Exploration of the pore structure of a peptide-gated Na+ channel

The FMRF-amide-activated sodium channel (FaNaC), a member of the ENaC/Degen erin family, is a homotetramer, each subunit containing two transmembrane s egments. We changed independently every residue of the first transmembrane segment (TMI) into a cysteine and tested each position's accessibility to t he cysteine covalent reagents MTSET and MTSES. Eleven mutants were accessib le to the cationic MTSET, showing that TMI faces the ion translocation path way. This was confirmed by the accessibility of cysteines present in the ac id-sensing ion channels and other mutations introduced in FaNaC TM1. Modifi cation of accessibilities for positions 69, 71 and 72 in the open state sho ws that the gating mechanism consists of the opening of a constriction clos e to the intracellular side. The anionic MTSES did not penetrate into the c hannel, indicating the presence of a charge selectivity filter in the outer vestibule. Furthermore, amiloride inhibition resulted in the channel occlu sion in the middle of the pore. Summarizing, the ionic pore of FaNaC includ es a large aqueous cavity, with a charge selectivity filter in the outer ve stibule and the gate close to the interior.