Use of Trpc Channel for the Treatment of a Cardiovascular Disease

Abstract

The invention refers to the use of a TRPC channel, an inactivating mutant thereof, or a nucleotide sequence coding for the TRPC channel or for the inactivating mutant for the production of a medicament for the treatment of a cardiovascular disease and a method of screening a modulator of the TRPC channel or an inactivating mutant thereof.

1 . A method for improving the vascular function of a mammal comprising the step of administering to said mammal an pharmaceutically effective amount of a TRPC channel protein or an inactivating mutant of said TRPC channel protein. 
     
     
         2 . The method according to  claim 1 , wherein the TRPC channel protein is selected from the group consisting of TRPC3 channel, TRPC6 channel and TRPC7 channel. 
     
     
         3 . The method according to  claim 2 , wherein the amino acid sequence of TRPC3 consists of the amino acid sequence of SEQ ID NO: 2, the amino acid sequence of TRPC6 consists of the amino acid sequence of SEQ ID NO: 5, and the amino acid sequence of TRPC7 consists of the amino acid sequence of SEQ ID NO: 9. 
     
     
         4 . The method according to  claim 2 , wherein the inactivating mutant of said TRPC channel protein is selected from the group consisting of TRPC3 DN  with the amino acid sequence of SEQ ID NO: 3, TRPC6 DN  with the amino acid sequence of SEQ ID NO: 7 and TRPC7 DN  with the amino acid sequence of SEQ ID NO: 11. 
     
     
         5 . The method according to  claim 34 , wherein nucleotide sequence coding for TRPC3 is the nucleotide sequence of SEQ ID NO: 2, the nucleotide sequence of TRPC6 is the nucleotide sequence of SEQ ID NO: 6, and the nucleotide sequence of TRPC7 is the nucleotide sequence of SEQ ID NO: 10. 
     
     
         6 . The method according to  claim 34 , wherein the nucleotide sequence coding for TRPC3 DN  is the nucleotide sequence of SEQ ID NO: 4, the nucleotide sequence coding for TRPC6 DN  is the nucleotide sequence of SEQ ID NO: 8, and the nucleotide sequence coding for TRPC7 DN  is the nucleotide sequence of SEQ ID NO: 12. 
     
     
         7 - 13 . (canceled) 
     
     
         14 . The method according to  claim 1  wherein the cardiovascular disease is atherosclerosis. 
     
     
         15 . A method of screening a modulator of a TRPC channel protein or an inactivating mutant of a TRPC channel protein, or a modulator of expression of a nucleotide sequence coding for a TRPC channel protein or the inactivating mutant of a TRCP channel protein, wherein the method comprises the steps of:
 (a) contacting a cell expressing a TRPC or an inactivating mutant,   (b) stimulating Ca 2+  influx by a channel activator before, simultaneously or after contacting the cell with a test compound, and   (c) measuring or detecting a change of the TRPC channel activity.   
     
     
         16 . The method of  claim 15  further comprising the additional step of selecting a test compound with an activity against a cardiovascular disease by comparing the changes of the TRPC channel activity in the absence of the test compound. 
     
     
         17 . The method according to  claim 15  wherein the expression of the TRPC channel or inactivating mutant in the cell is controlled is an inducible promoter. 
     
     
         18 . The method according to  claim 17 , wherein the inducible promoter is selected from a tetracycline-inducible promoter. 
     
     
         19 . The method according to  claim 15  wherein the cell is a fluorescent cell. 
     
     
         20 . The method according to  claim 15  wherein the cell is selected from a MDCK, HEK 293, HEK 293 T, BHK, COS, NIH3T3, Swiss3T3 or CHO cell. 
     
     
         21 . The method according to  claim 15  wherein the TRPC channel activity is measured or detected by measuring or detecting a change in ion fluxes, in particular Ca 2+  fluxes, by preferably patch clamp techniques, whole cell currents radiolabeled ion fluxes, or in particular fluorescence preferably using voltage-sensitive dyes or ion-sensitive dyes. 
     
     
         22 . The method according to  claim 15  wherein the channel activator is a compound selected from diacylglycerols, in particular 1-Oleyl-2acetyl-sn-glycerol (OAG), G q -coupled receptor agonists, especially phenylephrine and in particular trypsin, an agonist that stimulates receptor tyrosine kinases, especially epidermal growth factor (EGF), or diacylglycerol generating enzymes, especially phospholipases or activators thereof. 
     
     
         23 . The method according to  claim 15  wherein the TRPC is selected from TRPC3, TRPC6, TRPC7, or an inactivating mutant thereof. 
     
     
         24 . The method according to  claim 23 , wherein the inactivating mutant is selected from the group consisting of TRPC3 DN  with the amino acid sequence of SEQ ID NO: 3, TRPC6 DN  with the amino acid sequence of SEQ ID NO: 7, and TRPC7 DN  with the amino acid sequence of SEQ ID NO: 11. 
     
     
         25 . The method according to  claim 15  wherein the test compound is provided in the form of a chemical compound library. 
     
     
         26 . The method according to  claim 15  wherein the method is carried out on an array. 
     
     
         27 . The method according to  claim 26  wherein the method is carried out in a robotics system. 
     
     
         28 . The method according to  claim 27  wherein the method is a method of high-through put screening of the test compound. 
     
     
         29 . The method according to  claim 15  wherein the test compound detected is an inhibitor. 
     
     
         30 . The method according to  claim 15  wherein the cells are seeded onto a well of a multi-well test plate. 
     
     
         31 . A method for producing a medicament for the treatment of a cardiovascular disease, wherein the method comprises the steps of:
 a. carrying out the method according to  claim 15 ,   b. isolating a detected test compound suitable for the treatment of a cardiovascular disease, and   c. formulating the detected test compound with one or more pharmaceutically acceptable carriers or auxiliary substances.   
     
     
         32 . The method according to  claim 15  wherein the cardiovascular disease is atherosclerosis. 
     
     
         33 . A method for improving the vascular function of a mammal having a cardiovascular disease by genetically transferring to said mammal a nucleotide sequence coding for a TRPC channel protein or an inactivating mutant of said TRPC channel protein, whereby said nucleotide sequence is expressed in said mammal. 
     
     
         34 . The method use according to  claim 33  wherein the TRPC channel protein or inactivating mutant of said TRPC channel protein is selected from the group consisting of TRPC3 channel, TRPC6 channel and TRPC7 channel.