Article
- The EMBO Journal (2001) 20, 5595 - 5602
- doi:10.1093/emboj/20.20.5595
Exploration of the pore structure of a peptide-gated Na+ channel
Mallorie Poët1, Michel Tauc1, Eric Lingueglia2, Peggy Cance1, Philippe Poujeol1, Michel Lazdunski2 and Laurent Counillon1
- Laboratoire de Physiologie Cellulaire et Moléculaire, CNRS UMR6548, Université de Nice–Sophia Antipolis, Faculté des Sciences, Parc Valrose, 06108 Nice, France
- Institut de Pharmacologie Moléculaire et Cellulaire, CNRS UMR6097, Université de Nice–Sophia Antipolis, 660 route des Lucioles, 06560 Valbonne, France
Correspondence to:
Laurent Counillon, E-mail: counillo@unice.fr
Received 21 August 2000; Accepted 31 August 2001; Revised 2 July 2001
Abstract
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.
Keywords:
- ENac,
- Degenerins,
- FMRF-amide,
- ligand-gated channel,
- pore structure,
- substituted cysteine accessibility method
