Human calcium channel compositions and methods

Abstract

Isolated DNA encoding each of human calcium channel α 1 -, α 2 -, β- and γ-subunits, including subunits that arise as splice variants of primary transcripts, is provided. Cells and vectors containing the DNA and methods for identifying compounds that modulate the activity of human calcium channels are also provided.

What is claimed is:  
     
         1 . An isolated nucleic acid fragment that encodes a subunit of a human calcium channel, comprising a sequence of nucleotides that encodes the subunit, wherein the sequence of nucleotides encoding the subunit is selected from among: 
 (a) a sequence of nucleotides that encodes a human calcium channel subunit and includes the sequence of nucleotides set forth in any of SEQ ID Nos. 1-38;    (b) a sequence of nucleotides that encodes the subunit and hybridizes under conditions of high stringency to DNA that is complementary to an mRNA transcript present in a human cell that encodes a subunit that includes a sequence of nucleotides set forth in any of SEQ ID Nos. 1-38;    (c) a sequence of nucleotides that encodes the subunit that includes a sequence of amino acids encoded by any of SEQ ID Nos. 1-38; and    (d) a sequence of nucleotides that encodes a subunit that includes a sequence of amino acids encoded by a sequence of nucleotides that encodes such subunit and hybridizes under conditions of high stringency to DNA that is complementary to an mRNA transcript present in a human cell that encodes the subunit that includes the sequence of nucleotides set forth in any of SEQ ID Nos. 1-38.    
     
     
         2 . An isolated fragment of  claim 1 , comprising a sequence of nucleotides that encodes an α 1 -subunit of a human calcium channel.  
     
     
         3 . The fragment of  claim 1 , wherein the α 1 -subunit is a human neural calcium channel α 1  subunit.  
     
     
         4 . The fragment of  claim 1 , wherein the α 1  subunit is an α 1D -subunit, α 1C -subunit, α 1B -subunit, α 1E -subunit or an α 1A -subunit.  
     
     
         5 . The fragment of  claim 2 , comprising a sequence of nucleotides that encodes an α 1  subunit selected from the group consisting of α 1A-1 , α 1A-2 , α 1E-1 , α 1C-2  and α 1E-3 .  
     
     
         6 . An isolated fragment, comprising a sequence of nucleotides that encodes a β-subunit of a human calcium channel.  
     
     
         7 . The fragment of  claim 6 , comprising a sequence of nucleotides that encodes a β 2 -subunit.  
     
     
         8 . The DNA fragment of  claim 7 , wherein the subunit is a β 2C , β 2D  or β 2E  subunit.  
     
     
         9 . The fragment of  claim 6 , that encodes a β 3 -subunit.  
     
     
         10 . The fragment of  claim 7 , that encodes a β 4 -subunit.  
     
     
         11 . A eukaryotic cell, comprising heterologous DNA that encodes at an α 1 -subunit, wherein the α 1 -subunit is encoded by the DNA of  claim 2 .  
     
     
         12 . The cell of  claim 11 , further comprising heterologous DNA that encodes a β-subunit of a human calcium channel.  
     
     
         13 . The eukaryotic cell of  claim 12  that has a functional heterologous calcium channel that contains at least one subunit encoded by the heterologous DNA.  
     
     
         14 . The eukaryotic cell of  claim 11  selected from the group consisting of HEK 293 cells, Chinese hamster ovary cells, African green monkey cells, and mouse L cells.  
     
     
         15 . A eukaryotic cell with a functional, heterologous calcium channel, produced by a process comprising: 
 introducing into the cell heterologous nucleic acid that encodes at least one subunit of a calcium channel, wherein the subunit is encoded by the DNA of  claim 1 .    
     
     
         16 . The eukaryotic cell of  claim 15  that is an amphibian oöcyte.  
     
     
         17 . A method for identifying a compound that modulates the activity of a calcium channel, comprising; 
 suspending the eukaryotic cell of  claim 13  in a solution containing the compound and a calcium channel selective ion:    depolarizing the cell membrane of the cell; and    detecting the current flowing into the cell, wherein: 
 the heterologous calcium channel includes at least one human calcium channel subunit encoded by DNA or RNA that is heterologous to the cell,  
 the current that is detected is different from that produced by depolarizing the same or a substantially identical cell in the presence of the same calcium channel selective ion but in the absence of the compound.  
   
     
     
         18 . The method of  claim 17 , wherein prior to the depolarization step the cell is maintained at a holding potential which substantially inactivates calcium channels that are endogenous to the cell.  
     
     
         19 . The method of  claim 18 , wherein: 
 the cell is an amphibian oöcyte;    the heterologous subunits are encoded by RNA injected into the oöcyte; and    the heterologous subunits include an α 1 -subunit.    
     
     
         20 . The method of  claim 19 , wherein the subunits encoded by the RNA further comprise a β-subunit.  
     
     
         21 . The method of  claim 17 , wherein the cell is an HEK cell and the heterologous subunit is encoded by heterologous DNA.  
     
     
         22 . A substantially pure α 1 -subunit encoded by the DNA fragment of  claim 2 .  
     
     
         23 . A substantially pure β-subunit of a human calcium channel encoded by the DNA of  claim 6 .  
     
     
         24 . The DNA fragment of  claim 9 , wherein the subunit is a β 3−1  subunit.  
     
     
         25 . The eukaryotic cell of  claim 11 , comprising heterologous DNA that encodes an α 1  subunit selected from the group of subunits consisting of α 1A-1 , α 1A-2 , α 1C-2 , α 1E-1 , and α 1E-3 ; wherein: 
 the heterologous calcium channel contains at least one subunit encoded by the heterologous nucleic acid; and  
 the only heterologous ion channels are calcium channels.  
 
     
     
         26 . The eukaryotic cell of  claim 12 , wherein the β-subunit is a β 2C , β 2D , β 2E , β 3−1  or a β 4  subunit.  
     
     
         27 . The eukaryotic cell of  claim 12 , wherein the β subunit is a β 2  subunit.  
     
     
         28 . The eukaryotic cell of  claim 12 , wherein the β subunit is a β 4  subunit.  
     
     
         29 . The eukaryotic cell of  claim 15  with a functional, heterologous calcium channel, produced by a process comprising: 
 introducing into the cell nucleic acid that encodes an α 1  subunit of a human calcium channel and introducing into the cell nucleic acid that encodes a β 1  subunit of a human calcium channel, wherein: 
 at least one of the subunits is selected from the group consisting of α 1A-1 , α 1A-2 , α 1E-1 , α 1E-3 , β 2C , β 2D , β 2E , a β 3  and a β 4  subunit;  
 the heterologous calcium channel contains at least one subunit encoded by the heterologous nucleic acid; and  
 the only heterologous ion channels are calcium channels.  
 
 
     
     
         30 . The eukaryotic cell of  claim 29  selected from the group consisting of HEK 293 cells, Chinese hamster ovary cells, African green monkey cells, mouse L cells and amphibian oöcytes.  
     
     
         31 . The method of  claim 17 , wherein: 
 the heterologous calcium channel includes at least one human calcium channel subunit encoded by DNA or RNA that is heterologous to the cell;    at least one subunit is selected from the group consisting of α 1A-1 , α 1A-2 , α 1E-1 , α 1E-3 , α 1C-2 , β 2C , β 2D , β 2E , a β 3  subunit and a β 4  subunit;    the current that is detected is different from that produced by depolarizing the same or a substantially identical cell in the presence of the same calcium channel selective ion but in the absence of the compound.    
     
     
         32 . A subunit-specific antibody selected from the group consisting of antibodies that bind to an α subunit subtype or a β subunit subtype of a human calcium channel.  
     
     
         33 . The antibody of  claim 32 , wherein the antibody is subtype specific and the α 1  subunit is α 1A , α 1E  and α 1B .  
     
     
         34 . An RNA or single-stranded DNA probe of at least 16 bases in length, comprising at least 16 substantially contiguous nucleic acid bases from the sequence of nucleotides of  claim 1  that encodes an α 1  or β subunit of a human calcium channel.  
     
     
         35 . The RNA or single-stranded DNA probe of claim  54 , wherein the subunit is selected from the group of subunits consisting of α 1A-1 , α 1A-2 , α 1E-1 , α 1C-2 , α 1E-3 , α 1C-2 , β 2C , β 2D , β 2E , a β 3  and β 4 .  
     
     
         36 . The probe of  claim 34  that contains at least 30 nucleic acid bases that encode the subunit of a human calcium channel.  
     
     
         37 . A method for identifying nucleic acids that encode a human calcium channel subunit, comprising hybridizing under conditions of at least low stringency a probe of  claim 34  to a library of nucleic acid fragments, and selecting hybridizing fragments.  
     
     
         38 . A method for identifying cells or tissues that express a calcium channel subunit-encoding nucleic acid, comprising hybridizing under conditions of at least low stringency a probe of  claim 34  with mRNA expressed in the cells or tissues or cDNA produced from the mRNA, and thereby identifying cells or tissue that express mRNA that encodes the subunit.  
     
     
         39 . A substantially pure human calcium channel subunit selected from the group consisting of α 1A-1 , α 1A-2 , α 1E-1 , α 1C-2 , α 1E-3 , β 3−1 , β 2C , β 2D , β 2E  and β 4 .  
     
     
         40 . A method for producing a subunit of a human calcium channel, comprising introducing the fragment of  claim 1  into a prokaryotic or eukaryotic host cell, under conditions whereby the encoded subunit is expressed.