it is a discovery of the present inventors that the channel inhibitory properties of the r domain of cftr protein can be separated from the channel activating properties . thus activating polypeptides can be used to treat cftr defective cells , without concern for inhibition at certain concentrations . activating polypeptides may also be used to enhance the activity of normal cftr , including that delivered by gene transfer . a polypeptide for use in treating cftr - defective cells contains a 22 amino acid sequence , glxisxxinxxxlkxxffxxxx , as shown in seq id no : 6 . the amino terminal residue is acetylated and the carboxy terminal residue is amidated . the residue x , at positions 3 , 6 , 7 , 10 , and 11 is either glutamic acid or glutamine ; at position 12 is aspartic acid or asparagine ; at position 15 is glutamic acid or glutamine ; at position 16 is cysteine or serine ; at positions 19 or 20 is aspartic acid or asparagine ; at position 21 is methionine or norleucine ; at position 22 is either glutamic acid or glutamine . the amino acid residue at position 16 is more preferably serine . the amino residue at position 21 is more preferable norleucine . the polypeptide of seq id no : 6 has a net negative charge . the net negative charge is preferably within the ranges of 1 - 8 , 2 - 8 , 3 - 8 , 4 - 8 , 5 - 8 , 6 - 8 , or 7 - 8 . the polypeptide more preferably has the sequence of seq id no : 1 , gleiseqinqqnlkqsffndle , wherein l at position 21 is norleucine . the amino terminal residue of the polypeptide is preferably acetylated and the carboxy terminal residue is preferably amidated . the polypeptide may also be present in a composition with a pharmaceutically acceptable carrier . pharmaceutically acceptable carriers are well known to those in the art . pharmaceutically acceptable carriers include , but are not limited to , large , slowly metabolized macromolecules , such as proteins , polysaccharides , polylactic acids , polyglycolic acids , polymeric amino acids , amino acid copolymers , and inactive virus particles . the composition can also contain liquids , such as water , saline , glycerol , and ethanol , as well as substances such as wetting agents , emulsifying agents , or ph buffering agents . buffering agents include hanks &# 39 ; solution , ringer &# 39 ; s solution , or physiologically buffered saline . it may be desirable that the polypeptide be fused to another polypeptide to provide additional functional properties . for example , fusion to another protein such as keyhole limpet hemocyanin can be used to increase immunogenicity . another desirable fusion partner is a membrane - penetrating peptide . such peptides include vp - 22 ( seq id no : 3 ), as well as the peptides shown in seq id no : 4 and seq id no : 5 . such peptides can be used to facilitate the uptake of the polypeptide by target cells . the polypeptides of the invention may also be fused to proteins that cause specific targeting to lung epithelial cells . for instance , the peptide thalwht directs dna to human airway epithelial cells . single chain antibody variable domains may be used to do the same . a cftr protein can be activated by the polypeptide . the cftr protein can be in a cell , preferably in the cell membrane and the cftr protein forms a camp - regulated chloride channel . an effective amount of a polypeptide that comprises the sequence of seq id no : 6 can be administered to the cell , and administration of the polypeptide activates the cftr protein . the polypeptide administered more preferably comprises the sequence of seq id no : 1 . the cells may be any cells that contain or express a cftr protein . the cells may naturally express the cftr protein , such as lung epithelial cells , or the cells may express the cftr protein after transient or stable transformation . the cells may be primary cells isolated from individuals that express a wild - type cftr protein , or may be primary cells isolated from individuals that express a mutant cftr protein . the cells may also be of a stable cell line . the cells may also exist in the body . the cftr protein is a wild type or a mutant cftr protein . the mutant cftr protein is a cftr protein that is expressed by the cells and that is transported to the cell surface . the mutant cftr protein also forms a camp - regulated chloride channel . the mutant cftr protein may contain alterations that are known and characterized , or may contain alterations that have not yet been discovered . a mutant cftr protein that fails to undergo full activation is a cftr protein that does not conduct ions to the same degree as wild - type cftr . the mutant cftr protein may not conduct ions at all . the mutant protein may also conduct ions to a similar extent as wild type cftr but be present in the membrane in substantially lower amounts than is true for normal individuals . activated is defined as any increase in conductance by the cftr protein . an increase in conductance may result when the opening of the cftr channel occurs with greater frequency than previously observed . an increase in cftr conductance may result when the duration of opening is increased each time the cftr channel opens . an increase in conductance may also result due to greater ability to conduct ions each time the cftr protein channel is open . the increase in open probability of the cftr protein is preferably at least 25 %, at least 50 %, at least 75 %, at least 100 %, at least 125 %, at least 150 %, at least 175 %, at least 200 %, or at least 300 %. an effective amount is any amount of polypeptide that is sufficient to activate the cftr protein , as activate is defined above . preferably , the polypeptide is administered to achieve a concentration of 0 . 5 to 14 μm . more preferably , the polypeptide is administered to achieve a concentration of 4 - 6 μm . the polypeptide may be administered by any means acceptable in the art . for instance , the polypeptide may be administered in vitro , or to cells in culture , by addition to the medium . the polypeptide may be administered in vivo , to a patient , by any route including intravenous , intrathecal , oral , intranasal , transdermal , subcutaneous , intraperitoneal , parenteral , topical , sublingual , or rectal . most preferably , the polypeptide is administered to a patient in an aerosol . the aerosolized polypeptide can be co - administered with an expression vector that encodes wild type cftr protein . an expression vector may be linear dna that encodes wild type cftr protein , or a plasmid or human artificial chromosome that expresses wild type cftr protein . the vector may be administered as naked dna or may be administered complexed to lipid molecules such as with liposomes , short polypeptides such as the thalwht polypeptide , or polycations such as polylysine , with or without stabilizing agents and / or receptor ligands . the dna may also be administered in a viral vector . viral vectors are known in the art . several nonlimiting examples include retroviruses , adenoviruses , adeno - associated viruses , lentiviruses , and herpes simplex virus . the gene encoding the wild type cftr protein may additionally comprise a promoter sequence to drive expression of the cftr gene . any promoter known in the art may be used . promoters include strong promoters such as the promoters of cytomegalovirus , sv40 , or rous sarcoma virus . the promoter may also be a tissue specific promoter . preferably the tissue specific promoter is a lung specific promoter . lung specific promoters include the promoters of surfactant protein a , keratin 18 , du clara cell secretory protein , and the promoter of cftr . a cftr protein can also be activated by applying an effective amount of a polypeptide to a cftr protein in a lipid bilayer . the polypeptide comprises the amino acid sequence of seq id no : 6 . the polypeptide more preferably comprises the amino acid sequence of seq id no : 1 . activating a cftr protein in a lipid bilayer is useful to the art for screening agents for the treatment of cystic fibrosis . a cftr protein in a lipid bilayer may be a cftr protein that is expressed in cells in culture . the cells may express the cftr protein without manipulation , or may be stably or transiently transfected to express the cftr protein . the lipid bilayer may also be such artificial preparations as , without limitation , a microsome preparation , a lipid - bilayer vesicle preparation , or liposomes . the polypeptide may be applied to the protein by its addition to cell culture media , or solution in which the lipid bilayers are maintained . a change in conductance may be measured by any means known in the art , such as patch clamping . a cftr activating polypeptide can be synthesized by sequentially linking units of one or more amino acid residues to form a polypeptide comprising the amino acid sequence of seq id no : 6 . preferably the polypeptide has the amino acid sequence of seq id no : 1 . synthesis of the cftr polypeptide can be performed using solid - phase synthesis , liquid - phase synthesis , semisynthesis , or enzymatic synthesis techniques . preferably the polypeptides are synthesized by solid - phase synthesis . more preferably the peptides are synthesized by f - moc synthesis . the polypeptide of the invention may alternatively comprise the sequence of seq id no : 2 , gleiseqinqqnlkqsffndme . the polypeptide of seq id no : 2 is not modified . it is similar to the sequence of seq id no : 1 , but for a methionine at position 21 , rather than a norleucine . like seq id no : 1 and seq id no : 6 , it may be fused to a membrane penetrating polypeptide . nucleic acid molecules comprise a nucleotide sequence that encodes the polynucleotide sequence of seq id no : 2 . one of skill in the art will recognize that many sequences will encode the polypeptide , as more than one codon can specify a given amino acid . the nucleic acid may further comprise regulatory sequences that enhance the expression of the polypeptide . promoters may be strong constitutive promoters , as discussed above , or may be tissue - specific promoters . preferably the tissue - specific promoter is a lung - specific promoter . the nucleic acid molecules may further comprise a vector . the vector can be any suitable vector for the delivery of the polynucleotide sequence into the lungs of a patient , resulting in expression of the polypeptide in the lungs of the patient . a cftr protein can be activated by expression of a polynucleotide . a nucleic acid comprising a sequence encoding a polypeptide according to seq id no : 2 is administered to a cell comprising the cftr protein . the polypeptide is expressed and the cftr protein is thereby activated . the polynucleotide may be administered by any acceptable means in the art . preferably the polynucleotide is administered as an aerosol . the administration of the polypeptides of the present invention are most useful in treatment of a class of mutations that encode cftr proteins that are properly delivered to the plasma membrane but that are residually or minimally active . minimally or residually active cftr proteins have the ability to mediate or modulate channel conductance . however , channel conductance is insufficient to sustain the healthy , not cystic fibrotic phenotype . residually or minimally active includes proteins for which the activity of the cftr can be recorded but may be at a level that is barely detectable . this invention will also be useful for cftr mutants that are , to a large extent , misprocessed and thus reach the plasma membrane in much lower quantities than normally processed cftr , and for cftr mutants that are , to a large extent , improperly spliced , but retain production of some properly spliced cftr . known mutants of cftr are listed in table 1 . in addition to its utility in the activation of mutant forms of cftr , this invention will be a useful adjunct to gene therapy for cystic fibrosis . by enhancing the per - cftr molecule chloride transport activity , this peptide will increase the chloride transport activity obtained at any level of expression of cftr , thereby increasing its effective efficacy . the above disclosure generally describes the present invention . 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 . development of a polypeptide that exerts only an activating effect on cftr the activating peptide of q4n2neg2 was created by substituting glutamine residues for glutamic acid residues at four sites and asparagines for aspartic acid residues at two sites of the authentic neg2 peptide sequence gleisfeineedlkecffddme ( seq id no : 7 ). in addition , a serine residue was substituted for cysteine , to prevent peptide dimerization , and norleucine was substituted for methionine , to prevent oxidation . these changes create a peptide with reduced chemical reactivity and high predicted helical structure , confirmed by circular dichroism , as well as reduced net negative charge ( from − 9 to − 3 ). attempts to eliminate negative charge completely resulted in an insoluble peptide . when this peptide was added to the cis ( intracellular ) side of cftr channels captured in the planar lipid bilayer , at concentration ranging 0 . 5 to 14 μm , marked dose - related stimulation of channel activity was observed . at concentrations of 4 - 6 μm po of cftr doubles . no inhibitory activity was seen in any experiment at any concentration of peptide . to test whether the q4n2neg2 polypeptide is responsible for increasing the open probability of the cftr channel , synthetic q4n2neg2 , a 22 amino acid peptide , was added to the cis - intracellular side of single cftr channels captured in the planar lipid bilayer ( fig1 ). the diary plot of open probability as a function of time shows the activity of a single wt - cftr channel during the course of the experiment ( fig1 a ). during stimulation , the open probability doubles and more transitions are observed between the open and closed states ( fig1 b ). the open probability observed in 5 experiments at 4 μm concentration q4n2neg2 is shown to be increased by about two - fold in the graph ( fig1 c ). the q4 n2 neg2 peptide sequence has been tested on one mutant form of cftr , g551d , which reaches the plasma membrane . in the planar lipid bilayer , q4n2neg2 increased the open probability of g551 by about threefold . thus , this peptide is useful to stimulate channel activity in mutant forms of cftr that reach the plasma membrane . the neg2 sequence can also be rendered inhibitory , with no stimulatory activity , by scrambling the sequence such that the resulting peptide is predicted to not have helical tendencies , as confirmed by circular dichroism measurements , but retains the full net negative charge of − 9 . this peptide , called scrambled neg2 , inhibits channel activity by about 90 % at 6 μm concentration , with no stimulation observed at any concentration . in addition , insertion of a proline residue into the middle of the neg2 sequence also results in a peptide which inhibits channel activity by about 60 %, but does not stimulate . proline residues are known to disrupt helical structures . the wt cftr cdna was subcloned into an epstein - barr virus - based episomal eukaryotic expression vector , pcep4 ( invitrogen , san diego , calif . ), between the nhe1 and xho1 restriction sites . a human embryonic kidney cell line ( 293 - ebna hek ; invitrogen ) was used for transfection and expression of the cftr proteins ( ma et al ., 1997 , ma et al ., 1996 , xie et al ., 1995 ). the hek - 293 cell line contains a pcmv - ebna vector , which constitutively expresses the epstein - barr virus nuclear antigen - 1 ( ebna - 1 ) gene product and increases the transfection efficiency of epstein - barr virus - based vectors . the cells were maintained in dulbecco &# 39 ; s modified eagle medium with 10 % fbs and 1 % l - glutamine . geneticin ( g418 , 250 ( g / ml ) was added to the cell culture medium to maintain selection of the cells containing the pcmv - ebna vector . lipofectamine reagent ( life technologies , inc ) in optimem media ( serum - free ) was used to transfect the hek - 293 cells with pcep4 ( wt ). after 5 hours , serum was added to the media ( 10 % final serum concentration ). twenty - four hours after transfection , the transfection media was replaced with fresh media . the cells were harvested two days after transfection and microsomal membrane vesicles were prepared for single channel measurements in the lipid bilayer reconstitution system . hek - 293 cells transfected with pcep4 ( cftr ) were harvested and homogenized using a combination of hypotonic lysis and dounce homogenization in the presence of protease inhibitors ( ma et al ., 1997 , ma et al ., 1996 , xie et al ., 1995 ). microsomes were collected by centrifugation of postnuclear supernatant ( 4500 × g , 15 min ) at 100 , 000 × g for 20 min and resuspended in a buffer containing 250 mm sucrose , 10 mm hepes , ph 7 . 2 . the membrane vesicles were stored at − 75 ° c . until use . lipid bilayer membranes were formed across an aperture of ˜ 200 ( m diameter with a mixture of phosphatidylethanolamine : phosphatidylserine : cholesterol in a ratio of 5 : 5 : 1 . the lipids were dissolved in decane at a concentration of 33 mg / ml . the recording solutions contained : cis ( intracellular ), 200 mm cscl , 1 mm mgcl 2 , 2 mm atp , and 10 mm hepes - tris ( ph 7 . 4 ); trans ( extracellular ), 50 mm cscl , 10 mm hepes - tris ( ph 7 . 4 ). vesicles ( 1 - 4 ( 1 ) containing wild - type cftr were added to the cis solution . the pka catalytic subunit was present at a concentration of 50 units / ml in the cis solution unless noted otherwise . single channel currents were recorded with an axopatch 200a patch clamp unit ( axon instruments ). the currents were sampled at 1 - 2 . 5 ms / point . single channel data analyses were performed with pclamp and tips softwares . anderson , m . p ., berger , h . a ., rich , d . p ., gregory , r . j ., smith , a . e ., and welsh , m . j . ( 1991 ). nucleoside triphosphates are required to open the cftr chloride channel . cell 67 , 775 - 784 . bear , c . e ., li , c ., kartner , n ., bridges , r . j ., jensen , t . j ., ramjeesingh , m ., and riordan , j . r . ( 1992 ). purification and functional reconstitution of the cystic fibrosis transmembrane conductance regulator ( cftr ). cell 68 , 809 - 818 . carson , m . r ., travis , s . m ., and welsh , m . j . ( 1995 ). the two nucleotide - binding domains of cystic fibrosis transmembrane conductance regulator ( cftr ) have distinct functions in controlling channel activity . j . biol . chem . 270 , 1711 - 1717 . cheng , s . h ., rich , d . p ., marshall , j ., gregory , r . j ., welsh , m . j ., and smith , a . e . ( 1991 ). phosphorylation of the r domain by camp - dependent protein kinase regulates the cftr chloride channel . cell 66 , 1027 - 1036 . cotten , j . f . and welsh , m . j . ( 1997 ). covalent modification of the regulatory domain irreversibly stimulates cystic fibrosis transmembrane conductance regulator . j . biol . chem . 272 , 25617 - 25622 . dulhanty , a . m . and riordan , j . r . ( 1994 ). phosphorylation by camp - dependent protein kinase causes a conformational change in the r domain of the cystic fibrosis transmembrane conductance regulator . biochemistry 22 , 4072 - 4079 . gadsby , d . c . and nairn , a . c . ( 1994 ). regulation of cftr channel gating . trends biochem . sci . 19 , 513 - 518 . geourjon , c . and deleage , g . ( 1995 ). sopma : significant improvements in protein secondary structure prediction by consensus prediction from multiple alignments . cabios 11 , 681 - 684 . gunderson , k . l . and kopito , r . r . ( 1995 ). conformational states of cftr associated with channel gating : the role of atp binding and hydrolysis . cell 82 , 231 - 239 . higgens , c . f . ( 1992 ). abc transporters : from microorganisms to man . annu . rev . cell biol . 8 , 67 - 113 . ma , j . and davis , p . b . ( 1998 ). what we know and what we do not know about cystic fibrosis transmembrane conductance regulator . clinics in chest medicine 19 , 459 - 471 . ma , j ., tasch , j . e ., tao , t ., zhao , j ., xie , j ., drumm , m . l ., and davis , p . b . ( 1996 ). phosphorylation - dependent block of cystic fibrosis transmembrane conductance regulator chloride channel by exogenous r domain protein . j . biol . chem . 271 , 7351 - 7356 . ma , j ., zhao , j ., drumm , m . l ., xie , j ., and davis , p . b . ( 1997 ). function of the r domain in the cystic fibrosis transmembrane conductance regulator chloride channel . j . biol . chem . 272 , 28133 - 28141 . picciotto , m . r ., cohn , j . a ., bertuzzi , g ., greengard , p ., and nairn , a . c . ( 1992 ). phosphorylation of the cystic fibrosis transmembrane conductance regulator . j . biol . chem . 267 , 12742 - 12752 . quinton , p . m . ( 1986 ). missing cl − conductance in cystic fibrosis . am . j . physiol . 251 , c649 - c652 . rich , d . p ., berger , h . a ., cheng , s . h ., travis , s . m ., saxena , m ., smith , a . e ., and welsh , m . j . ( 1993 ). regulation of the cystic fibrosis transmembrane conductance regulator cl − channel by negative charge in the r domain . j . biol . chem . 268 , 20259 - 20267 . rich , d . p ., gregory , r . j ., anderson , m . p ., manavalan , p ., smith , a . e ., and welsh , m . j . ( 1991 ). effect of deleting the r domain on cftr - generated chloride channels . science 253 , 205 - 207 . riordan , j ., rommens , j ., kerem , b .- s ., noa , a ., rozmahel , r ., grzelczak , z ., zielenski , j ., lok , s ., plavsic , n ., chou , j .- l ., drumm , m ., iannuzzi , m ., collins , f ., and tsui , l .- c . ( 1989 ). identification of the cystic fibrosis gene : cloning and characterization of complementary dna . science 245 , 1066 - 1073 . rost , b . and sander , c . ( 1993 ). prediction of protein structure at better than 70 % accuracy . j . mol . biol . 232 , 584 - 599 . rost , b . and sander , c . ( 1994 ). combining evolutionary information and neural networks to predict protein secondary structure . proteins 19 , 55 - 72 . tabcharani , j . a ., chang , x .- b ., riordan , j . r . and hanrahan , j . w . ( 1991 ). phosphorylation - regulated c − channel in cho cells stably expressing the cystic fibrosis gene . nature 352 , 628 - 631 . tao , t ., xie , j ., drumm , m . l ., zhao , j ., davis , p . b ., and ma , j . ( 1996 ). slow conversions among subconductance states of cystic fibrosis transmembrane conductance regulator chloride channel . biophys . j . 70 , 743 - 753 . vankeerberghen , a ., wei , l ., jaspers , m ., cassiman , j .- j ., nilius , b ., and cuppens , h . ( 1998 ). characterization of 19 disease - associated missense mutations in the regulatory domain of the cystic fibrosis transmembrane conductance regulator . hum . mol . genet . 7 , 1761 - 1769 . welsh , m . j . and smith , a . e . ( 1993 ). molecular mechanisms of cftr chloride channel dysfunction in cystic fibrosis . cell 73 , 1251 - 1254 . winter , m . c . and welsh , m . j . ( 1997 ). stimulation of cftr activity by its phosphorylated r domain . nature 389 , 294 - 296 . xie , j ., drumm , m . l ., ma , j ., and davis , p . b . ( 1995 ). intracellular loop between transmembrane segments iv and v of cystic fibrosis transmembrane conductance regulator is involved in regulation of chloride channel conductance state . j . biol . chem . 270 , 28084 - 28091 . zielenski , j . and tsui , l . c . ( 1995 ). cystic fibrosis : genotypic and phenotypic variations . annu . rev . genetics 29 , 777 - 807 . ile arg val thr val cys glu gly lys asn leu leu gln arg ala asn
phosphorylation of the cystic fibrosis transmembrane conductance regulator by cyclic amp - dependent protein kinase is essential for opening the cftr chloride channel . a short segment containing many negatively charged amino acids within the regulatory domain of cftr is a critical regulator of the chloride channel activity . an isolated neg2 polypeptide may be expressed as a separate sequence that stimulates cftr channel openings at lower concentrations , but that inhibits cftr channel openings at higher concentrations . residues in the neg2 sequence were substituted to produce a polypeptide that exerts only an activating effect on cftr . one such polypeptide is the q4n2neg2 polypeptide . exogenous q4n2neg2 exerts stimulatory effects on both wild - type and mutant g551d cftr function , without exhibiting inhibitory activity at any concentration .