Abstract
Prokaryotic voltage-gated sodium channels (Na(V)s) are homotetramers and are thought to inactivate through a single mechanism, named C-type inactivation. Here we report the voltage dependence and inactivation rate of the NaChBac channel from Bacillus halodurans, the first identified prokaryotic Na(V), as well as of three new homologues cloned from Bacillus licheniformis (Na(V)BacL), Shewanella putrefaciens (Na(V)SheP), and Roseobacter denitrificans (Na(V)RosD). We found that, although activated by a lower membrane potential, Na(V)BacL inactivates as slowly as NaChBac. Na(V)SheP and Na(V)RosD inactivate faster than NaChBac. Mutational analysis of helix S6 showed that residues corresponding to the "glycine hinge" and "PXP motif" in voltage-gated potassium channels are not obligatory for channel gating in these prokaryotic Na(V)s, but mutations in the regions changed the inactivation rates. Mutation of the region corresponding to the glycine hinge in Na(V)BacL (A214G), Na(V)SheP (A216G), and NaChBac (G219A) accelerated inactivation in these channels, whereas mutation of glycine to alanine in the lower part of helix S6 in NaChBac (G229A), Na(V)BacL (G224A), and Na(V)RosD (G217A) reduced the inactivation rate. These results imply that activation gating in prokaryotic Na(V)s does not require gating motifs and that the residues of helix S6 affect C-type inactivation rates in these channels.
Highlights
Iary subunits to form the native channel [2, 3]
Searching bacterial genomic data bases, we found 26 sequences of putative NaChBac homologues from bacteria living in various environments
We found an unexpected feature in the primary structure of NaVSheP, namely a lack of glycine residues in helix S6
Summary
The identified primary sequence data were obtained from. 6121) were obtained from the RIKEN BioResource Center. (National Institute of Technology and Evaluation Biological Resource Center number. 101722), closely related to O. alexandrii by 16 S rRNA sequence analysis, were obtained from the National Institute of Technology and Evaluation. Main linkers of NaVs or the N termini of voltage-gated potas- The following primers were used for cloning: 5Ј-CGCGGAsium channels, a mechanism described by the “ball and chain” TCCATGAACACTCATCAAAATCGA-3Ј and 5Ј-CGCGGA-. C-type inactivation is common in TCCATCTTTCTTTTCATTTTGGGT-3Ј for B. licheniformis; tetrameric cation channels, its molecular mechanism remains 5Ј-CGCGGATCCATGAGTACATCTTTACTT-3Ј and 5Ј-. Mutational analysis of these new NaV homologues provide AGTCG-3Ј for L. vestfoldensis; 5Ј-CGCGGATCCATGACAGevidence that the rate of C-type inactivation is affected by the TGCGGGAAATGGTA-3Ј and 5Ј-CGCGGATCCGCCGCG-
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