Subunits of glutamate receptors, their preparation and their use

Details Subunits of glutamate receptors, their preparation and their use Subunits of glutamate receptors, their preparation and their use

1996-08-05

1998-05-26

Jones, W. Gary

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

536243, 530350, C07H 2102, C07H 2104

Patent

active

057566976

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BRIEF SUMMARY
This application is a 371 of PCT/EP95/00290 filed 27 Jan. 95, published as WO95/21188 Aug. 10, 1995. The invention relates to the expression of novel variants of ionotropic glutamate receptor subunits in eukaryotic cells and to methods for finding functional ligands for corresponding glutamate receptor channels.
Glutamate is the most important excitatory neurotransmitter in the central nervous system (TIPS 11, 1990, 126-132; Pharmacological Reviews 40, 1989, 143-210; TIPS 13, 1992, 291-296) and is involved in numerous pathophysiological processes such as epilepsy, schizophrenia, ischemia. Glutamate receptors are therefore potential sites of attack for appropriate drugs.
To date, the primary structure has been elucidated for some subunits of AMPA, kainate and NMDA receptors and some metabotropic receptors (Nature 342, 1989, 643; Science 249, 1990, 556; Neuron 8, 1992, 169; Science 256, 1992, 1217; Nature 358, 1992, 36).
Four AMPA-glutamate receptor subunits of the rat have hitherto been described in the literature, GluRA, GluRB, GluRC and GluRD, each of which occurs in two splicing variants "flip" and "flop" (Science 249, 1990, 1580). In addition, RNA editing which affects the Q/R site of the second transmembrane domain has been shown for mouse and rat GluRB. These two GluRB variants differ considerably in their electrophysiological properties (Cell 67, 1991, 11-19; Neuron 8; 1992, 189-198). The human cDNA for GluRAflip and GluRAflop has likewise been published (PNAS U.S.A. 88, 1991, 7557-7561; PNAS USA 89, 1992, 1443-1447).
We have now found variants of the human glutamate receptor subunits A, B, C and D, as well as DNA sequences which code for such subunits. These subunits lead to GluR channels with specific electrophysiological properties.
We have found that the first amino acid of the flip/flop region of GluRA, GluRB, GluRC and GluRD can be in the form of glycine (G) or arginine (R) due to RNA editing. The names of the corresponding subunits are as follows:
In the case of GluRB the RNA editing known for the rat has been taken into account in the naming of the corresponding variants.
Furthermore, a variant of GluRA produced by alternative splicing, in which a 240 bp fragment is missing in the 5' region of the GluRA cDNA and thus the corresponding protein is truncated by 80 amino acids, has been found. The names of the corresponding subunits are as follows:
The following DNA and amino-acid sequences all relate to human glutamate receptor subunits. sequence (SEQ ID NO: 2) derived therefrom;
Compared with the GluRAflipG cDNA, the GluRAflipR cDNA has a base exchange at position bp 2269 which converts a glycine codon (GGA) into an arginine codon (AGA). A corresponding statement applies to GluRAflopR.
The GluRAdel240 variants correspond to the said GluRA variants but have a deletion: bp 221-460 relative to SEQ ID NO: 1 and 3.
SEQ ID NO: 5 depicts the cDNA sequence of GluRBflipQ-G and the polypeptide sequence derived therefrom (SEQ ID NO: 6); SEQ ID NO: 7 depicts the cDNA sequence of GluRBflopQ-G, and SEQ ID NO: 8 depicts the polypeptide sequence derived therefrom.
Compared with GluRBflipQ-G, the cDNA for GluRBflipQ-R has a base exchange at position bp 2290 which converts a glycine codon (GGA) into an arginine codon (AGA). A corresponding statement applies to GluRBflopQ-R.
Compared with the abovementioned GluRB variants, the cDNA molecules for GluRBflipR-G, GluRBflipR-R, GluRBflopR-G and GluRBflopR-R have a base exchange at position bp 1820 which converts a glutamine codon (CAG) into an arginine codon (CGG).
Compared with GluRCflipG and GluRCflopG, respectively, the cDNA molecules for GluRCflipR and GluRCflopR have a base exchange at position bp 2377 which converts a glycine codon (GGA) into an arginine codon (AGA).
Compared with GluRDflipG and GluRDflopG, respectively, the cDNA molecules for GluRDflipR and GluRDflopR have a base exchange at position bp 2293 which converts a glycine codon (GGA) into an arginine codon (AGA).
Other suitable DNA sequences are those which although they have a different nucleotide s

REFERENCES:
patent: 5202257 (1993-04-01), Heinemann et al.
patent: 5385831 (1995-01-01), Mulvihill et al.
patent: 5521297 (1996-05-01), Daggett et al.
patent: 5547855 (1996-08-01), Kamboj et al.
patent: 5585479 (1996-12-01), Hoke et al.
J. NeuroScience, vol. 12, 1010-23, 1992.
Science, vol. 249, 556-60 (1990).
Nature, vol. 342, 643-48 (1989).
Poc. Natl. Acad. Sci, vol. 88, 7557-61 (1991).
Proc. Natl. Acad. Sci., vol. 89, 1443-47 (1992).

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