Two-Sided Simultaneous Block of a F- Channel (FLUC)

Abstract

The recently discovered family of F- channels, the Flucs, are highly selective for F- and function to rescue microorganisms from F- toxicity in acidic environments. Structurally, Fluc is a four pass transmembrane protein that assembles as a dual-topology homodimer. This architecture, where two Fluc subunits orient antiparallel with respect to each other, is unique among ion channels and requires a two-fold symmetry axis parallel to the membrane plane. The symmetry axis further suggests that Fluc may present identical interfaces on both sides of a membrane.In fact, using fibronectin III domain “monobody” blockers specifically selected from a phage display library for nano-molar binding affinity, it was shown that a single Fluc channel is blocked on both sides of a planar lipid bilayer [Stockbridge et. al. Nature Comm., in press]. This leaves open the question of whether the sites of block on either side of the channel can be occupied simultaneously. We approach this question by experimentally testing a two site block model O B1 B2 by analysis of monobody dependent block times in single channel electrophysiology recordings. In the case of symmetric block, this scheme postulates that the dwell times of the B1 and B2 composite blocked state should exhibit monobody concentration dependence. In addition, information about cooperativity, whether negative or positive, between the two blocking sites can be quantitatively determined.