Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
1999-03-12
2002-03-05
McGarry, Sean (Department: 1635)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C530S300000
Reexamination Certificate
active
06353091
ABSTRACT:
FIELD OF THE INVENTION
The invention pertains to human N-type calcium channel &agr;
1B
subunit isoforms.
BACKGROUND OF THE INVENTION
Voltage gated calcium channels, also known as voltage dependent calcium channels (VDCCs) are multisubunit membrane spanning proteins which permit controlled calcium influx from an extracellular environment into the interior of a cell. Several types of voltage gated calcium channel have been described in different tissues, including N-type, P/Q-type, L-type and T-type channels. A voltage gated calcium channel permits entry into the cell of calcium upon depolarization of the membrane of the cell, which is a lessening of the difference in electrical potential between the outside and the inside of the cell.
A voltage gated calcium channel contains several proteins, including &agr;
1
, &agr;
2
, &bgr;, and &ggr; subunits. Subtypes of the calcium channel subunits also are known. For instance, &agr;
1
subtypes include &agr;
1A
, &agr;
1B
, &agr;
1C
, &agr;
1D
, &agr;
1E
and &agr;
1S
. Each subunit may have one or more isoforms which result from alternative splicing of RNA in the formation of a completed messenger RNA which encodes the subunit. For example, at least four isoforms of the rat N-type &agr;
1B
subunit are known (see, e.g., Lin et al.,
Neuron
18:153-166, 1997).
Isoforms of calcium channel &agr;
1
subunits may be expressed differently in different tissues (see, e.g., Lin et al., 1997). Differential expression of subunits isoforms raises the possibility of developing therapeutics which are specific for distinct isoforms of the &agr;
1
subunits, thereby lessening side effects resulting from the use of therapeutics which are effective for more than one calcium channel isoform. Two isoforms of the human N-type calcium channel &agr;
1B
subunit were published by Williams et al in 1992 (
Science
257:389-395). Given the existence of several additional rat isoforms in a highly conserved gene family, it is surprising that additional human isoforms of the N-type calcium channel &agr;
1B
subunit have not been discovered. Such isoforms would be useful for developing isoform-specific therapeutics.
SUMMARY OF THE INVENTION
The invention provides isolated nucleic acid molecules, unique fragments of those molecules, expression vectors containing the foregoing, and host cells transfected with those molecules. The invention also provides isolated polypeptides and inhibitors of the foregoing nucleic acids and polypeptides which reduce voltage-gated calcium influx. The foregoing can be used in the diagnosis or treatment of conditions characterized by increased or decreased human N-type calcium channel h&agr;
1B+SFVG
subunit activity and can be used in methods in which it is therapeutically useful to increase or decrease human N-type calcium channel h&agr;
1B+SFVG
subunit activity such as treatments for stroke, pain (e.g., neuropathic pain), traumatic brain injury and conditions characterized by increased or decreased voltage regulated calcium influx. Here, we present the identification of a novel human N-type calcium channel &agr;
1B
subunit, h&agr;
1B+SFVG
, which plays a role in voltage-gated calcium influx.
It was discovered that a brain &agr;
1B
calcium channel subunit isoform (splice variant) contains a four amino acid insert relative to published human &agr;
1B
calcium channel isoforms (SEQ ID NO:5 [GenBank accession number M94172], SEQ ID NO:7 [GenBank accession number M94173]). Surprisingly, this insert, SFVG (SEQ ID NO:2, encoded by SEQ ID NO:1), is similar but not identical to an insert found in a rat &agr;
1B
channel (GenBank accession number M92905). A significant proportion of the human N-type calcium channel &agr;
1B
subunit mRNA in brain was found to be the h&agr;
1B+SFVG
sub-type; given the abundance of its expression the isolation of this sub-type so long after the identification of other &agr;
1B
isoforms is unexpected. The SFVG-containing human N-type calcium channel h&agr;
1B+SFVG
subunit also lacks an amino acid sequence, ET, which is present in published human N-type calcium channel h&agr;
1B+SFVG
subunit isoforms (amino acids 1557-1558 of SEQ ID NOs:5 and 7).
The invention involves in one aspect an isolated human N-type calcium channel &agr;
1B
subunit polypeptide which includes the amino acid sequence of SEQ ID NO:2 (an h&agr;
1B+SFVG
polypeptide). In one embodiment, the polypeptide comprises the amino acid sequence of SEQ ID NO:4, and preferably consists of the amino acid sequence of SEQ ID NO:4. In another embodiment the h&agr;
1B+SFVG
calcium channel polypeptide is a fragment or variant of the foregoing polypeptides, wherein the fragment or variant includes the amino acid sequence of SEQ ID NO:2 or additions, deletions or substitutions thereof which confer the same function as SEQ ID NO: 2. Preferred variants include those having additions, substitutions or deletions relative to the human N-type calcium channel h&agr;
1B+SFVG
subunit polypeptide sequence disclosed herein, particularly those variants which retain one or more of the activities of the human N-type calcium channel h&agr;
1B+SFVG
subunit, including subunits with or without the ET exon sequence.
According to another aspect of the invention, an isolated nucleic acid molecule which encodes any of the foregoing human N-type calcium channel h&agr;
1B+SFVG
subunit polypeptide is provided. In certain embodiments, the nucleic acid molecule includes SEQ ID NO:1. In one preferred embodiment, the human N-type calcium channel h&agr;
1B+SFVG
subunit polypeptides is encoded by a nucleic acid molecule which comprises the nucleotide sequence of SEQ ID NO:3 (Williams et al. sequence +SFVG, −ET), and which preferably consists of the nucleotide sequence of SEQ ID NO:3. In another embodiment the nucleic acid is an allele of the nucleic acid sequence of SEQ ID NO:3.
In another aspect the invention is an expression vector comprising the human N-type calcium channel h&agr;
1B+SFVG
subunit nucleic acid molecule operably linked to a promoter. Also included within the invention is a host cell transformed or transfected with the expression vector.
According to another aspect of the invention, an agent which selectively binds the human N-type calcium channel h&agr;
1B+SFVG
subunit polypeptide or a nucleic acid that encodes the human N-type calcium channel h&agr;
1B+SFVG
subunit polypeptide is provided. By “selectively binds” it is meant that the agent binds the human N-type calcium channel h&agr;
1B+SFVG
subunit polypeptide or nucleic acid, or any fragment thereof which retains the amino acids of SEQ ID NO:2 or the nucleotides of SEQ ID NO:1, to a greater extent than the agent binds other human N-type calcium channel &agr;
1B
subunit isoforms, and preferably does not bind other human N-type calcium channel &agr;
1B
subunit isoforms. In one embodiment, the agent is a polypeptide which binds selectively to the human N-type calcium channel h&agr;
1B+SFVG
subunit polypeptide. The polypeptide can be a monoclonal antibody, a polyclonal antibody, or an antibody fragment selected from the group consisting of a Fab fragment, a F(ab)
2
fragment and a fragment including a CDR3 region. In another embodiment, the agent is an antisense nucleic acid which selectively binds to a nucleic acid encoding the human N-type calcium channel h&agr;
1B+SFVG
subunit polypeptide. Preferably the foregoing agents are inhibitors (antagonists) or agonists of the calcium channel activity of the human N-type calcium channel h&agr;
1B+SFVG
subunit polypeptide.
According to another aspect of the inventions, a dominant negative human N-type calcium channel h&agr;
1B+SFVG
subunit polypeptide is provided. The dominant negative polypeptide is an inhibitor of the function of the calcium channel.
The invention also provides compositions including any of the foregoing polypeptides, nucleic acids or agents in combination with a pharmaceutically acceptable carrier.
In another as
Lipscombe Diane
Schorge Stephanie
Brown University Research Founddation
McGarry Sean
Wolf Greenfield & Sacks P.C.
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