NMR investigation of the isolated second voltage-sensing domain of human Nav1.4 channel

Voltage-gated Na⁺ channels are essential for the functioning of cardiovascular, muscular, and nervous systems. The α-subunit of eukaryotic Na⁺ channel consists of ~2000 amino acid residues and encloses 24 transmembrane (TM) helices, which form five membrane domains: four voltage-sensing (VSD) and one pore domain. The structural complexity significantly impedes recombinant production and structural studies of full-sized Na⁺ channels. Modular organization of voltage-gated channels gives an idea for studying of the isolated second VSD of human skeletal muscle Nav1.4 channel (VSD-II). Several variants of VSD-II (~150a.a., four TM helices) with different N- and C-termini were produced by cell-free expression. Screening of membrane mimetics revealed low stability of VSD-II samples in media containing phospholipids (bicelles, nanodiscs) associated with the aggregation of electrically neutral domain molecules. The almost complete resonance assignment of ¹³C,¹⁵N-labeled VSD-II was obtained in LPPG micelles. The secondary structure of VSD-II showed similarity with the structures of bacterial Na⁺ channels. The fragment of S4 TM helix between the first and second conserved Arg residues probably adopts 3₁₀-helical conformation. Water accessibility of S3 helix, observed by the Mn²⁺ titration, pointed to the formation of water-filled crevices in the micelle embedded VSD-II. ¹⁵N relaxation data revealed characteristic pattern of μs-ms time scale motions in the VSD-II regions sharing expected interhelical contacts. VSD-II demonstrated enhanced mobility at ps-ns time scale as compared to isolated VSDs of K⁺ channels. These results validate structural studies of isolated VSDs of Na⁺ channels and show possible pitfalls in application of this 'divide and conquer' approach.