The C-terminal coiled-coil of the bacterial voltage-gated sodium channel NaChBac is not essential for tetramer formation, but stabilizes subunit-to-subunit interactions.
The NaChBac is a prokaryotic homologue of the voltage-gated sodium channel found in the genome of the alkalophilic bacterium Bacillus halodurans C-125. Like a repeating cassette of mammalian sodium channel, the NaChBac possesses hydrophobic domains corresponding to six putative transmembrane segments and a pore loop, and exerts channel function by forming a tetramer although detailed mechanisms of subunit assembly remain unclear. We generated truncated mutants from NaChBac, and investigated their ability to form tetramers in relation to their channel functions. A mutant that deletes almost all of the C-terminal coiled-coil structure lost its voltage-dependent ion permeability, although it was properly translocated to the cell surface. The mutant protein was purified as a tetramer using a reduced concentration of detergent, but the association between the subunits was shown to be much weaker than the wild type. The chemical cross-linking, blue native PAGE, sedimentation velocity experiments, size exclusion chromatography, immunoprecipitation, and electron microscopy all supported its tetrameric assembly. We further purified the C-terminal cytoplasmic domain alone and confirmed its self-oligomerization. These data suggest that the C-terminal coiled-coil structure stabilizes subunit-to-subunit interactions of NaChBac, but is not critical for their tetramer formation.
Mio
Kazuhiro
K
Biomedicinal Information Research Center, National Institute of Advanced Industrial Science and Technology, Aomi 2-3-26, Koto-ku, Tokyo 135-0064, Japan. kazu.mio@aist.go.jp
Mio
Muneyo
M
Arisaka
Fumio
F
Sato
Masahiko
M
Sato
Chikara
C
D001407:Bacillus / Q000378:metabolism*; D001710:Biotin / Q000737:chemistry / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000737:chemistry; D003593:Cytoplasm / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004586:Electrophoresis; D004594:Electrophysiology; D057927:Hydrophobic and Hydrophilic Interactions; D047468:Immunoprecipitation; D015640:Ion Channel Gating / Q000235:genetics / Q000502:physiology*; D009154:Mutation; D055503:Protein Multimerization; D055550:Protein Stability; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015222:Sodium Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014461:Ultracentrifugation
D003432:Cross-Linking Reagents; D021122:Protein Subunits; D015222:Sodium Channels; D001710:Biotin