Human neuronal nicotinic acetylcholine receptor compositions and methods employing same

Abstract

DNA encoding human neuronal nicotinic acetylcholine receptor alpha and beta subunits, mammalian and amphibian cells containing said DNA, methods for producing alpha and beta subunits and recombinant (i.e., isolated or substantially pure) alpha subunits (specifically α 6 ) and beta subunits (specifically β 3 ) are provided. In addition, combinations of a plurality of subunits (i.e., one or more of α 1 , α 2 , α 3 , α 4 , α 5 , α 6 and/or α 7 subunits in combination with one or more of β 3 subunits are provided.

1 . Isolated DNA comprising a sequence of nucleotides encoding an α 6  subunit of a human neuronal nicotinic acetylcholine receptor.  
     
     
         2 . DNA according to  claim 1  wherein 
 said DNA encodes the amino acid sequence set forth in SEQ ID NO:1, or    said DNA encodes the amino acid sequence encoded by clone pcDNA3-KEalpha6.3.    
     
     
         3 . DNA according to  claim 1  wherein 
 said DNA hybridizes to substantially the entire coding sequence (nucleotides 143-1624) set forth in SEQ ID NO:1 under high stringency conditions, or    said DNA hybridizes under high stringency conditions to substantially the entire sequence of the α6-encoding insert of clone pcDNA3-KEalpha6.3.    
     
     
         4 . DNA according to  claim 1  wherein 
 said DNA has substantially the same nucleotide sequence as nucleotides 143-1624 set forth in SEQ ID NO:1, or    said DNA has substantially the same nucleotide sequence as the α6-encoding insert of clone pcDNA3-KEalpha6.3.    
     
     
         5 . Isolated DNA comprising nucleotides encoding a substantially full length β3 subunit of a human neuronal nicotinic acetylcholine receptor.  
     
     
         6 . DNA according to  claim 5  wherein the nucleotides of said DNA encode the amino acid sequence set forth in SEQ ID NO:4.  
     
     
         7 . DNA according to  claim 5  wherein the nucleotides of said DNA hybridize under high stringency conditions to substantially the entire coding sequence (nucleotides 98-1472) set forth in SEQ ID NO:3.  
     
     
         8 . DNA according to  claim 5  wherein the nucleotides of said DNA have substantially the same nucleotide sequence as nucleotides 98-1472 set forth in SEQ ID NO:3.  
     
     
         9 . Cells containing at least one DNA according to  claim 1 , wherein said cells are bacterial cells, eukaryotic cells or amphibian oöcytes.  
     
     
         10 . Cells according to  claim 9 , further containing at least one DNA encoding a β subunit of human neuronal nicotinic acetylcholine receptor.  
     
     
         11 . Cells according to  claim 10  wherein said β subunit is selected from β 2 , β 3  or β 4 .  
     
     
         12 . Cells according to  claim 10  wherein said P subunit is β 3 .  
     
     
         13 . Cells according to  claim 9  wherein said cells express functional neuronal nicotinic acetylcholine receptors that contain one or more subunits encoded by said DNA.  
     
     
         14 . Cells containing at least one DNA according to  claim 5 , wherein said cells are bacterial cells, eukaryotic cells or amphibian oöcytes.  
     
     
         15 . Cells according to  claim 14 , further containing at least one DNA encoding an α subunit of human neuronal nicotinic acetylcholine receptor.  
     
     
         16 . Cells according to  claim 15  wherein said α subunit is selected from α 2 , α 3 , α 4 , α 5 , α 6 , or α 7 .  
     
     
         17 . Cells according to  claim 20  wherein said α subunit is α 6 .  
     
     
         18 . Cells according to  claim 14  wherein said cells express functional neuronal nicotinic acetylcholine receptors that contain one or more subunits encoded by said DNA.  
     
     
         19 . A method of screening compounds to identify compounds which modulate the activity of human neuronal nicotinic acetylcholine receptors, said method comprising determining the effect of a compound on the neuronal nicotinic acetylcholine receptor activity in test cells according to  claim 10 , compared to the effect on control cells or to the neuronal nicotinic acetylcholine receptor activity of the cells in the absence of the compound, 
 wherein control cells are substantially identical to the test cells, but control cells do not express nicotinic acetylcholine receptors.    
     
     
         20 . A method of screening compounds to identify compounds which modulate the activity of human neuronal nicotinic acetylcholine receptors, said method comprising determining the effect of a compound on the neuronal nicotinic acetylcholine receptor activity in test cells according to  claim 15 , compared to the effect on control cells or to the neuronal nicotinic acetylcholine receptor activity of the cells in the absence of the compound, 
 wherein control cells are substantially identical to the test cells, but control cells do not express nicotinic acetylcholine receptors.    
     
     
         21 . Recombinant human neuronal nicotinic acetylcholine receptor α 6  subunit.  
     
     
         22 . A recombinant human neuronal nicotinic acetylcholine receptor comprising one or more of the subunits of  claim 21 .  
     
     
         23 . Human neuronal nicotinic acetylcholine receptor according to  claim 22 , further comprising at least one human neuronal nicotinic acetylcholine receptor beta subunit.  
     
     
         24 . Recombinant human neuronal nicotinic acetylcholine receptor β 3  subunit.  
     
     
         25 . A recombinant human neuronal nicotinic acetylcholine receptor comprising the subunit of  claim 24 .  
     
     
         26 . Human neuronal nicotinic acetylcholine receptor according to  claim 25 , further comprising at least one human neuronal nicotinic acetylcholine receptor alpha subunit.  
     
     
         27 . A method for identifying functional neuronal nicotinic acetylcholine receptor subunits and combinations thereof, said method comprising: 
 (a) introducing at least one DNA according to  claim 1 , or RNA complementary thereto, and optionally DNA encoding at least one beta subunit of a human neuronal nicotinic acetylcholine receptor, or RNA complementary thereto, into eukaryotic cells; and    (b) assaying for neuronal nicotinic acetylcholine receptor activity in cells of step (a), wherein the activity is mediated by a receptor containing one or more of the subunits encoded by said introduced DNA.    
     
     
         28 . A method for identifying functional neuronal nicotinic acetylcholine receptor subunits and combinations thereof, said method comprising: 
 (a) introducing at least one DNA according to  claim 5 , or RNA complementary thereto, and DNA encoding at least one alpha subunit of a neuronal nicotinic acetylcholine receptor, or RNA complementary thereto, into eukaryotic cells; and    (b) assaying for neuronal nicotinic acetylcholine receptor activity in cells of step (a), wherein the activity is mediated by a receptor containing one or more of the subunits encoded by said introduced DNA.    
     
     
         29 . Isolated mRNA encoded by the DNA of  claim 1 .  
     
     
         30 . Isolated mRNA encoded by the DNA of  claim 5 .  
     
     
         31 . Cells containing mRNA according to  claim 29 .  
     
     
         32 . Cells containing mRNA according to  claim 30 .  
     
     
         33 . Cells according to  claim 31  wherein said cells further contain mRNA encoding a beta subunit of a human neuronal nicotinic acetylcholine receptor.  
     
     
         34 . Cells according to  claim 32  wherein said cells further contain mRNA encoding an alpha subunit of a human neuronal nicotinic acetylcholine receptor.