The NaChBac pore: creation and characterisation of a KcsA-like sodium channel

Powl, A.M., de Planque, M.R.R., Morgan, H. and Wallace, B.A. (2010) The NaChBac pore: creation and characterisation of a KcsA-like sodium channel. Biophysical Journal, 98 (3), supplement S1, p.7a. (doi:10.1016/j.bpj.2009.12.043).

Abstract

Voltage-gated sodium channels (VGSC) are integral membrane proteins responsible for the transient flux of sodium ions across cell membranes in response to changes in membrane potential. In humans as well as lower eukaryotes they are essential for homeostasis and normal functioning, and mutations in them are associated with a range of disease states. Although potassium channels, which are members of the same large family of voltage-gated channels have been well characterized, much less known about the structural features of sodium channels. For potassium ion channels, an important advance in understanding resulted from the determination of the three dimensional structure of the bacterial potassium channel KcsA, a simplified channel composed only of two transmembrane segments per subunit present in the tetrameric structure. In 2001, Ren et al found that bacteria also possess simplified versions of sodium channels, although in this case the individual subunits of all the homologues that have been identified thus far possess six transmembrane segments, which include both a pore-forming subdomain (S5-S6) and a voltage-sensing subdomain (S1-S4). Here we report on the creation of a smaller KcsA-like pore-only version of a sodium channel from the B. halodurans VGSC (pNaChBac), engineered to contain S5-S6 plus the C-terminal region of the NaChBac channel. The NaChBac pore has been expressed and purified from E. coli membranes, solubilised in detergent micelles, reconstituted into lipid vesicles and characterized for its secondary structure and thermal stability, as well as its electrophysiological properties from single-channel recordings, providing new insight into features of sodium channel structure and function.

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Contributors

Author: M.R.R. de Planque

Author: H. Morgan

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