BIOCHIM BIOPHYS ACTA, 2017, 1859(3):1-33

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

V. 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 (~ 150 a.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 of13C,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.

IBCH: 3325
Ссылка на статью в журнале: https://linkinghub.elsevier.com/retrieve/pii/S0005273617300044
Кол-во цитирований на 10.2020: 9
Данные статьи проверены модераторами 2017-03-01