Russ. J. Bioorganic Chem., 2017, 43(6):634-643

“Divide and conquer” approach to the structural studies of multidomain ion channels by the example of isolated voltage sensing domains of human Kv2.1 and Nav1.4 channels

Voltage-gated K+and Na+channels are involved in diverse physiological processes including excitability of heart, muscular and neuronal cells, as well as release of hormones and neurotransmitters. These channels have modular structure and contain five membrane domains: four voltage-sensing domains (VSDs) and one pore domain. VSDs of different channels contain unique ligand-binding sites and are considered as potential pharmacological targets. Modular organization of ion channels points to the possibility of NMR structural studies of isolated VSDs apart from the pore. Here, the feasibility of such studies is considered by the example of VSD of human Kv2.1 channel and VSD-I of human Nav1.4 channel. Cell-free protein expression systems based on the S30 bacterial extract from E. coli, which allow us to produce milligram quantities of VSD samples, including their analogues labeled with stable isotopes, were developed. The choice of membrane- mimicking media that provide long-term stability of the native structure of the membrane protein and high-quality of NMR spectra is a crucial step in NMR studies. Screening of various environments showed that the domains of the Kv2.1 and Nav1.4 channels are unstable in media containing phospholipids: micelles of short-chain lipid DC7PC and lipid-detergent bicelles based on zwitterionic or anionic saturated lipids (DMPC and DMPG). It was demonstrated that the optimal media for NMR studies are the mixtures of zwitterionic and weakly cationic detergents (FOS-12/LDAO). The VSD sample of the Nav1.4 channel in FOS- 12/LDAO environment aggregated irreversibly within a few days despite the high-quality spectra. It is likely that VSDs of human K+and Na+channels are not completely autonomous membrane domains and the contacts with other domains of the channel are required for their stabilization.

IBCH: 3434
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Данные статьи проверены модераторами 2017-11-01