fmrfamide and (2-sulfonatoethyl)methanethiosulfonate

The FMRFamide-gated Na(+) channel (FaNaC) is a unique peptide-gated sodium channel and a member of the epithelial sodium channel/degenerin family. Previous studies have shown that an aspartate residue (Asp(552)) in the second transmembrane domain is involved in activation of the FaNaC. To examine the significance of a negative charge at position 552, we used a cysteine-modification method. Macroscopic currents of a cysteine mutant (D552C) were potentiated or inhibited by use of positively or negatively charged sulfhydryl reagents ([2-(trimethylammonium)ethyl]methanethiosulfonate bromide, MTSET, and sodium (2-sulfonatoethyl)methanethiosulfonate, MTSES, respectively). Dose-response analysis showed that treatment with MTSET increased the potency of the FMRFamide in the FaNaC whereas treatment with MTSES reduced the maximum response. Negative charge at position 552 was necessary for the characteristic inward rectification of the FaNaC. These results suggest that negative electric charge at position 552 is important to the activation and permeation properties of the FaNaC.

Topics: Animals; Aplysia; Cysteine; Dose-Response Relationship, Drug; FMRFamide; Ion Channel Gating; Membrane Potentials; Mesylates; Models, Molecular; Mutation; Nerve Tissue Proteins; Oocytes; Permeability; Protein Conformation; Sodium; Sodium Channels; Static Electricity; Surface Properties; Xenopus laevis

The FMRF-amide-activated sodium channel (FaNaC), a member of the ENaC/Degenerin family, is a homotetramer, each subunit containing two transmembrane segments. We changed independently every residue of the first transmembrane segment (TM1) into a cysteine and tested each position's accessibility to the cysteine covalent reagents MTSET and MTSES. Eleven mutants were accessible to the cationic MTSET, showing that TM1 faces the ion translocation pathway. This was confirmed by the accessibility of cysteines present in the acid-sensing ion channels and other mutations introduced in FaNaC TM1. Modification of accessibilities for positions 69, 71 and 72 in the open state shows that the gating mechanism consists of the opening of a constriction close to the intracellular side. The anionic MTSES did not penetrate into the channel, indicating the presence of a charge selectivity filter in the outer vestibule. Furthermore, amiloride inhibition resulted in the channel occlusion in the middle of the pore. Summarizing, the ionic pore of FaNaC includes a large aqueous cavity, with a charge selectivity filter in the outer vestibule and the gate close to the interior.

Topics: Amino Acid Sequence; Amino Acid Substitution; Animals; Cell Line; Cysteine; DNA, Complementary; Female; FMRFamide; Humans; Ion Channel Gating; Ion Transport; Mesylates; Models, Molecular; Molecular Sequence Data; Multigene Family; Mutagenesis, Site-Directed; Oocytes; Protein Conformation; Protein Structure, Tertiary; Recombinant Fusion Proteins; Sequence Alignment; Sequence Homology, Amino Acid; Sodium; Sodium Channels; Static Electricity; Sulfhydryl Reagents; Xenopus laevis