Patent Application: US-27205005-A

Abstract:
membrane proteins represent ˜ 30 % of the proteome of both prokaryotes and eukaryotes . unique to cell surface receptors is their biogenesis pathway , which involves vesicular trafficking from the endoplasmic reticulum through the golgi apparatus and to the cell surface . increasing evidence suggests specific regulation of biogenesis for different membrane receptors , hence affecting their surface expression . a pulse - chase assay can be used to monitor function recovery after chemobleaching to probe the transit time of the cell surface receptors to reach the cell surface . this method distinguishes molecular density from functional density . the ability of the reported method to access the biogenesis pathways in a high - throughput manner facilitates the identification and evaluation of molecules affecting receptor trafficking .

Description:
the inventors have developed a strategy in which the activity of targeted cell surface receptors is modified , inhibited , or changed . by monitoring replenished activity of the receptors after modification , inhibition , or change , the assay allows for determination of transit time for receptor recovery on the cell surface . the procedure is dubbed “ function recovery after chemobleaching ” ( frac ), although either chemical or biological agents can be used to accomplish the “ bleaching .” the methodology takes advantage of a modification strategy applicable to almost all cell surface proteins . the function or activity of the cell surface proteins can be any detectable readout , such as ligand binding , enzymatic activity , epitope accessibility , ion conduction , fluorescence , and other physical properties . to assay the transit time of nacently surfaced receptor , we first modify , inhibit , or change ( pulse ) accessible functional molecules on the cell surface and then monitor the recovery of the original function / activity at subsequent time points ( chase ). one thus may determine the time required to populate the cell surface with newly inserted cell surface molecules ( fig1 ). designing versions of this assay involves consideration of at least four factors . ( i ) a functional assay preferably monitors activity predominantly or only from molecules on the cell surface . ( ii ) preferably the assay does not affect protein half - lives on the cell surface . ( iii ) the activity detection should be relatively short compared with the time required to repopulate the cell surface with functional molecules . ( iv ) the pulse should be rapid , quantitative , and optionally irreversible , thereby allowing detection of the activity contributed only by the newly arrived ( or newly activated ) proteins on the cell surface . these are considerations only , rather than mandatory properties . deviation from one of these considerations may involve compensation by another . furthermore , one of ordinary skill in the art will appreciate that treatment of cell surfaces can be restricted to a desirable area , such as the apical surface of epithelial cells . thus , spatially restricted populations of cell surface receptors can be affected . the activity or function on the cell surface which is monitored exists in two states . the first state is before the treatment with a chemical or biological agent and the second state is after treatment . the two states differ in magnitude or quality . for example , the second state may be an inhibited form , which is less active or exhibits no activity . the second state may have an altered activity , such as a change in binding properties from one ligand or substrate to another . alternatively , the binding properties may change so that ligand or substrate binding becomes more or less selective . another option is that the first and second states may differ epitopically , i . e ., they are differentially bound by an antibody . in another option , the second state of the cell surface receptor may be more active then the first state . in another alternative , the second state results in a different subcellular location or a different oligomerization state . for example , in the second state the cell surface receptor may be internal to the cell . chemical or biological agents for treating the cell surface to effect a change in state of a receptor &# 39 ; s activity or function can be any known in the art . the agents may bind or react reversibly or irreversibly with the cell surface receptor . the agents may covalently modify the cell surface receptor . the agents do not , however , destroy the cells or compromise the integrity of the cells . thus the agents only have access to proteins which are external ( on the cell surface ) at the time of treatment . the agents may be toxins , antibodies , or pharmaceutical agents . reactive agents such as mercury , diazonium ions , and dyes can be covalently linked to dextran to render them nonpermeant to cells and thus reagents suitable for treating cell surfaces . suitable agents include methanethiosulfonates , including 2 - trimethylammoniumethyl methanethiosulfonate bromide . pore blockers such as teteraethylammonium can be used for potassium channels , and mk801 can be used for nmda ( glutamate ) receptors . agents which bind to , but do not block , pore - forming proteins can also be used . in one particular assay , the biological agent is an antibody which specifically binds to a cell surface receptor . the same or a different antibody to the cell surface receptor is used to detect the second population of cell surface receptors . the antibodies , whether the same or different , can optionally be differentially labeled so that first and second populations are readily distinguishable . alternatively , an antibody can be used in an assay of the first and second state . for example , a chemical agent may modify one or more residues in an epitope . the antibody can then be used to detect the original ( first state ) or the modified ( second state ) epitope . the cell surface receptor may , for example , be a specific ligand binding receptor or may be an ion channel . assays for the binding of ligand or for the uptake of the ion can be performed as are known in the art . any means of detecting the activity of the cell surface receptor can be used . preferably the detection will be relatively quick compared to the transit time of the receptor and / or compared to the time that the first population of cell surface receptors remains in the second state . as discussed below , if potassium ion channels are being assayed , a rubidium ion flux assay can be performed . potassium channel activity can be measured by other means including voltage - sensitive dyes , fluorescence , and conventional electrophysiological recordings ( 15 ). the cell surface receptors may be native to the cell or they may be introduced by recombinant means from the same or a different species . the receptors may be mutant , engineered , or wild - type . examples of receptors which can be used are k + channels , kir2 . 1 channels , kir2 . 1y , channels , herg channels , ca 2 + channels , na + channels , glutamate channels , and cl − channels . the receptors which are assayed may form a homogenous population or may be heterogeneous , i . e ., comprising two or more types with similar or coordinated functions . the assay can be performed on two sets of cells which differ in one or more aspects , and the results can be compared . the two sets of cells can be , e . g ., from a normal and a diseased or pathological sample . the two sets of cells can be , e . g ., a parental type ( wild - type ) and a genetically modified type . the genetic modification can be , for example , a mutation , an additional gene or copy of a gene , a viral infection , a bacterial infection , a recombinant gene , etc . alternatively , the two sets of cells can be subjected to two different environmental conditions . for example , one set of cells can be subjected to a chemical agent , a biological agent , radiation , or illumination , or to an environmental condition of a particular temperature , pressure , or cell density , and the effect of the environmental condition can be determined oh the recovery of the cell surface receptor function . any chemical or biological agent can be tested . these can be purified compounds , mixtures of compounds , libraries of compounds , natural products , or synthetic products . they can be tested individually or in groups . cells used in the assays of the invention are any membrane enclosure which provides insulation to internal components from chemical and / or biological treatments . a cell may be a native or recombinant cell , and may be from any organism . a cell may be in a specific state , e . g ., a sperm or an oocyte , a fusion product of more than one cell , an aggregation of identical or heterogeneous cells formed as the result of growth in culture or isolated from tissue , or a vesicle of insulated lipid layers . a cell may be from a particular organ such as heart , brain , kidney , liver , colon , skin , etc . cells may be treated and assayed in vitro in cell culture or in vivo in whole animals . recovery of the function of the cell surface receptor ( or the first state of the activity of the cell surface receptor ) can be determined at a fixed time point , at multiple time points , or continuously . a rate can be determined or an amount of recovery at a fixed time point can be determined . the above disclosure generally describes the present invention . all references disclosed herein are expressly incorporated by reference . a more complete understanding can be obtained by reference to the following specific examples which are provided herein for purposes of illustration only , and are not intended to limit the scope of the invention . materials . the methanethiosulfonate reagents , such as [ 2 -( trimethylammonium ) ethyl ] methanethiosulfonate bromide ( mtset ), were purchased from toronto research chemicals ( downsview , on , canada ). brefeldin a ( bfa ), sodium butyrate , and rbcl were purchased from sigma . transfection and stable cell lines . expression vectors were constructed using pcdna3 . 1 (+) ( invitrogen ), and the kir2 . 1 cdna was cloned into hindiii and noti sites . for the two kir2 . 1 cdna clones used in the study , one has the hemagglutinin ( ha ) epitope inserted at the first extracellular loop between m1 and p - loop to facilitate surface detection ( 13 ), referred to as kir2 . 1 . the other has both the ha epitope and a peptide , frgrswty , fused at the c - terminus to facilitate surface expression , which is referred to as kir2 . 1y . the transfection into hek 293 cells was carried out with fugene 6 ( roche diagnostics ). stable clones were selected for neomycin resistance . stable clones with high expression level of channel proteins were selected using immunoprecipitation and flow cytometry . these stable cell lines were maintained in 50 / 50 dmem / f12 medium containing 10 % fbs , penicillin / streptomycin , l - glutamine , and 500 μg / ml g418 . flow cytometry . hek 293 cells stably expressing kir2 . 1 or kir2 . 1y channel were seeded at ˜ 3 × 10 5 cells per well in a six - well plate and allowed to grow for 16 - 20 h in complete growth medium with 5 mm sodium butyrate at 37 ° c . and 5 % co 2 . cells were washed with 1 × pbs and harvested by incubation with 0 . 5 mm edta in 1 × pbs for 5 - 10 min at room temperature . cells then were washed twice with hanks &# 39 ; balanced salt solution ( hbss ) plus 5 mm hepes ( ph 7 . 3 ) and 2 % fbs and incubated with rat anti - ha monoclonal antibody ( roche diagnostics ) on ice for 1 h . cells then were washed twice again with hbss staining medium and incubated with fitc - labeled goat anti - rat igg antibody ( jackson immunoresearch ) for 15 min on ice . finally , the cells were washed twice with hbss staining medium , and the channel - surface expression was measured by facscalibur ( becton dickinson ) with cellquest software ( becton dickinson ). rb + flux assay and atomic absorption spectrometry . all assays shown were performed at room temperature in poly ( l - lysine ) ( 0 . 1 mg / ml )- coated 24 - well microplates . for experiments with transiently transfected cells , ˜ 1 × 10 5 cells were seeded in each well the day before transfection ( 0 . 3 μg of dna per well ). sodium butyrate at 5 mm was added 6 - 8 h after transfection . the assays were performed 24 h after transfection . hek 293 cells stably expressing either kir2 . 1 or kir2 . 1y channel were seeded with a cell density of 2 × 10 5 cells per well . to enhance expression , 5 mm sodium butyrate was added 4 - 6 h after seeding . cells then were cultured continuously for 16 - 20 h before experiments . rb + influx . to perform the assay , cells first were incubated in 50 / 50 dmem / f12 complete growth medium containing 5 mm rbcl for the indicated time periods before the medium was quickly aspirated . cells then were washed twice quickly with non - rb + dmem / f12 medium and lysed with 0 . 5 ml of 0 . 1 % sds per well . the rb + concentration in cell lysates was measured by atomic absorption spectrophotometry ( icr8000 , aurora biomed , vancouver ), according to the manufacturer &# 39 ; s user manual . rb + efflux . to load rb + into cells , we incubated cells in 0 . 5 ml per well 50 / 50 dmem / f12 complete growth medium containing ˜ 5 mm rbcl at 37 ° c . for 3 - 4 h . cells then were washed twice quickly with 1 ml per well non - rb + medium to remove residual rb + . to perform the assay , 0 . 5 ml of non - rb + complete growth medium per well was added to the cells , which were incubated at room temperature for 15 min unless otherwise indicated in the legends of fig1 , 3 , 4 , 5 . the supernatants from each well were transferred quickly to a new 24 - well plate at the end of incubation . cells then were lysed with 0 . 5 ml of 0 . 1 % sds per well . the supernatants and cell lysates were diluted further with distilled water , and the diluted samples were transferred to a 96 - well microplate . the rb + concentration in each sample was analyzed using the icr 8000 . rb + efflux (%) was determined as : where [ rb + ] sup represents the rb + concentration in the supernatant , and [ rb + ] lysate represents the rb + concentration in the cell lysate . kir2 . 1 encodes an inward rectifier k + channel consisting of four subunits that align to form a centrally positioned hydrophilic pore to conduct k + ions ( 14 ). one of the attractive features of the kir2 . 1 channel is that it has an open probability of nearly 100 % at resting potential , thereby permitting an option of monitoring the activity without depolarization . assays for detecting k + channel activities include electrophysiological recording , fluorescence - based measurements , and rb + flux assay ( for review , see ref . 15 ). all three types of assays can be achieved within minutes . the primary distinctions concern whether the assay reads signals from individual cells or a population of cells and whether the cells remain intact during the entire assay procedure . we chose the rb + flux assay because of its ability to assay a large population of cells for extended time periods without rupture of cell membrane . the flux activity linearly reflects the conducting ion passage . rb + is a nonphysiological ion but is permeable to almost all k + channels , which affords general applicability . in addition , the rb + assays described here were carried out under conditions that do not tamper with the membrane potential , hence avoiding a stimulation of membrane fusion that could be caused by depolarization - induced ca 2 + influx . to modify , nullify , or change the k + channels already expressed on the cell surface , several approaches have been considered . these include antibody binding induced inhibition ( 16 ), pore blockers ( such as tetraethylammonium ), and covalent modifications by , for example , methanethiosulfonate ( mts ) reagents ( 17 ). of the three possibilities , covalent modification is advantageous because mts reagents are irreversible under typical physiological redox conditions and would not cause clustering that often leads to other subsequent events , including endocytosis . other reagents may be preferred for other cell surface receptors or applications . earlier reports using electrophysiological recording have shown that membrane - nonpermeable mts reagents such as mtset reduce or abolish the current of kir2 . 1 by covalently modifying the reduced form of cysteine residues already present in the channel protein or introduced by site - directed mutagenesis ( 18 ). it is not known whether or to what extent the modifications by mts reagents will affect channel activity measured by a rb + flux assay . we tested several cysteine positions of kir2 . 1 mutants using mtset , a positively charged cysteinyl sulfhydryl - specific reagent . our results show that wild - type kir2 . 1 may be quantitatively nullified by mtset . notably , a mutation of c149 , the only cysteine on the extracellular side , leads to a loss of sensitivity to mtset both by electrophysiological recording and rb + assay ( data not shown ). this data is consistent with results from electrophysiological recording ( 18 ), further confirming the fact that mtset acts on the extracellular cysteine at the 149 position . to monitor the specificity of the rb + assay and the effectiveness of rb + detection by atomic absorption spectrometry ( 19 ), we measured the rb + uptake signals of transiently transfected cells at different times of incubation . comparison of transfected cells with nontransfected cells revealed an ˜ 2 - fold signal - to - noise ratio ( fig2 a ) in a 10 - min uptake . treatment with mtset before the assay abolished & gt ; 80 % of the specific signal . prolonging uptake time increased the total signal but did not improve the signal - to - noise ratio . it should be noted that the influx measures the absolute level ( mg / l ) of rb + inside the cell . thus , it is also a function of cell number in a given assay and dependent on na + — k + atpase . we then compared the uptake ( i . e ., influx ) with the efflux assay , which reflects the ratio of rb + released in the supernatant to total rb + in the loaded cells ( see methods and fig2 b ). to further improve the signal - to - noise ratio , cell lines were generated that stably express either the kir2 . 1 or kir2 . 1y channels ( see methods ). the efflux assay using a stable kir2 . 1y line displayed a significant improvement of a nearly 4 - to 6 - fold signal - to - noise ratio ( fig2 c ). when cells were treated with mtset , only background signals were observed . to determine the optimal conditions , we performed a titration using different concentrations and time courses of the mtset treatment . the results suggest an optimal treatment with 2 . 5 mm mtset for 5 min ( fig2 d and 2 e ). these results demonstrate the functional expression of kir2 . 1 channels , the sensitivity of the rb + efflux assay , and the feasibility of nullifying the channel activity on the cell surface by mtset . to evaluate frac , we generated two cell lines expressing either the kir2 . 1 or kir2 . 1y where the protein expression level of kir2 . 1y is ˜ 2 - fold higher than that of kir2 . 1 ( data not shown ). both cell lines were treated first with mtset to nullify the channels on the cell surface . after its removal , the treated cells were assayed after incubations of the indicated periods of time for recovery . the half - maximal functional recovery time ( fr 1 / 2 ) at 37 ° c . for both kir2 . 1 and kir2 . 1y was 1 h ( fig3 a ). the functional expression reached 90 % of the initial level 5 h after chemobleaching with mtset . during the chase period , the rb + loaded cells without mtset treatment gave rise to similar signals ( fig3 a ). the background rb + efflux of nontransfected cells remained consistent with or without mtset treatment ( fig3 a ). the result shows that recovery was specific to the cells expressing the recombinant channels . to monitor the level of kir2 . 1y protein on the cell surface , flow cytometry analyses were carried out using live cells stained with anti - ha antibody ( see methods ). the total amount of protein signals remained essentially constant throughout the entire experimental time period of 5 h ( fig3 b ). the source of functional recovery may originate from “ reactivation ” of the existing surface channel protein and / or from newly arrived vesicles . to test whether vesicular transport was responsible for delivering the recovered activity , the frac experiments were carried out in parallel but at different recovery temperatures ( fig3 a and 4 a - 4 c ). it is known that lowering the temperature can stall the er - to - golgi or cis - to trans - golgi transitions ( 20 ). in either case , only limited recovery should be observed , presumably caused by the vesicles in transit between trans - golgi and the cell surface . fig4 a - 4 c shows that the recovery of activity was progressively slowed when temperatures decreased from 37 ° c . ( fr 1 / 2 = 1 h ) to 30 ° c . ( fr 1 / 2 = 2 h ) to 22 ° c . ( fr 1 / 2 = 3 . 5 h ). at 15 ° c ., which typically blocks the er - to - golgi transition ( 20 ), the activity was recovered partially , up to 40 % of the initial level ( fig4 a ). normalized recovery suggests a residual activity at 0 h ( fig4 d ). the activity may be contributed by a rapid recovery of the functional channels during the rb + assay , because all rb + efflux assays were performed at 22 ° c . these experiments support the notion that reduction of vesicular transport at progressively lower temperatures was causal to the different fr 1 / 2 values . vesicular trafficking is sensitive to a variety of pharmacological agents . bfa is a hydrophobic membrane - permeable fungal toxin , which was reported to inhibit multiple steps of vesicular trafficking by causing disruption of golgi apparatus and , to some extent , er - to - golgi transition ( 21 ). bfa has been used to block protein secretion ( 22 ), and it is thought to act on gtp exchange factors , which activate a family of small gtpases known as adp - ribosylation factors ( 23 - 25 ). hence , the application of bfa in the above frac assay could test the role of vesicular transport in the recovery of channel activity on the cell surface . experiments were carried out to compare the extent of activity recovery in 3 h under conditions of no treatment , mtset only , and treatment with mtset followed by bfa ( fig5 a ). the activity of nontreated cells remained consistent over a 3 - h incubation . the mtset - treated preparations displayed ˜ 70 % recovery , consistent with data shown in fig3 a . when bfa was added immediately after the mtset treatment , & lt ; 20 % recovery was observed . the bfa effect was specific to the recovery because without mtset treatment , the 3 - h incubation with bfa did not affect the activity for channels that were already on the cell surface ( fig5 a ). fig5 b shows the dose - dependent effect of bfa treatment on the recovery of activity at 3 h . the results suggest that 0 . 1 - 0 . 2 μm bfa treatment gave intermediate effects . prolonged incubation with 0 . 2 μm bfa for up to 8 h did not yield further improvements in recovery ( data not shown ). the bfa effect reaches a plateau at concentrations of & gt ; 0 . 6 μm . in the absence of mtset treatment , the surface channel activity displayed no sensitivity to bfa concentrations ( fig5 b ), demonstrating the specificity of bfa effects for recovery . incubation of bfa for the extended time of 3 h did not result in a detectable reduction of rb + efflux activity in the absence of mtset treatment for kir2 . 1y ( fig5 ). in the case of kir2 . 1 , we observed reproducible ˜ 30 % reduction over the same period of incubation ( data not shown ). the difference may stem from rate of endocytosis and total number of channel protein on cell surface . for kir2 . 1y , the combination of quantitative elimination of surface channel activity by mtset , the & gt ; 90 % activity recovery , and little detectable reduction of rb + efflux in the presence of bfa then would predict a 2 - fold increase of protein signal on the cell surface . to the contrary , fig3 b provides no support of a comparable increase at the protein level . thus , the source of the recovered activity likely originated from newly surfaced channel molecules , which represent only a small fraction of total channel protein on the cell surface . it would be particularly interesting to investigate , especially in polarized cells , any potential spatial distribution preference for the freshly replenished channel protein coupled with frac . the reported method allows for a direct measurement of the transition for repopulating functional channel protein on the cell surface . of particular interest is the observation of consistent channel activity over a long time period in the absence of er and golgi vesicular transport ( fig5 a - 5 b ). when the surfaced channel molecules were irreversibly nullified , the cells were able to repopulate the surface with newly arrived functional channels within hours . in the 3 - h recovery experiments , ˜ 70 % of activity was recovered , but it was not accompanied by a significant increase of channel protein on the cell surface ( fig3 a - 3 c ). together , these data provide evidence that the rb + efflux by kir2 . 1y channels on the cell surface is contributed by only a small fraction of the channel protein , suggesting the existence of a substantial fraction of “ sleeping ” channels . these sleeping channels are detectable at the protein level but functionally null to the rb + assay . the frac experiments reported here allow for specific determination of the time required for a cell to populate its surface with functional channels and receptors . the electrophysiological measurement has allowed for the determination of overall conductance of the two cell lines , hence permitting an estimation of 5 , 000 - 7 , 000 conducting channels per cell for kir2 . 1y and 3 , 000 - 4 , 000 conducting channels for kir2 . 1 ( data not shown ). it is known that different membrane proteins express with much variation in terms of molecular and functional density on the cell surface . the total recovery time likely reflects different incremental steps of biogenesis . the transit time measurement could provide a key parameter to differentiate their trafficking properties and potentially even discrete steps . for example , the fraction of bfa - insensitive recovery may represent vesicles that have exited er and cis - golgi compartments ( fig5 a ). similarly , application of cycloheximide in frac experiments , which inhibits new protein synthesis , an earlier step than that inhibited by bfa , resulted in only 40 % activity recovery ( data not shown ), compared with 20 % activity recovery found with bfa ( fig5 a ). hence , assays may provide resolution to isolate and evaluate compounds and cdnas that affect these pathways , which could be useful for clinical intervention and mechanistic studies . it is well known that cysteine substitution is a rather tolerable mutation . site - directed mutagenesis allows for engineering a site into receptors of interest to confer sensitivity to the mts reagent treatment ( 17 ). one can also use other covalent chemistry including various compounds that react with a primary amine group . there are certainly other available methods in addition to mts agents that may be used to “ inactivate ” receptors . for example , some receptors have antagonists with extremely high affinity and / or an unusually long off - rate . dizocilpine ( mk - 801 ) is an open channel blocker for the n - methyl - d - aspartate ( nmda ) receptor . it is conceivable that mk - 801 combined with a calcium - based assay would allow for determination of the rate by which nmda receptors repopulate the neuron surface . the spatial resolution of ca 2 + - based imaging technologies may offer a comparison of rates for recovery in different subcellular domains of a single cell . of the & gt ; 400 ion channel genes in the human genome , at least 167 are annotated to encode k + channels . k + channels are critical to a variety of biological processes ranging from neuronal excitability to oncogenesis . high - throughput assays to monitor channel activities and trafficking are of great value ( 15 ). recently , a few cardiac k + channels , particularly human ether - ago - go - related gene ( herg )- encoded k + channels , have become subjects of recommended safety testing for all drug candidates , because many approved drugs have been found to inhibit herg , which causes acquired long qt syndrome . these include therapeutic agents such as antiarrhythmics , antihistamines , antipsychotics , and antibiotics ( 26 ). the interactions of some of these compounds with the herg k + channels prolong cardiac repolarization , hence qt prolongation ( long qt ). in some cases , long qt induces torsade de pointes , which potentially could cause cardiac sudden death ( 27 ). the activity of herg channels can be monitored by the rb + assay ( 28 ). there is an increasing demand of profiling compounds &# 39 ; 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