Mutated voltage-gated sodium channel nav alpha subunit for...

Details Mutated voltage-gated sodium channel nav alpha subunit for... Mutated voltage-gated sodium channel nav alpha subunit for...

C - Chemistry, Metallurgy

07

K

C07K 14/705 (2006.01) C07K 14/47 (2006.01) C12N 15/12 (2006.01) C12Q 1/02 (2006.01) G01N 33/567 (2006.01) G01N 21/64 (2006.01)

Patent

CA 2745371

Reagents, methods and kits for screening for compounds that modulate the activity of voltage-gated sodium channels (NaV), such as human NaV1.5/SCN5A/hH1 are described. The reagents, methods and kits are based on mutated NaV alpha subunit polyptides of SEQ ID NO:5 with mutations at positions 372, 898, 1419 and 1711 (the DEKA motif) and at positions 11485, 1486 and 1487 (the IFM motif) resulting in increased permeability for a group IIA divalent cation (Ca++) and decreased inactivation rate. The mutant polypeptide is used in a method and kit for determining whether a test compound modulates the channel activity, preferably using a chimeric polypeptide (chameleon polypeptide) comprising calmodulin, a calmodulin binding protein (M13), and two fluorescent agents.

L'invention concerne des réactifs, des procédés et des kits pour la sélection de composés qui modulent l'activité des canaux sodiques sensibles au voltage (NaV), tels que le NaV1.5/SCN5A/hH1 humain. Les réactifs, les procédés et les kits sont basés sur des polypeptides mutés de sous-unité alpha NaV de SEQ ID NO:5 avec des mutations aux positions 372, 898, 1419 et 1711 (motif DEKA) et aux positions 11485, 1486 et 1487 (motif IFM), ce qui entraîne une perméabilité accrue pour un cation divalent (Ca++) du groupe IIA et un taux d'inactivation réduit. Le polypeptide mutant est utilisé dans un procédé et un kit pour déterminer si un composé de test module l'activité du canal, en utilisant de préférence un polypeptide chimère (polypeptide caméléon) comprenant de la calmoduline, une protéine de liaison à la calmoduline (M13), et deux agents fluorescents.

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