Predicting a double mutant in the twilight zone of low homology modeling for the skeletal muscle voltage-gated sodium channel subunit beta-1 ( Nav1.4β1)

The molecular structure modeling of the beta 1 subunit of the skeletal muscle voltage-gated sodium channel (Na(v)1.4) 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 Na(v)1.4 alpha subunit with (ii) site-directed mutagenesis (SDM), as well as (iii) electrophysiological experiments to study the structure and function of the beta 1 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 Na(v)1.4 channels. Thismutant 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 beta 1 subunit would not be in complex with the a subunit. Exhaustive and unbiased sampling of all beta 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 beta 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 beta 1-like voltage dependence) and it is safe to utter that the Na(v)1.4 alpha/beta 1 interface is likely to be of reversible nature. (C) 2015 Scior et al. Published by Elsevier B.V. on behalf of the Research Network of Computational and Structural Biotechnology. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).