Expression system for human brain-specific voltage-gated sodium channel, type 1

• US 7,125,676 B2
• Filed: 02/25/2003
• Issued: 10/24/2006
• Est. Priority Date: 02/25/2002
• Status: Expired due to Fees

• Current Assignee: Vanderbilt University
• Original Assignee: Vanderbilt University
• Inventors: Lossin, Christoph, George, Alfred L. Jr.
• Primary Examiner(s): Spector, Lorraine
• Assistant Examiner(s): LOCKARD, JON MCCLELLAND
• Application Number:
  US10/374,954
• Publication Number:
  US 20050260576A1
• Time in Patent Office: 1,337 Days
• Field of Search: None
• US Class Current: 435/7.1
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: C07K 14/705 Receptors; Cell surface antigens; Cell surface determinants tumour specific antigens C07K14/4748

Abstract

Isolated nucleic acids encoding human SCN1A polypeptides, recombinantly expressed and isolated human SCN1A polypeptides, heterologous expression systems for recombinant expression of human SCN1A polypeptides, assay methods employing the same, and methods and compositions for modulation of sodium channel function.

Claims

1. An isolated and modified human sodium channel type 1 alpha subunit (SCN1A) nucleic acid molecule comprising:

(a) a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO;

1 comprising one or more mutations within nucleotides 1203–

1209;

or

(b) a nucleotide sequence at least about 95% identical to SEQ ID NO;

1, and further comprising one or more mutations within nucleotide positions corresponding to nucleotides 1203–

1209 of SEQ ID NO;

1

wherein the nucleotide sequences of (a) and (b) encode an SCN1A voltage-gated sodium channel polypeptide that regulates Na⁺ ion membrane permeation.

2. An isolated and modified human sodium channel type 1 alpha subunit (SCN1A) nucleic acid molecule comprising:

(a) a nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO;

3;

or

(b) a nucleic acid molecule at least about 95% identical to SEQ ID NO;

3, and wherein the nucleic acid molecule comprises a T to C transition at each of the nucleotide positions corresponding to nucleotides 1206 and 1209 of SEQ ID NO;

3 and encodes an SCN1A voltage-gated sodium channel polypeptide that regulates Na⁺ ion membrane permeation.

3. An isolated host cell for heterologous expression of a human sodium channel type 1 alpha subunit (SCN1A) polypeptide, the host cell comprising:

(a) a polypeptide encoded by a nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO;

1 comprising one or more mutations within nucleotides 1203–

1209;

or

(b) a polypeptide encoded by a nucleic acid molecule at least about 95% identical to SEQ ID NO;

1 and further comprising one or more mutations within nucleotide positions corresponding to nucleotides 1203–

1209 of SEQ ID NO;

1

wherein the polypeptides of (a) and (b) each comprise an SCN1A voltage-gated sodium channel polypeptide that regulates Na⁺ ion membrane permeation.

5. The host cell of claim 3 or 4, wherein the host cell comprises a mammalian cell.

6. The host cell of claim 5, wherein the mammalian cell comprises a human cell.

7. The host cell of claim 3 or 4, wherein the host cell is from a stable cell line.

8. The host cell of claim 3 or 4, further comprising a human sodium channel β

-subunit, wherein the sodium channel β

-subunit is expressed in the host cell.

9. The host cell of claim 8, wherein the human sodium channel β

-subunit comprises a recombinantly expressed human sodium channel β

-subunit.

10. The host cell of claim 8, wherein the human sodium channel β

-subunit comprises human sodium channel type 1 beta subunit (SCN1B).

11. The host cell of claim 8, further comprising at least one other human sodium channel β

-subunit, wherein the at least one other sodium channel β

-subunit is expressed in the host cell.

12. The host cell of claim 11, wherein the at least one other human sodium channel β

-subunit comprises human sodium channel type 2 beta subunit (SCN2B).

4. An isolated host cell for heterologous expression of a human sodium channel type 1 alpha subunit (SCN1A) polypeptide, the host cell comprising:

(a) a polypeptide encoded by a nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO;

3;

or

(b) a polypeptide that regulates Na⁺ ion membrane permeation, which is encoded by a nucleic acid molecule at least about 95% identical to SEQ ID NO;

3, and wherein the nucleic acid molecule comprises a T to C transition at each of the nucleotide positions corresponding to nucleotides 1206 and 1209 of SEQ ID NO;

3.

13. An isolated host cell for heterologous expression of a recombinant human sodium channel type 1 alpha subunit (SCN1A) polypeptide, the host cell comprising a construct comprising:

(a) a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO;

1 that comprises one or more mutations within nucleotides 1203–

1209;

or

(b) a nucleotide sequence at least about 95% identical to SEQ ID NO;

1, and further comprising one or more mutations within nucleotide positions corresponding to nucleotides 1203–

1209 of SEQ ID NO;

1

wherein the nucleotide sequences of (a) and (b) encode an SCN1A voltage-gated sodium channel polypeptide that regulates Na⁺ ion membrane permeation.

15. The host cell of claim 13 or 14, wherein the host cell comprises a mammalian cell.

16. The host cell of claim 15, wherein the mammalian cell comprises a human cell.

17. The host cell of claim 13 or 14, wherein the host cell is from a stable cell line.

18. The host cell of claim 13 or 14, further comprising a human sodium channel β

-subunit, wherein the sodium channel β

-subunit is expressed in the host cell.

19. The host cell of claim 18, wherein the human sodium channel β

-subunit comprises a recombinantly expressed sodium channel β

-subunit.

20. The host cell of claim 18, wherein the human sodium channel β

-subunit comprises human sodium channel type 1 beta subunit (SCN1B).

21. The host cell of claim 18, further comprising at least one other human sodium channel β

-subunit, wherein the at least one other sodium channel β

-subunit is expressed in the host cell.

22. The host cell of claim 21, wherein the at least one other human sodium channel β

-subunit comprises human sodium channel type 2 beta subunit (SCN2B).

14. An isolated host cell for heterologous expression of a recombinant human sodium channel type 1 alpha subunit (SCN1A) polypeptide, the host cell comprising a construct comprising:

(a) a nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO;

3;

or

(b) a nucleic acid molecule at least about 95% identical to SEQ ID NO;

3, and wherein the nucleic acid molecule comprises a T to C transition at each of the nucleotide positions corresponding to nucleotides 1206 and 1209 of SEQ ID NO;

3 and encodes an SCN1A voltage-gated sodium channel polypeptide that regulates Na⁺ ion membrane permeation.

23. A method for identifying a sodium channel modulator, the method comprising:

(a) providing a heterologous expression system whereby a human SCN1A polypeptide is expressed from a modified SCN1A nucleic acid molecule in a host cell, wherein the nucleic acid molecule encoding a human SCN1A polypeptide comprises;

(1) a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO;

1 comprising one or more mutations within nucleotides 1203–

1209;

or

(2) a nucleotide sequence at least 95% identical to SEQ ID NO;

1, and further comprising one or more mutations within nucleotide positions corresponding to nucleotides 1203–

1209 of SEQ ID NO;

1;

or

(3) a nucleotide sequence of SEQ ID NO;

3;

or

(4) a nucleotide sequence at least 95% identical to SEQ ID NO;

3, and wherein the nucleic acid molecule comprises a T to C transition at each of the nucleotide positions corresponding to nucleotides 1206 and 1209 of SEQ ID NO;

3, wherein the nucleotide sequences of (1), (2), (3), and (4) encode an SCN1A voltage-gated sodium channel polypeptide that regulates Na+ ion membrane permeation;

(b) providing a test substance to the system of (a);

(c) assaying sodium channel conductance in the presence of the test substance;

(d) comparing sodium channel conductance in the presence of the test substance with sodium channel conductance in the absence of the test compound; and

(e) identifying a test substance as a sodium channel modulator by measuring an increase or decrease in sodium channel conductance in the presence of the test substance as compared to sodium channel conductance in the absence of the test substance.

24. The method of claim 23, wherein the host cell comprises a mammalian cell.

25. The method of claim 24, wherein the mammalian cell comprises a human cell.

26. The method of claim 23, wherein the host cell is from a stable cell line.

27. The method of claim 23, wherein the host cell further comprises a human sodium channel β

-subunit.

28. The method of claim 27, wherein the human sodium channel β

-subunit comprises a recombinantly expressed human sodium channel β

-subunit.

29. The method of claim 27, wherein the human sodium channel β

-subunit comprises human SCN1B.

30. The method of claim 27, wherein the host cell comprises at least one other human sodium channel β

-subunit, wherein the at least one other sodium channel β

-subunit is expressed in the host cell.

31. The method of claim 30, wherein the at least one other human sodium channel β

-subunit comprises human sodium channel type 2 beta subunit (SCN2B).