Ion channel

Abstract

The present invention relates to a novel 1,957 amino acid tetrodotoxin-insensitive voltage-gated sodium channel specifically located in mammalian sensory neurons. Nucleic acid sequences coding for the novel sodium channel, vectors, host cells and methods of identifying modulators of the novel sodium channel for use in treatment of pain are also provided.

1 . An antibody which binds specifically to a voltage gated TTX resistant sodium channel, 
 wherein said channel is encoded by an nucleic acid that specifically hybridizes to the complement of SEQ ID NO: 1 under conditions of 50-60° C., 5×SSC for 30 minutes.    
     
     
         2 . The antibody of  claim 1  wherein said channel is encoded by a nucleic acid that specifically hybridises to the complement of SEQ ID NO:1 under conditions of 0.2×SSC, 0.5% SDS at 68° C. for 20 minutes.  
     
     
         3 . The antibody of  claim 2  wherein the channel has the sequence of SEQ ID NO: 2.  
     
     
         4 . The antibody of  claim 3  which binds specifically to an extracellular loop of the sodium channel of SEQ ID NO: 2.  
     
     
         5 . The antibody of  claim 3  which binds specifically to a peptide selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20.  
     
     
         6 . The antibody of  claim 1  which is a polyclonal antibody.  
     
     
         7 . The antibody of  claim 1  which is a monoclonal antibody.  
     
     
         8 . The antibody of  claim 7 , wherein said antibody selected from the group consisting of chimeric, single chain and humanised antibodies.  
     
     
         9 . The antibody of  claim 7 , which is a human monoclonal antibody.  
     
     
         10 . The antibody of  claim 1  which is an intact antibody molecule.  
     
     
         11 . The antibody of  claim 1 , which is a fragment of an intact antibody molecule, wherein said fragment comprises the antibody binding region.  
     
     
         12 . The antibody of  claim 11  which is a Fab or F(ab) 2  fragment.  
     
     
         13 . A method of producing an antibody comprising; 
 administering a voltage gated TTX resistant sodium channel encoded by an nucleic acid that specifically hybridizes to the complement of SEQ ID NO: 1 under conditions of 50-60° C., 5×SSC for 30 minutes, or a fragment thereof to a non-human animal, and 
 obtaining an antibody from said animal.  
   
     
     
         14 . The method of  claim 13  wherein said fragment comprises an extracellular loop of said polypeptide.  
     
     
         15 . The method of  claim 13  wherein the fragment is contained in a fusion protein with glutathione-S-transferase.  
     
     
         16 . The method of  claim 13  wherein said fragment is a peptide selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20.  
     
     
         17 . The method of  claim 13  comprising determining the binding of the antibody to the channel  
     
     
         18 . The method of  claim 13  comprising determining the binding of the antibody to sensory neurons.  
     
     
         19 . A method of preparing a monoclonal antibody which binds specifically to a voltage gated TTX resistant sodium channel encoded by an nucleic acid that specifically hybridizes to the complement of SEQ ID NO: 1 under conditions of 50-60° C., 5×SSC for 30 minutes, the method comprising; 
 culturing a cell line which produces said antibody.    
     
     
         20 . The method of  claim 19  wherein the cell line is selected from the group consisting of hybridoma, trioma, human B cell hybridoma and EBV hybridoma.  
     
     
         21 . A method of binding an antibody comprising; 
 contacting a voltage gated TTX resistant sodium channel encoded by an nucleic acid that specifically hybridizes to the complement of SEQ ID NO: 1 under conditions of 50-60° C., 5×SSC for 30 minutes with an antibody which binds specifically to said channel,    whereby said antibody binds to said channel.    
     
     
         22 . The method of  claim 21  wherein the channel is in a tissue which expresses said channel.  
     
     
         23 . The method of  claim 21  further comprising detecting the binding of the antibody to the channel.  
     
     
         24 . A method of identifying an antibody which modulates a mammalian sensory neuron sodium channel, comprising; 
 contacting an antibody with a host cell comprising a voltage gated TTX resistant sodium channel encoded by an nucleic acid that specifically hybridizes to the complement of SEQ ID NO: 1 under conditions of 50-60° C., 5×SSC for 30 minutes, and    detecting the binding of said antibody to the channel,    binding of said antibody to the channel being indicative that the antibody modulates the mammalian sensory neuron sodium channel.    
     
     
         25 . A method of identifying an antibody which modulates a mammalian sensory neuron sodium channel, comprising; 
 contacting an antibody with a host cell comprising a voltage gated TTX resistant sodium channel encoded by an nucleic acid that specifically hybridizes to the complement of SEQ ID NO: 1 under conditions of 50-60° C., 5×SSC for 30 minutes, and    detecting the activity of said channel,    a change in activity in the presence of the antibody being indicative that the antibody modulates the mammalian sensory neuron sodium channel.    
     
     
         26 . The method of  claim 25  wherein said antibody is identified as a blocker of the activity of said channel.  
     
     
         27 . The method of  claim 25  wherein said host cell and said test compound are contacted under depolarising conditions.  
     
     
         28 . The method of  claim 25  wherein changes in the activity of the sodium channel are detected by determining the ion flux through said channel.  
     
     
         29 . The method of  claim 28  wherein changes in the activity of the sodium channel are detected by determining the sodium ion flux through said channel.  
     
     
         30 . The method of  claim 29  wherein the sodium ions are radiolabelled.  
     
     
         31 . The method of  claim 27  wherein changes in the activity of the sodium channel are detected by determining the flux of guanidine through said channel.  
     
     
         32 . The method of  claim 25  comprising detecting changes in the activity of the sodium channel using electrophysiology.  
     
     
         33 . The method of  claim 32  comprising detecting changes in the activity of the sodium channel using a patch clamp.  
     
     
         34 . A method of treating pain comprising administering to an individual in need thereof an antibody which binds specifically to a sensory neuron specific sodium channel encoded by an nucleic acid that specifically hybridizes to the complement of SEQ ID NO: 1 under conditions of 50-60° C., 5×SSC for 30 minutes.  
     
     
         35 . A method according to  claim 34  wherein the channel has the sequence of SEQ ID NO: 2.