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US 5,846,757 AGranted
Human calcium channel .alpha..sub.1, .alpha..sub.2, and .beta. subunits and assays using them
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.
Claims42
A method for identifying a compound that modulates the activity of a calcium channel, comprising;
- suspending a eukaryotic cell which has a functional, heterologous calcium channel, in a solution containing a test compound and a calcium channel selective ion;
- depolarizing the cell membrane of said cell;
- detecting the current flowing into said cell;
- and
- comparing the current flowing into the cell with the current that flows into the same or a substantially identical cell suspended in the presence of the same calcium channel selective ion but in the absence of the compound, whereby test compounds that modulate the activity of a calcium channel are identified, wherein:
- the heterologous calcium channel includes one or more human calcium channel subunits expressed by nucleic acid that is heterologous to said cell;
- the only heterologous ion channels that are expressed by the cells are calcium channels;
- the current that is detected in the presence of a compound that modulates the activity of the heterologous calcium channel is different from that produced when the same or a substantially identical cell in the presence of the same calcium channel selective ion but in the absence of said compound is depolarized;
- the one or more subunits comprise .alpha..sub.1 -subunit selected from the group consisting of:
- an .alpha..sub.1C -subunit comprising the sequence of amino acids encoded by the sequence nucleotides set forth in SEQ ID NO. 3;
- an a.sub.1D subunit comprising the sequence of amino acids encoded by the sequence of nucleotides set forth in SEQ ID NO:
- 1 or SEQ ID NO. 29;
- a human calcium channel .alpha..sub.1 -subunit that is encoded by a DNA molecule 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 .alpha..sub.1 -subunit-encoding DNA molecule is capable of hybridizing under conditions of high stringency to the DNA that is fully complementary to the mRNA transcript;
- an .alpha..sub.1C -subunit encoded by an mRNA transcript native to a human cell, wherein DNA that is fully complementary to the transcript is capable of hybridizing under conditions of high stringency to a probe having the sequence of nucleotides set forth in SEQ ID No. 4, SEQ ID No. 5 or SEQ ID No. 6;
- and
- an .alpha..sub.1D -subunit encoded by an mRNA transcript native to a human cell, wherein DNA that is fully complementary to the transcript is capable of hybridizing under conditions of high stringency to a probe having the sequence of nucleotides set forth in SEQ ID No. 2.
13 more elements
A substantially pure .alpha..sub.1 -subunit of a human calcium channel, wherein:
- the .alpha..sub.1 -subunit is selected from the group consisting of:
- an .alpha..sub.1C -subunit comprising the sequence of amino acids encoded by the sequence nucleotides set forth in SEQ ID NO. 3;
- an .alpha..sub.1D -subunit comprising the sequence of amino acids encoded by the sequence of nucleotides set forth in SEQ ID NO:
- 1 or SEQ ID NO. 29;
- an .alpha..sub.1A -subunit comprising the sequence of amino acids encoded by the .alpha..sub.1A -encoding DNA in a phage having all of the identifying characteristics of the phage deposited as ATCC Accession No. 75293;
- a human calcium channel .alpha..sub.1 -subunit that is encoded by a DNA molecule 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 .alpha..sub.1 -subunit-encoding DNA molecule is capable of hybridizing under conditions of high stringency to the DNA that is fully complementary to the mRNA transcripts;
- an .alpha..sub.1A -subunit encoded by an mRNA transcript native to a human cell, wherein DNA that is fully complementary to the transcript is capable of hybridizing under conditions of high stringency to a probe having the sequence of nucleotides set forth in SEQ ID No. 21;
- an .alpha..sub.1C -subunit encoded by an mRNA transcript native to a human cell, wherein DNA that is fully complementary to the transcript is capable of hybridizing under conditions of high stringency to a probe having the sequence of nucleotides set forth in SEQ ID No. 4, SEQ ID No. 5 or SEQ ID No. 6;
- and
- an .alpha..sub.1D -Subunit encoded by an mRNA transcript native to a human cell, wherein DNA that is fully complementary to the transcript is capable of hybridizing under conditions of high stringency to a probe having the sequence of nucleotides set forth in SEQ ID No. 2.
8 more elements
A substantially pure .alpha..sub.1 -subunit of a human calcium channel that is encoded by nucleic acid native to a human cell, wherein:
- (a) the .alpha..sub.1 -subunit is selected from the group consisting of:
- an .alpha..sub.1c -subunit encoded by a DNA molecule comprising the sequence nucleotides set forth in SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5 or SEQ ID NO. 6;
- an .alpha..sub.1D subunit comprising the sequence of amino acids encoded by the sequence of nucleotides set forth in SEQ ID NO:
- 1 or SEQ ID NO. 29;
- an .alpha..sub.1A -subunit encoded by a DNA molecule comprising the sequence of nucleotides set forth in SEQ ID NO. 21 or encoded by the .alpha..sub.1A -encoding DNA in a phage having all of the identifying characteristics of the phage deposited as ATCC Accession No. 75293;
- 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 the mRNA transcript native to a human cell;
- and
- (b) the .alpha..sub.1 -subunit binds to at least one compound selected from the group consisting of a dihydropyridines, a phenylalkylamines, .omega.-CgTx, and a pyrazonoylguanidines.
6 more elements
A substantially pure .alpha..sub.2 -subunit of a human calcium channel selected from the group consisting of:
- an .alpha..sub.2a -subunit comprising the sequence of amino acids encoded by the sequence nucleotides set forth in SEQ ID NO. 22;
- an .alpha..sub.2c subunit comprising the sequence of amino acids encoded by the sequence of nucleotides set forth in SEQ ID NO. 23;
- an .alpha..sub.2d subunit comprising the sequence of amino acids encoded by the sequence of nucleotides set forth in SEQ ID NO. 24;
- and
- an .alpha..sub.2e subunit comprising the sequence of amino acids encoded by the sequence of nucleotides set forth in SEQ ID NO. 25.
2 more elements
An isolated .alpha..sub.2b -subunit of a human calcium channel comprising the sequence of amino acids encoded by the sequence of nucleotides set forth in SEQ ID NO. 11.
A substantially pure .beta..sub.1 -subunit of a human calcium channel selected from the group consisting of:
- a .beta..sub.1-1 -subunit comprising the sequence of amino acids encoded by the sequence of nucleotides set forth in SEQ ID NO. 26;
- a .beta..sub.1-2 -subunit comprising the sequence of amino acids encoded by the sequence of nucleotides set forth in SEQ ID NO. 9;
- a .beta..sub.1-3 -subunit comprising the sequence of amino acids encoded by the sequence of nucleotides set forth in SEQ ID NO.10;
- a .beta..sub.1-4 -subunit comprising the sequence of amino acids encoded by the sequence of nucleotides set forth in SEQ ID NO. 27;
- a .beta..sub.1-5 -subunit comprising the sequence of amino acids encoded by the sequence of nucleotides set forth in SEQ ID NO. 28;
- and
- a .beta..sub.1 subunit encoded by an mRNA transcript native to a human cell, wherein DNA that is fully complementary to the transcript is capable of hybridizing under conditions of high stringency to a probe having a sequence corresponding to at least 30 contiguous nucleotides present in SEQ ID NO:
- 26. SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:27 or SEQ NO:
- 28;
- and
- a human calcium channel .beta..sub.1 -subunit that is encoded by a DNA molecule 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 .beta..sub.1 -subunits;
- such that any probe that contains at least 14 contiguous bases from the coding portion of the .beta..sub.1 -subunit-encoding DNA molecule is capable of hybridizing under conditions of high stringency to the DNA molecule that is fully complementary to the mRNA transcript.
9 more elements
A substantially pure peptide, comprising the sequence of amino acids encoded by SEQ ID No. 3 or 6.
A substantially pure peptide, comprising the sequence of amino acids encoded by SEQ ID No. 2.
Dependencies are inferred from the claim text.