Ligand binding assays for vanilloid receptors

Abstract

The present invention describes an assay in which pH alters RTX affinity to VR1 receptors. The RTX affinity was increased with increasing pH from 4.1 to 8.6. Both the RTX affinity and apparent number of RTX binding sites were decreased with increasing pH from 8.6 to 10.6. The high pH may be causing the cell membranes to denature. These pH conditions (pH 8.0 to pH 9.0) provide a high signal to noise ratio, give a more robust assay and require use of less experimental materials.

1 . A method for measuring ligand binding to a vanilloid receptor comprising the steps of: 
 (a) forming in an aqueous solution having a pH in the range of about 7.5 to about 10.0 a liquid composition comprising a test compound, a labeled ligand, and at least a ligand-interacting portion of a vanilloid receptor protein;    (b) incubating the solution for a time sufficient to permit the test compound and labeled ligand to contact the vanilloid receptor;    (c) measuring the amount of labeled ligand bound to the protein; and    (d) determining if the test compound bound to the receptor by observing a reduction in the amount of expected labeled ligand.    
     
     
         2 . The method of  claim 2  wherein the ligand-interacting portion of a vanilloid receptor protein is an intact vanilloid receptor protein.  
     
     
         3 . The method of  claim 1  wherein the vanilloid receptor is a human vanilloid receptor.  
     
     
         4 . The method of  claim 1  wherein the pH is in the range of about 8.0 to about 9.5.  
     
     
         5 . The method of  claim 1  wherein the pH is in the range of about pH 8.1 to about 9.1.  
     
     
         6 . The method of  claim 1  wherein the labeled ligand is a radiolabeled ligand.  
     
     
         7 . The method of  claim 6  wherein the radiolabeled ligand is tritiated resiniferatoxin.  
     
     
         8 . The method of  claim 1  additionally comprising the steps after the incubating step of: 
 removing unbound labeled ligand from the solution; and    isolating the receptor protein;    
     
     
         9 . The method of  claim 1  wherein the aqueous buffer further comprises a divalent cation selected from the group consisting of: 
 (a) magnesium at a final concentration of between about 1 to about 5 mM; and    (b) calcium at a final concentration of about 0.1 mM to about 2 mM    
     
     
         10 . The method of  claim 9  wherein the magnesium concentration is about 2 mM.  
     
     
         11 . The method of  claim 9  wherein the calcium concentration is about 0.8 mM  
     
     
         12 . The method of  claim 9  wherein the vanilloid receptor is a human vanilloid receptor.  
     
     
         13 . The method of  claim 1  wherein the removing step comprises adding a sufficient quantity of alpha 1 acid glycoprotein to the aqueous solution to adsorb unbound labeled ligand.  
     
     
         14 . The method of  claim 1  wherein the steps are performed in order.  
     
     
         15 . The method of  claim 9  wherein the isolating step is performed before the removing step.  
     
     
         16 . A method to measure ligand binding to a vanilloid receptor comprising the steps, in order 
 (a) combining in an aqueous solution having a pH in the range of about 7.5 to about 10.0, a test compound, a labeled ligand, and vanilloid receptor protein, said protein being associated with a portion of a cell membrane;    (b) incubating the solution for sufficient time for the test compound and ligand to contact the vanilloid receptor;    (c) adding a sufficient quantity of alpha 1 acid glycoprotein to the solution to adsorb unbound labeled ligand;    (d) isolating the membrane from the aqueous solution;    (e) measuring the amount of labeled ligand bound to the protein in the membrane; and    (f) determining if the test compound bound to the receptor by observing a reduction in the amount of expected labeled ligand.    
     
     
         17 . A method to measure compound binding to a vanilloid receptor comprising the steps, in order 
 (a) combining in an aqueous solution having a pH of about 8.6, a test compound, a radiolabeled resiniferatoxin, and a human vanilloid receptor-1 (VR1) protein, said protein being a portion of a cell membrane;    (b) incubating the solution for sufficient time for the test compound and the resiniferatoxin to contact the vanilloid receptor;    (c) adding a sufficient quantity of alpha 1 acid glycoprotein to the solution to adsorb unbound resiniferatoxin;    (d) isolating the membrane from the aqueous solution;    (e) measuring the amount of resiniferatoxin bound to the protein in the membrane; and    (f) determining if the test compound bound to the receptor by observing a reduction in the amount of expected resiniferatoxin.    
     
     
         18 . The method of  claim 17  wherein the buffer also contains a divalent cation selected from the group consisting of: 
 (a) magnesium at a final concentration of about 2 mM; and    (b) calcium at a final concentration of about 0.8 mM.