Biochemical, biophysical and electrophysiological characterisation of the recombinant voltage-gated sodium channel from the marine bacterium Maricaulis Maris MCS10

The eukaryotic voltage-gated sodium channel Nav is an integral membrane protein that transports sodium across the cell membrane. While the Nav has a four-domain structure with six α-helical transmembrane segments in each domain, the first bacterial homologue, which was identified in 2001 in Bacillus halodurans (NaChBac), consists of only one domain. Its simple constitution makes NaChBac and prokaryotic homologues interesting targets for structural analysis. The present study is focused on the production, biochemical and biophysical characterisation as well as crystallisation of the voltage-gated sodium channel from Maricaulis maris MCS10 (NaChMM). Thus, an optimised protocol for the production of milligram quantities (6 mg/L) of highly pure recombinant NaChMM was established. The integrity of the protein was demonstrated by SDS-PAGE/Western-blot and further evaluated by N-terminal sequencing and mass spectrometry. While the gel-filtration trials suggest the presence of a homogenous population in several tested detergents, cross-linking and Blue-Native gel experiments provide insight into the inhomogeneity of the sample represented by the detection of monomeric, dimeric and tetrameric states. Furthermore, extensive crystallisation trials (~ 8,000) were performed using robotics and potential preliminary crystallisation conditions were discussed. In comparison, an attempt was made to produce and further characterise the recently described sodium channels from Bacillus halodurans C-125 and Silicibacter pomeroyi DSS-3, which led to the conclusion that both sodium channels can only be produced at a low scale and are less stable in a solution. The in vivo activity of NaChMM was investigated using the whole-cell patch clamp method. In contrast to NaChBac, NaChMM demonstrates a fast activation and inactivation kinetics. Surprisingly, NaChMM presents a double mechanism of inactivation, which has been to date observed only in the mammalian Nav. Due to the differences in the constitution of Nav vs. NaChMM, the NaChMM inactivation mechanisms cannot be classified as the so-called C-type or N-type mechanisms which are only present in Nav. A further investigation by site-directed mutagenesis is therefore required.