Human calcium channel .alpha.-1C/.alpha.-1D, .alpha.-2, .beta.-1, and .gamma.subunits and cells expressing the DNA
AssigneeSibia Neurosciences, Inc.
InventorsMichael M. Harpold, Steven B. Ellis, Mark E. Williams, Daniel H. Feldman, Ann F. McCue, Robert Brenner
Life of this patent
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Abstract
Isolated DNA encoding each of human calcium channel .alpha..sub.1 -, .alpha..sub.2 -, .beta.- and .gamma.-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.
Claims64
An isolated DNA molecule, comprising a sequence of nucleotides that encodes an .alpha..sub.1 -subunit of a human calcium channel selected from the group consisting of:
- an .alpha..sub.1C subunit comprising the sequence of amino acids set forth in SEQ ID NO. 45;
- an .alpha..sub.1D subunit comprising the sequence of amino acids of the .alpha..sub.1 -subunit set forth in SEQ ID NO. 49;
- or comprising the sequence of amino acids of the .alpha..sub.1 -subunit set forth in SEQ ID NO. 151;
- and
- a human calcium channel .alpha..sub.1 -subunit that is encoded by DNA capable of hybridizing under conditions of high stringency with DNA that is fully complementary to an mRNA transcript native to a human cell that encodes one of the aforesaid human calcium channel .alpha..sub.1 -subunits, wherein:
- the .alpha..sub.1 -subunit is an .alpha..sub.1C - or .alpha..sub.1D -subunit;
- such that any probe that contains at least 14 contiguous bases from the coding portion of the .alpha..sub.1 -encoding DNA is capable of hybridizing under conditions of high stringency to the DNA that if fully complementary to the mRNA transcript.
An isolated eukaryotic cell with a functional, heterologous calcium channel, produced by a process comprising introducing into the cell at least one RNA transcript selected from the group consisting of a first RNA which is translatable in said cell into an .alpha..sub.1 -subunit of a human calcium channel, a second RNA which is translatable in said cell into a .beta.-subunit of a human calcium channel, a third RNA which is translatable in said cell into an .alpha..sub.2 -subunit of a human calcium channel, and a fourth RNA which is translatable in said cell into a .gamma.-subunit of a human calcium channel, wherein the cell is a mammalian cell, yeast cell, or amphibian oocyte, and wherein:
- the .alpha..sub.1 -subunit is selected from the group consisting of:
- an .alpha..sub.1C -subunit comprising the sequence of amino acids set forth in SEQ ID NO:
- 45,
- an .alpha..sub.1D -subunit comprising the sequence of amino acids set forth in SEQ ID NO:
- 49 or SEQ ID NO:
- 51,
- an .alpha..sub.1D -subunit having the sequence of an .alpha..sub.1D -subunit native to a human cell that comprises the sequence of amino acids set forth in SEQ ID NO:
- 50, and
- a human calcium channel .alpha..sub.1 -subunit, wherein the .alpha..sub.1 -subunit is an .alpha..sub.1C - or .alpha..sub.1D -subunit encoded by DNA capable of hybridizing under conditions of high stringency with DNA that is fully complementary to an mRNA transcript native to a human cell that encodes one of the aforesaid human calcium channel .alpha..sub.1 -subunits, such that any probe that contains at least 14 contiguous bases from the coding portion of the DNA that encodes the .alpha..sub.1 -subunit hybridizes under conditions of high stringency to the DNA that is fully complementary to the mRNA transcript that encodes an .alpha..sub.1 -subunit;
- the .alpha..sub.2 -subunit is selected from the group consisting of:
- an .alpha..sub.2 -subunit comprising the sequence set forth in SEQ ID NO:
- 53,
- an .alpha..sub.2 -subunit comprising the sequence set forth in SEQ ID NO:
- 54,
- an .alpha..sub.2 -subunit comprising the sequence set forth in SEQ ID NO:
- 55,
- an .alpha..sub.2 -subunit comprising the sequence set forth in SEQ ID NO:
- 56, and
- an .alpha..sub.2 -subunit comprising the amino acid sequence of an .alpha..sub.2a, .alpha..sub.2c, .alpha..sub.2d, or .alpha..sub.2e subunit polypeptide, wherein DNA encoding the .alpha.2-subunit is capable of hybridizing under conditions of high stringency with DNA that is fully complementary to an mRNA transcript native to a human cell that encodes the amino acid sequence set forth in SEQ ID NO:
- 53, 54, 55, or 56, such that any probe that contains at least 14 contiguous bases from the coding portion of the DNA that encodes the .alpha.2-subunit hybridizes under conditions of high stringency to the DNA that is fully complementary to the mRNA transcript that encodes an .alpha..sub.2 -subunit;
- the .beta.-subunit is selected from the group consisting of:
- a .beta.-subunit comprising the sequence of amino acids set forth in SEQ ID NO:
- 40,
- a .beta.-subunit comprising the sequence of amino acids set forth in SEQ ID NO:
- 41,
- a .beta.-subunit comprising the sequence of amino acids set forth in SEQ ID NO:
- 42,
- a .beta.-subunit comprising the sequence of amino acids set forth in SEQ ID NO:
- 43,
- a .beta.-subunit comprising the sequence of amino acids set forth in SEQ ID NO:
- 44, and
- a human calcium channel .beta.-subunit encoded by DNA capable of hybridizing under conditions of high stringency with DNA that is fully complementary to an mRNA transcript native to a human cell that encodes one of the aforesaid .beta. subunits, such that any probe that contains at least 14 contiguous bases from the coding portion of the DNA that encodes the .beta.-subunit hybridizes under conditions of high stringency to the DNA that is fully complementary to the mRNA transcript that encodes a .beta.-subunit;
- the .gamma.-subunit is selected from the group consisting of:
- a .gamma.-subunit native to a human cell which comprises the sequence of amino acids set forth in SEQ ID NO:
- 57, and
- a human calcium channel .gamma.-subunit encoded by DNA capale of hybridizing under conditions of high stringency with DNA that is fully complementary to an mRNA transcript native to a human cell that comprises the sequence of nucleotides set forth in SEQ ID NO:
- 14, wherein T residues are replaced by U, such that any probe that contains at least 14 contiguous bases from the coding portion of the DNA that encodes the .gamma.-subunit hybridizes under conditions of high stringency to the DNA that is fully complementary to the mRNA transcript that encodes a .gamma.-subunit;
- and
- the only heterologous ion channels that are expressed by the cell are calcium channels.
An isolated eukaryotic cell with a functional heterologous calcium channel, produced by a process comprising introducing into the cell at least one RNA transcript that is translatable in the cell into an .alpha..sub.2 -subunit of a human calcium channel, and optionally introducing into the cell one or more RNA transcripts selected from the group consisting of an RNA that is translatable in the cell into an .alpha..sub.1 -subunit of a human calcium channel, an RNA that is translatable in the cell into a .beta.-subunit of a human calcium channel, and an RNA that is translatable in the cell into a .gamma.-subunit of a human calcium channel, wherein:
- the cell is a mammalian cell, yeast cell, or amphibian oocyte;
- the only heterologous ion channels that are expressed by the cells are calcium channels;
- and
- the .alpha..sub.2 subunit is a human calcium channel .alpha..sub.2b -subunit comprising the amino acid sequence set forth in SEQ ID NO:
- 52 or a human calcium channel .alpha..sub.2b subunit encoded by DNA capable of hybridizing under conditions of high stringency with DNA that is fully complementary to an mRNA transcript native to a human cell that encodes the aforesaid human calcium channel .alpha..sub.2b subunit such that any probe that contains at least 14 contiguous bases from the coding portion of the DNA that encodes the .alpha..sub.2b -subunit hybridizes under conditions of high stringency to the DNA that is fully complementary to the mRNA transcript.
An isolated DNA molecule, comprising a sequence of nucleotides that encodes an .alpha..sub.1 -subunit of a human calcium channel native to a human cell selected from the group consisting of:
- an .alpha..sub.1C subunit comprising the sequence of nucleic acids set forth in SEQ ID NO:
- 45 or 46,
- an .alpha..sub.1D subunit comprising the sequence of amino acids set forth in SEQ ID NO:
- 49, 50, or 51, and
- a human calcium channel .alpha..sub.1 -subunit that is encoded by DNA capable of hybridizing under conditions of high stringency with DNA that is fully complementary to an mRNA transcript native to a human cell that encodes one of the aforesaid human calcium channel .alpha..sub.1 -subunits;
- wherein the .alpha..sub.1 -subunit binds to at least one compound selected from the group consisting of a dihydropyridine, a phenylalkylamine, .omega.-CgTx, and a pyrazonoylguanidine.
An isolated DNA molecule, comprising a sequence of nucleotides that encodes an .alpha..sub.1 -subunit of a human calcium channel selected from the group consisting of:
- an .alpha..sub.1C subunit comprising the sequence of nucleic acids set forth in SEQ ID NO:
- 45 or 46,
- an .alpha..sub.1D subunit comprising the sequence of amino acids set forth in SEQ ID NO:
- 49, 50, or 51, and
- a human calcium channel .alpha..sub.1 -subunit that is encoded by DNA capable of hybridizing under conditions of high stringency with DNA that is fully complementary to an mRNA transcript native to a human cell that encodes one of the aforesaid human calcium channel .alpha..sub.1 -subunits;
- wherein the .alpha..sub.1 -subunit is a naturally occurring .alpha..sub.1C or .alpha..sub.1D -subunit.
An isolated DNA molecule, comprising a sequence of nucleotides encoding the sequence of amino acids set forth in SEQ ID NO:
- 45.
An isolated DNA molecule, comprising a sequence of nucleotides encoding a human calcium channel .alpha..sub.1C subunit native to a human cell that comprises the sequence of amino acids set forth in SEQ ID NO:
- 46.
An isolated DNA probe, comprising the sequence of nucleotides set forth in SEQ ID NO:
- 2.
An isolated DNA probe, comprising a sequence of nucleotides that encodes the sequence of amino acids set forth in SEQ ID NO:
- 50.
An isolated DNA molecule, comprising a sequence of nucleotides encoding a human calcium channel .alpha..sub.1D subunit native to a human cell that comprises the sequence of amino acids set forth in SEQ ID NO:
- 50.
An isolated DNA molecule, comprising a sequence of nucleotides encoding the sequence of amino acids set forth in SEQ ID NO:
- 49.
An isolated DNA molecule, comprising a sequence of nucleotides encoding the sequence of amino acids set forth in SEQ ID NO:
- 51.
An isolated DNA molecule, comprising a sequence of nucleotides that encodes an .alpha..sub.2 -subunit of a human calcium channel selected from the group consisting of:
- an .alpha..sub.2 -subunit comprising the sequence set forth in SEQ ID NO:
- 53,
- an .alpha..sub.2 -subunit comprising the sequence set forth in SEQ ID NO:
- 54,
- an .alpha..sub.2 -subunit comprising the sequence set forth in SEQ ID NO:
- 55,
- an .alpha..sub.2 -subunit comprising the sequence set forth in SEQ ID NO:
- 56, and
- an .alpha..sub.2 -subunit comprising the amino acid sequence of an .alpha..sub.2a, .alpha..sub.2c, .alpha..sub.2d, or .alpha..sub.2e subunit polypeptide, wherein the DNA molecule is capable of hybridizing under conditions of high stringency with DNA that is fully complementary to an mRNA transcript native to a human cell that encodes the amino acid sequence set forth in SEQ ID NO:
- 53, 54, 55, or 56, such that any probe that contains at least 14 contiguous bases from the coding portion of the DNA molecule hybridizes under conditions of high stringency to the DNA that is fully complementary to the mRNA transcript.
An isolated DNA molecule, comprising a sequence of nucleotides that encodes an .alpha..sub.2b -subunit of a human calcium channel comprising the amino acid sequence set forth in SEQ ID NO:
- 52 or is a human calcium channel .alpha..sub.2b subunit encoded by DNA capable of hybridizing under conditions of high stringency with DNA that is fully complementary to an mRNA transcript native to a human cell that encodes the aforesaid human calcium channel .alpha..sub.2b subunit such that any probe that contains at least 14 contiguous bases from the coding portion of the DNA that encodes the .alpha..sub.2b -subunit hybridizes under conditions of high stringency to the DNA that is fully complementary to the mRNA transcript.
An isolated DNA molecule, comprising a sequence of nucleotides that encodes:
- an .alpha..sub.2 -subunit of a human calcium channel comprising the amino acid sequence set forth in SEQ ID NO:
- 53, 54, 55, 56, or
- a naturally occurring human calcium channel .alpha..sub.2 subunit, wherein the .alpha..sub.2 -subunit is an .alpha..sub.2a, .alpha..sub.2c, .alpha..sub.2d, or .alpha..sub.2e subtype and is encoded by DNA capable of hybridizing under conditions of high stringency with DNA that is fully complementary to an mRNA transcript native to a human cell that encodes one of the aforesaid human calcium channel .alpha..sub.2 -subunits.
An isolated DNA molecule, comprising the sequence of nucleotides set forth in SEQ ID No. 11.
An isolated DNA molecule, comprising the sequence of nucleotides set forth in SEQ ID Nos. 53, 54, 55 or 56.
An isolated DNA molecule, comprising a sequence of nucleotides that encodes a .beta.-subunit of a human calcium channel selected from the group consisting of:
- a .beta.-subunit comprising the sequence of amino acids set forth in SEQ ID NO:
- 40,
- a .beta.-subunit comprising the sequence of amino acids set forth in SEQ ID NO:
- 41,
- a .beta.-subunit comprising the sequence of amino acids set forth in SEQ ID NO:
- 42,
- a .beta.-subunit comprising the sequence of amino acids set forth in SEQ ID NO:
- 43,
- a .beta.-subunit comprising the sequence of amino acids set forth in SEQ ID NO:
- 44, and
- a human calcium channel .beta.-subunit that is encoded by DNA capable of hybridizing under conditions of high stringency with DNA that is fully complementary to an mRNA transcript native to a human cell that encodes one of the aforesaid .beta. subunits, such that any probe containing 14 contiguous bases from the coding portion of the DNA molecule hybridizes under conditions of high stringency to the DNA that is fully complementary to the mRNA transcript.
An isolated DNA molecule, comprising a sequence of nucleotides that encodes a naturally occurring .beta.-subunit of a human calcium channel selected from the group consisting of:
- a .beta.-subunit comprising the sequence of amino acids set forth in SEQ ID NO:
- 40,
- a .beta.-subunit comprising the sequence of amino acids set forth in SEQ ID NO:
- 41,
- a .beta.-subunit comprising the sequence of amino acids set forth in SEQ ID NO:
- 42,
- a .beta.-subunit comprising the sequence of amino acids set forth in SEQ ID NO:
- 43,
- a .beta.-subunit comprising the sequence of amino acids set forth in SEQ ID NO:
- 44, and
- a human calcium channel .beta.-subunit that is encoded by DNA capable of hybridizing under conditions of high stringency with DNA that is fully complementary to an mRNA transcript native to a human cell that encodes one of the aforesaid .beta. subunits.
An isolated DNA molecule, comprising a sequence of nucleotides that encodes the sequence of amino acids set forth in SEQ ID NO:
- 40.
An isolated DNA molecule, comprising a sequence of nucleotides that encodes the sequence of amino acids set forth in SEQ ID NO:
- 41.
An isolated DNA molecule, comprising a sequence of nucleotides that encodes the sequence of amino acids set forth in SEQ ID NO. 42.
An isolated DNA molecule, comprising a sequence of nucleotides that encodes the sequence of amino acids set forth in SEQ ID NO:
- 44.
An isolated DNA molecule, comprising the sequence of nucleotides set forth in SEQ ID Nos:
- 9, 10, 37, 38 or 39.
Dependencies are inferred from the claim text.