title: Predicting a double mutant in the twilight zone of low homology modeling for the skeletal muscle voltage-gated sodium channel subunit beta-1 (Nav1.4 β1).
abstract: The molecular structure modeling of the β1 subunit of the skeletal muscle voltage-gated sodium channel (Nav1.4) was carried out in the twilight zone of very low homology. Structural significance can per se be confounded with random sequence similarities. Hence, we combined (i) not automated computational modeling of weakly homologous 3D templates, some with interfaces to analogous structures to the pore-bearing Nav1.4 α subunit with (ii) site-directed mutagenesis (SDM), as well as (iii) electrophysiological experiments to study the structure and function of the β1 subunit. Despite the distant phylogenic relationships, we found a 3D-template to identify two adjacent amino acids leading to the long-awaited loss of function (inactivation) of Nav1.4 channels. This mutant type (T109A, N110A, herein called TANA) was expressed and tested on cells of hamster ovary (CHO). The present electrophysiological results showed that the double alanine substitution TANA disrupted channel inactivation as if the β1 subunit would not be in complex with the α subunit. Exhaustive and unbiased sampling of "all β proteins" (Ig-like, Ig) resulted in a plethora of 3D templates which were compared to the target secondary structure prediction. The location of TANA was made possible thanks to another "all β protein" structure in complex with an irreversible bound protein as well as a reversible protein-protein interface (our "Rosetta Stone" effect). This finding coincides with our electrophysiological data (disrupted β1-like voltage dependence) and it is safe to utter that the Nav1.4 α/β1 interface is likely to be of reversible nature.
authors.affiliation: Facultad de Ciencias Químicas, Universidad Autónoma de Puebla, Puebla, Mexico.
authors.forename: Thomas
authors.initials: T
authors.lastname: Scior
authors.affiliation: Facultad de Ciencias Químicas, Universidad Autónoma de Puebla, Puebla, Mexico.
authors.forename: Bertin
authors.initials: B
authors.lastname: Paiz-Candia
authors.affiliation: Laboratorio de Biofísica, Instituto de Fisiología, Universidad Autónoma de Puebla, Puebla, Mexico.
authors.forename: Ángel A
authors.initials: ÁA
authors.lastname: Islas
authors.affiliation: Laboratorio de Biofísica, Instituto de Fisiología, Universidad Autónoma de Puebla, Puebla, Mexico.
authors.forename: Alfredo
authors.initials: A
authors.lastname: Sánchez-Solano
authors.affiliation: Centro de Química, Instituto de Ciencias, Universidad Autónoma de Puebla, Puebla, Mexico.
authors.forename: Lourdes
authors.initials: L
authors.lastname: Millan-Perez Peña
authors.affiliation: Laboratorio de Biofísica, Instituto de Fisiología, Universidad Autónoma de Puebla, Puebla, Mexico.
authors.forename: Claudia
authors.initials: C
authors.lastname: Mancilla-Simbro
authors.affiliation: Laboratorio de Biofísica, Instituto de Fisiología, Universidad Autónoma de Puebla, Puebla, Mexico.
authors.forename: Eduardo M
authors.initials: EM
authors.lastname: Salinas-Stefanon
keywords: Analogy modeling; CDR1; Ig-like; MD-2; MD-2, myeloid differentiation factor 2 (MD-2); Nav1.4, skeletal muscle voltage-gated sodium channel; Patch-clamp; SDM, site-directed mutagenesis; Site-directed mutagenesis; TLR4, Toll-like receptor type 4; Trk, tyrosine receptor.