Potassium-dependent sodium-calcium exchanger
View Patent ↗A novel polypeptide, a polynucleotide encoding the polypeptide, an expression vector comprising the polynucleotide, a cell transfected by the expression vector, a method for producing the polypeptide, and a convenient screening system for obtaining a substance useful in treating cell injuries due to postischemic reperfusion or an inflammatory disease, are disclosed. The polypeptide is a potassium-dependent sodium-calcium exchanger expressed in peripheral leukocytes.
1. An isolated polypeptide (a) consisting of the amino acid sequence of SEQ ID NO: 2, or (b) exhibiting a potassium-dependent sodium-calcium exchange activity and consisting of an amino acid sequence in which 1 to 5 amino acids in total are substituted, deleted, inserted, and/or added at one or plural portions in the amino acid sequence of SEQ ID NO: 2.
2. The isolated polypeptide according to claim 1 , wherein the sodium-calcium exchange activity is a reverse sodium-calcium exchange activity.
3. An isolated polynucleotide encoding the polypeptide according to claim 1 or 2 .
4. An expression vector comprising the polynucleotide according to claim 3 .
5. A cell transfected with the expression vector according to claim 4 .
6. A method for producing the isolated polypeptide according to claim 1 or 2 , the method comprising expressing a polynucleotide encoding the polypeptide in a cell transfected with an expression vector comprising the polynucleotide.
7. A method for screening for an inhibitor of the polypeptide according to claim 1 or 2 , comprising the steps of:
(a) introducing into a cell an isolated polynucleotide encoding the polypeptide of claim 1 or 2 ,
(b) bringing the cell expressing the polypeptide into contact with a substance to be tested,
(c) analyzing whether or not a potassium-dependent sodium-calcium exchange activity in the polypeptide is inhibited, and
(d) selecting the substance that inhibits the potassium-dependent sodium-calcium exchange activity in the polypeptide.
8. A method for screening for an inhibitor of leukocyte activation, comprising the steps of:
(a) introducing into a cell an isolated polynucleotide encoding a polypeptide
(i) consisting of the amino acid sequence of SEQ ID NO: 2, or
(ii) exhibiting a potassium-dependent sodium-calcium exchange activity and consisting of an amino acid sequence in which 1 to 5 amino acids in total are substituted, deleted, inserted, and/or added at one or plural portions in the amino acid sequence of SEQ ID NO: 2,
(b) bringing the cell expressing the polypeptide into contact with a substance to be tested,
(c) analyzing whether or not a potassium-dependent sodium-calcium exchange activity in the polypeptide is inhibited,
(d) selecting the substance that inhibits the potassium-dependent sodium-calcium exchange activity in the polypeptide, and
(e) confirming that the selected substance inhibits leukocyte activation.
9. A method for screening for a therapeutic agent for postischemic reperfusion injury and/or an inflammatory disease, comprising the steps of:
(a) introducing into a cell an isolated polynucleotide encoding a polypeptide
(i) consisting of the amino acid sequence of SEQ ID NO: 2, or
(ii) exhibiting a potassium-dependent sodium-calcium exchange activity and consisting of an amino acid sequence in which 1 to 5 amino acids in total are substituted, deleted, inserted, and/or added at one or plural portions in the amino acid sequence of SEQ ID NO: 2,
(b) bringing the cell expressing the polypeptide into contact with a substance to be tested,
(c) analyzing whether or not a potassium-dependent sodium-calcium exchange activity in the polypeptide is inhibited, and
(d) selecting the substance that inhibits the potassium-dependent sodium-calcium exchange activity in the polypeptide for use as a therapeutic agent for postischemic reperfusion injury and/or an inflammatory disease.
10. A process for manufacturing a pharmaceutical composition for treating postischemic reperfusion injury and/or an inflammatory disease, comprising the steps of:
(a) introducing into a cell an isolated polynucleotide encoding a polypeptide
(i) consisting of the amino acid sequence of SEQ ID NO: 2, or
(ii) exhibiting a potassium-dependent sodium-calcium exchange activity and consisting of an amino acid sequence in which 1 to 5 amino acids in total are substituted, deleted, inserted, and/or added at one or plural portions in the amino acid sequence of SEQ ID NO: 2,
(b) bringing the cell expressing the polypeptide into contact with a substance to be tested,
(c) analyzing whether or not a potassium-dependent sodium-calcium exchange activity in the polypeptide is inhibited,
(d) selecting the substance that inhibits the potassium-dependent sodium-calcium exchange activity in the polypeptide, and
(e) preparing a pharmaceutical composition containing the substance.