Summary
Three synthetic peptides corresponding to parts of S4 of the first repeat of eel electroplax sodium channel were synthesized. The basic peptide was C +1 which corresponds to amino acids 210–223 (eel channel numbering) and two subfractions: an external fraction, C +1ex (amino acid 210–217); and an internal part, C +1in (amino acid 218–221). Peptide C +1 includes four of the charged amino acids of this domain; peptide C +1ex includes three of the charged amino acids and is closer to the external membrane surface (according to channel models) than peptide C +1in which includes the fourth charged amino acid alone.
Antibodies generated in rabbits against these peptides were shown to be site specific. Using the whole-cell patch-clamp technique, we found that in rat dorsal root ganglion (DRG) cells, the antibodies against C +1in but not against C +1ex had an effect on the gating parameters. They shifted the Na-channel inactivation curve towards hyperpolarization and decreased the slope of the Na-channel activation curve. These results demonstrate that during the conformational changes associated with channel gating, the fourth charged amino acid of S4 must be accessible to antibodies given to the external solution. Furthermore, they indicate a specific involvement of S4 in the voltage dependency of the gating processes.
This study was supported by a basic research grant of The Israel Academy of Sciences and Humanities (#430.87 to H.M. and G.S.).
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We wish to express our gratitude to Dr. M. Tosteson (Harvard Medical School) for providing us with samples of peptide S4IV to use in the ELISA assays. We thank Dr. R. Gordon (The Max Planck Institute for Biophysics, Frankfurt) for immunochemical advise and protocols. The advice of Drs. M. Sammar, M. Paizi, R. Schatzberger, I. Zeitoun and Y. Mika (Technion) was very useful. We thank Mrs. A. Schwartz (Technion) for participating in the experiments.
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Kra-Oz, Z., Spira, G., Palti, Y. et al. Involvement of different S4 parts in the voltage dependency of Na channel gating. J. Membarin Biol. 129, 189–198 (1992). https://doi.org/10.1007/BF00219514
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DOI: https://doi.org/10.1007/BF00219514