Comparative study of the gating motif and C-type inactivation in prokaryotic voltage-gated sodium channels.
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.
Irie
Katsumasa
K
From the Department of Biophysics, Graduate School of Science, Kyoto University, Oiwake, Kitashirakawa, Sakyo-ku, Kyoto 606-8502; the Japan Biological Informatics Consortium, Oiwake, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, and.
Kitagawa
Kazuya
K
From the Department of Biophysics, Graduate School of Science, Kyoto University, Oiwake, Kitashirakawa, Sakyo-ku, Kyoto 606-8502.
Nagura
Hitoshi
H
From the Department of Biophysics, Graduate School of Science, Kyoto University, Oiwake, Kitashirakawa, Sakyo-ku, Kyoto 606-8502.
Imai
Tomoya
T
the Research Institute for Sustainable Humanosphere (RISH), Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan.
Shimomura
Takushi
T
From the Department of Biophysics, Graduate School of Science, Kyoto University, Oiwake, Kitashirakawa, Sakyo-ku, Kyoto 606-8502.
Fujiyoshi
Yoshinori
Y
From the Department of Biophysics, Graduate School of Science, Kyoto University, Oiwake, Kitashirakawa, Sakyo-ku, Kyoto 606-8502. Electronic address: yoshi@em.biophys.kyoto-u.ac.jp.
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001407:Bacillus / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000145:classification / Q000235:genetics / Q000502:physiology*; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D004594:Electrophysiology; D006801:Humans; D015640:Ion Channel Gating / Q000235:genetics / Q000502:physiology*; D008564:Membrane Potentials; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010802:Phylogeny; D041904:Roseobacter / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D020614:Shewanella putrefaciens / Q000235:genetics / Q000378:metabolism; D015222:Sodium Channels / Q000145:classification / Q000235:genetics / Q000502:physiology*; D014162:Transfection
D001426:Bacterial Proteins; C443666:NaChBac protein, bacteria; D015222:Sodium Channels