Two-sided block of a dual-topology F⁻ channel

The Fluc family is a set of small membrane proteins forming F ⁻-specific electrodiffusive ion channels that rescue microorganisms from F ⁻ toxicity during exposure to weakly acidic environments. The functional channel is built as a dual-topology homodimer with twofold symmetry parallel to the membrane plane. Fluc channels are blocked by nanomolar-affinity fibronectin-domain monobodies originally selected from phage-display libraries. The unusual symmetrical antiparallel dimeric architecture of Flucs demands that the two chemically equivalent monobody-binding epitopes reside on opposite ends of the channel, a double-sided blocking situation that has never before presented itself in ion channel biophysics. However, it is not known if both sites can be simultaneously occupied, and if so, whether monobodies bind independently or cooperatively to their transmembrane epitopes. Here, we use direct monobody-binding assays and single-channel recordings of a Fluc channel homolog to reveal a novel trimolecular blocking behavior that reveals a doubly occupied blocked state. Kinetic analysis of single-channel recordings made with monobody on both sides of the membrane shows substantial negative cooperativity between the two blocking sites. Significance The study's significance is in its novelty along several lines. First, the ion channel studied, a Fluc F ⁻ channel, is recently discovered and has been only sparsely studied yet; this work adds a piece of the puzzle to the channel's mechanistic landscape. Second, this is the first channel, to our knowledge, for which two-sided blocking has been observed; this type of blocking is a consequence of the unusual dual-topology assembly of Fluc channels. Third, the mathematical analysis of this kind of block is novel (necessarily so). Fourth, the main result—that the two ends of the pore can be simultaneously occupied by blockers but with negative cooperativity in binding—provokes future structural and mechanistic experiments.