Enhancers of CFTR chloride channel function

Abstract

Phosphorylation of the cystic fibrosis transmembrane conductance regulator (CFTR) by cyclic AMP-dependent protein kinase (PKA) is essential for opening the CFTR chloride channel. A short segment containing many negatively charged amino acids (817-838, NEG2) within the regulatory (R) domain of CFTR is a critical regulator of the chloride channel activity. Deletion of NEG2 from CFTR completely eliminates the PKA dependence of the chloride channel. Exogenous NEG2 peptide interacts with the CFTR molecule and exhibits stimulatory effects on CFTR function. Our data suggest that NEG2 interacts with other cytosolic domains of CFTR to control the opening transitions of the chloride channel.

1 . An isolated polypeptide comprising a portion of CFTR (cystic fibrosis transmembrane conductance regulator) protein of between 10 and 100 amino acids, said portion comprising 18 amino acids as shown in SEQ ID NO: 1.  
     
     
         2 . The polypeptide of  claim 1  which comprises 22 amino acids as shown in SEQ ID NO: 2.  
     
     
         3 . The polypeptide of  claim 1  wherein the polypeptide is fused to a membrane-penetrating peptide.  
     
     
         4 . The polypeptide of  claim 2  wherein the polypeptide is fused to a membrane-penetrating peptide.  
     
     
         5 . The polypeptide of  claim 3  wherein the membrane-penetrating peptide is selected from the group consisting of: VP-22 (SEQ ID NO: 3), (SEQ ID NO: 4), and (SEQ ID NO: 5).  
     
     
         6 . The polypeptide of  claim 4  wherein the membrane-penetrating peptide is selected from the group consisting of: VP-22 (SEQ ID NO: 3), (SEQ ID NO: 4), and (SEQ ID NO: 5).  
     
     
         7 . The polypeptide of  claim 1  which is free of phosphorylation.  
     
     
         8 . A method of activating a CFTR protein comprising: 
 applying a polypeptide to a CFTR protein which forms a cAMP regulated chloride channel, said polypeptide comprising a portion of CFTR protein of between about 10 and 100 amino acids, said portion comprising 18 amino acids as shown in SEQ ID NO: 1, whereby the open probability of the channel formed by the CFTR increases by at least 25%.    
     
     
         9 . The method of  claim 8  wherein the open probability of the channel formed by the CFTR increases by at least 50%.  
     
     
         10 . The method of  claim 8  wherein the open probability of the channel formed by the CFTR increases by at least 75%.  
     
     
         11 . The method of  claim 8  wherein the open probability of the channel formed by the CFTR increases by at least 100%.  
     
     
         12 . The method of  claim 8  wherein the open probability of the channel formed by the CFTR increases by at least 125%.  
     
     
         13 . The method of  claim 8  wherein the open probability of the channel formed by the CFTR increases by at least 150%.  
     
     
         14 . The method of  claim 8  wherein the open probability of the channel formed by the CFTR increases by at least 200%.  
     
     
         15 . The method of  claim 8  wherein the CFTR protein is a mutant which reaches a cell's plasma membrane but fails to undergo full activation.  
     
     
         16 . The method of  claim 15  wherein the CFTR protein is listed at http://www.genet.sickkids.on.ca/cftr-cgi-bin/fulltable.  
     
     
         17 . The method of  claim 8  wherein the step of applying is performed by administering an aerosolized polypeptide to a patient with a mutant CFTR protein.  
     
     
         18 . The method of  claim 8  wherein the CFTR protein is in a lipid bilayer and a change in conductance is measured upon applying the polypeptide.  
     
     
         19 . The method of  claim 8  wherein the step of applying the polypeptide is accomplished by administering a nucleic acid encoding the polypeptide to a patient who expresses the CFTR protein, whereby the polypeptide is expressed  
     
     
         20 . The method of  claim 19  wherein the nucleic acid is administered as an aerosol to the patient's airways.  
     
     
         21 . A method of activating a CFTR protein comprising: 
 applying a polypeptide to a CFTR protein which forms a cAMP regulated chloride channel, said polypeptide comprising a portion of CFTR protein of between 10 and 100 amino acids, said portion comprising 22 amino acids as shown in SEQ ID NO: 1, whereby the open probability of the channel formed by the CFTR increases by at least 25%.    
     
     
         22 . The method of  claim 21  wherein the open probability of the channel formed by the CFTR increases by at least 50%.  
     
     
         23 . The method of  claim 21  wherein the open probability of the channel formed by the CFTR increases by at least 75%.  
     
     
         24 . The method of  claim 21  wherein the open probability of the channel formed by the CFTR increases by at least 100%.  
     
     
         25 . The method of  claim 21  wherein the open probability of the channel formed by the CFTR increases by at least 125%.  
     
     
         26 . The method of  claim 21  wherein the open probability of the channel formed by the CFTR increases by at least 150%.  
     
     
         27 . The method of  claim 21  wherein the open probability of the channel formed by the CFTR increases by at least 200%.  
     
     
         28 . The method of  claim 21  wherein the CFTR protein is a mutant which reaches a cell's plasma membrane but fails to undergo full activation.  
     
     
         29 . The method of  claim 28  wherein the CFTR protein is listed at http://www.genet.sickkids.on.ca/cftr-cgi-bin/fulltable.  
     
     
         30 . The method of  claim 21  wherein the step of applying is performed by administering an aerosolized polypeptide to a patient with a mutant CFTR protein.  
     
     
         31 . The method of  claim 21  wherein the CFTR protein is in a lipid bilayer and a change in conductance is measured upon applying the polypeptide.  
     
     
         32 . The method of  claim 21  wherein the step of applying the polypeptide is accomplished by administering a nucleic acid encoding the polypeptide to a patient who expresses the CFTR protein, whereby the polypeptide is expressed.  
     
     
         33 . The method of  claim 32  wherein the nucleic acid is administered as an aerosol to the patient's airways.  
     
     
         34 . The method of  claim 8  or  21  wherein the polypeptide is free of phosphorylation.