraliposomal potential , which the rb tracer follows ( heginbotham et al .
the rate of rb uptake increases with the density of channels in the reconstituted membranes ( fig .
( < 2 g protein / mg lipid ) , uptake increases linearly , as liposomes in excess are titrated with channel protein .
eventually , uptake levels off at high protein density > 5 g / mg ( fig .
, this effect can not be quantified , as has been done by poisson statistics in other liposome systems ( goldberg and miller , 1991 ; heginbotham et al . , 1998 ;
maduke et al . , 1999 ) , since the final steady level of rb uptake can not be accurately determined here ; nonetheless , the value of the half - saturation density ( 1 g / mg ) indicates that a large fraction of the protein is functionally active , since it is at this density that the number of liposomes is roughly equal to the number of protein molecules present in the reconstitution mix ( heginbotham et al . , 1998 ) .
( a ) valinomycin - normalized rb uptake time course for 0 ( white ) , 0.1 ( light gray ) , 1 ( dark gray ) , and 5 ( black ) g mlok1/mg lipid in the presence of 50 m camp .
each time point represents rb accumulation after 45 min in liposomes containing mlok1 at the indicated concentrations and in the presence of 50 m camp .
the appearance of a k selectivity sequence ( ttgygd ) between s5 and s6 suggests strongly that mlok1 should show a familiar k selectivity profile , permitting permeation by k and rb and effectively barring na and li . to test this expectation , we adapted the concentrative uptake assay to assess ion selectivity , comparing rb uptake into liposomes loaded with different test cations in the presence of camp ( fig .
uptake is similar in k and rb , while neither na nor li supports significant uptake .
these experiments do not allow precise estimates of ionic permeability ratios , but it is clear that rb influx rate is close to the k rate , and na and li permeate , if at all , at much lower rates .
each bar represents rb uptake into liposomes reconstituted with 5 g protein / mg lipid measured after 45 min of uptake and normalized to the total amount of radioactivity in the sample .
reconstituted liposomes were formed in the presence of the 400 mm of the cl salts of the indicated cations , along with of 50 m camp .
cyclic nucleotides bias the open  closed equilibrium of eukaryotic cng and hcn channels toward open conformations .
this behavior is mirrored in mlok1 , although the maximum stimulatory effect of camp is only twofold ( fig .
4 ) . to examine the ligand dependence of this effect , we reconstituted the channel into liposomes and used the uptake assay to determine dose  response curves for camp and cgmp .
rb flux time courses in the presence of varying cyclic nucleotide concentrations are shown in fig . 7 ( a and b ) .
both camp and cgmp stimulate the uptake , and both give similar approximately twofold maximal stimulation .
response curves were derived from these time courses by normalizing the uptake values measured at 15 or 45 min to the corresponding isochronal uptake at maximal ligand concentration .
these follow a simple langmuir - type concentration dependence ( hill coefficient of unity ) , with camp being 10-fold more effective than cgmp ( k1/2 = 60 and 600 nm , respectively ) .
flux time courses into liposomes reconstituted with 5 g mlok1/mg lipid in the presence of camp ( a ) and cgmp ( b ) , at 0 ( white ) , 0.1 ( light gray ) , 1 ( dark gray ) , and 10 ( black ) m concentration .
uptake values at 15 min ( squares ) and 45 min ( circles ) are normalized to both maximum ( saturating concentration of cnmp ) and minimum ( no cnmp ) values .
smooth curves are drawn according to single - site binding functions , with k1/2 = 60 nm for camp ( black ) and 600 nm for cgmp ( gray ) .
the uptake experiments above demonstrate that mlok1 mediates membrane transport for rb and k but do not distinguish between electrically conductive ion movement , as would be expected for a channel homologue , and an electroneutral mechanism .
the preferred way of demonstrating a conductive mechanism is to reconstitute the protein into planar lipid bilayers and record electrical currents ; however , we have been so far unable to do this
the method employs proton uptake to report on the electrical potential set up across the liposome membrane by a k gradient .
8 a ; liposomes loaded with high k are suspended in low k solution , and the ph of the suspension is recorded .
a k - selective conductance present in the membrane produces a negative - inside electrical potential that attracts protons into the intraliposomal space .
the proton - impermeable liposome membrane does not allow protons to  follow  this potential , but treatment with a proton ionophore removes this kinetic constraint and allows protons to enter , as may be observed by a ph increase in the external solution .
addition of the weak - acid uncoupler fccp , which permeabilizes the membranes specifically to protons , has no effect on the ph of the suspension by itself .
addition of the k - specific ionophore valinomycin polarizes the membrane and produces an immediate rise in ph , as protons are taken up against a ph gradient but down their electrical gradient .
when this experiment is repeated on liposomes reconstituted with mlok1 , however , fccp causes prompt h influx without addition of valinomycin ( fig .
thus , mlok1 substitutes functionally for valinomycin in setting up a negative internal membrane potential , and is therefore conductive , not electroneutral .
the experiment further shows that mlok1 is selective for k over both na and h. subsequent addition of valinomycin causes only a small additional ph increase , as though < 25% of the liposomes in the population are free of functional mlok1 protein .
these ph responses are completely abolished in the absence of a k gradient ( unpublished data ) .
liposomes are loaded with high k and suspended in low k , with a k - specific conductance present in the membrane . under these conditions ,
if a proton ionophore is present to circumvent the impermeability of the lipid bilayer , protons will be drawn into the liposomes to  follow  the electrical potential set up by the k gradient .
vesicles ( 20 mg / ml ) loaded with high k ( 450 mm ) were diluted 20-fold into lightly buffered low k ( 10 mm ) medium , and ph was recorded . at asterisk ,
fccp ( 0.050.5 g / ml ) , and at arrow , valinomycin ( 0.5 g / ml ) were added .
horizontal scale bar , 30 s ; vertical scale bar , 13 nmol h , 0.003 ph units .
all structures of ion channels determined so far have been prokaryotic in origin , mainly because of the empirical and tragic fact that overexpression of eukaryotic integral membrane proteins in e. coli has been so reliably unsuccessful . for this reason , if we wish to know what a eukaryotic channel protein looks like , we are currently forced into the unsatisfactory but practical necessity of seeking bacterial or archeal homologues of the channel of interest . in such a search we found five putative s4-type ion channels of the cyclic nucleotide - binding subclass . in striking contrast to the abundance of other channel families among bacterial genomes , such as kv and kir k channels and clc cl channels ,
these cnbd - containing homologues are rare , so far found only in five bacterial genomes , three of which are rhizobial symbionts . in no case is the biological function of any of these prokaryotic channels known .
we chose to focus on mlok1 , which expresses well in e. coli and may be readily purified in quantity . to maintain the protein in a homogenous tetrameric state , it is necessary to keep cyclic nucleotide present throughout all steps of the purification procedure .
the purified protein reconstituted into liposomes catalyzes rb uptake in a cyclic nucleotide - stimulated fashion .
these two phenomena , along with the low concentration range in which the ligands act , imply that the cooh - terminal cnbd is properly folded and functionally competent
. furthermore , the tetrameric nature and k - like ionic selectivity of conductive fluxes are as expected for a k channel . for these reasons
, we consider that the purified mlok1 protein , in fulfilling the expectations of its sequence , is correctly folded and functional .
to which subtype of cyclic nucleotide binding channel does mlok1 belong : cng or hcn ?
it is certainly not of the cng subtype , since it is k selective and shows a k signature sequence .
we are tempted to assign it to the hcn subfamily , on the basis of this k selectivity , as well as the substantial basal activity in the absence of cyclic nucleotides .
but in fact we can not definitively assert that the channel is of hcn subtype .
since we have not been able to record electrical currents from mlok1 , we can not gauge its voltage dependence , a fundamental defining characteristic of hcn channels .
moreover , the high selectivity of mlok1-mediated flux for k over na would not be expected for a conventional hcn channel , as these are only weakly selective for k. we are therefore unwilling to assign this channel firmly to either of the categories defined in eukaryotic channels . both camp and cgmp bind to this channel , as indicated by chromatographic behavior and ion fluxes . in the flux assay
, camp shows 10-fold higher potency than cgmp , with the two ligands giving similar maximal effects .
several ligand - selectivity determinants have been identified in cnbds from eukaryotic channels , from both electrophysiological analysis of cng channels ( altenhofen et al . , 1991 ; varnum et al . ,
1995 ) and direct structure determination of an isolated cnbd from an hcn channel ( zagotta et al . , 2003 ) .
all eukaryotic cnbds , regardless of ligand preference , have a conserved arginine residue that coordinates the cyclic nucleotide phosphate ( zagotta et al . , 2003 ) ,
a threonine is always found adjacent to this arginine in eukaryotic channels that bind both camp and cgmp ; in the structure of hcn2 cnbd , the hydroxyl group of this threonine hydrogen bonds to the n2 atom of the guanine ring , while camp - specific channel isoforms , such as the sea urchin hcn channel ( gauss et al . , 1998 ) , lack this hydroxyl group . in mlok1 , this position uses a serine ( s308 ) , the hydroxyl of which may play an equivalent h - bonding role , permitting cgmp to bind .
finally , cng channels that are highly selective for cgmp have an acidic residue closer to the cooh terminus ( fig . 1 , asterisk ) , and
neutralizing this residue removes the ligand selectivity ( varnum et al . , 1995 ) ; mlok1 has an alanine residue here , in harmony with responsiveness to both camp and cgmp . on several grounds , therefore ,
the ligand dependence of mlok1 is consistent with expectations based on molecular determinants in its cnbd .
one feature of mlok1 , however , is unexpected from precedent : the absence of a c - linker sequence , a motif found in all eukaryotic cng and hcn channels .
the c - linker domain has been proposed to transmit the ligand - binding signal to the channel 's gate in s6 .
this proposal arose from mutagenesis studies in cng and hcn channels ( gordon and zagotta , 1995 ; zong et al .
, 1998 ; paoletti et al . , 1999 ; johnson and zagotta , 2001 ; wang et al . , 2001 ) and
was strengthened by high - resolution structures of an isolated cnbd from an hcn isoform ( zagotta et al . , 2003 ) , where most of the intersubunit contacts in the tetramer are formed by the c - linker , which is composed of four -helices .
this structure suggested that cyclic nucleotide binding drives a rearrangement of the c - linker domains , which opens the channel 's ion permeation pathway .
our results refute this proposal , at least as a necessary , general mechanism of channel modulation by cyclic nucleotides , and they suggest that a minimal linking sequence is able to substitute for this function in the prokaryotic homologue . finally , we should ask whether the results here prove that mlok1 is a k channel . in fact
to be sure , the gross functional characteristics of the reconstituted protein are consistent with a channel mechanism : electrically conductive transport with k - like ionic selectivity . but without a direct measurement of unitary transport rate , it is impossible to rigorously distinguish channel from transporter mechanisms , and the liposome system on which we rely does not permit an estimate of this .
we note that mlok1-mediated rb uptake is 10100-fold slower than the rate expected for an always - open channel with a  typical
 unitary conductance in the range 10100 ps , or than that observed under similar conditions for kcsa ( heginbotham et al . , 1998 ) .
this low uptake rate might reflect a small unitary conductance or a low open probability , even when maximally activated by cyclic nucleotide , or a combination of both . in the absence of direct single - channel recording , this point must remain unresolved , but it is worth noting that eukaryotic hcn channels have extremely low single - channel currents ( difrancesco and mangoni , 1994 ) . in light of its tetrameric architecture , k - selective cyclic nucleotide - modulated ion fluxes , and unambiguous membership in the s4 superfamily of ion channels ,
 a search of prokaryotic genomes uncovered a gene from mesorhizobium loti homologous to eukaryotic k+ channels of the s4 superfamily that also carry a cyclic nucleotide binding domain at the cooh terminus . 
 the gene was cloned from genomic dna , and the protein , denoted mlok1 , was overexpressed in escherichia coli and purified . 
 gel filtration analysis revealed a heterogeneous distribution of protein sizes which , upon inclusion of cyclic nucleotide , coalesces into a homogeneous population , eluting at the size expected for a homotetramer . 
 as followed by a radioactive 86rb+ flux assay , the putative channel protein catalyzes ionic flux with a selectivity expected for a k+ channel . 
 ion transport is stimulated by camp and cgmp at submicromolar concentrations . since this bacterial homologue 
 does not have the  
 c - linker  sequence found in all eukaryotic s4-type cyclic nucleotide - modulated ion channels , these results show that this four - helix structure is not a general requirement for transducing the cyclic nucleotide - binding signal to channel opening . 
INTRODUCTION
MATERIALS AND METHODS
Biochemical
Flux Assays
RESULTS
DISCUSSION