Dimerization mechanism of an inverted-topology ion channel in membranes

Abstract

Many ion channels are multi-subunit complexes with a polar permeation pathway at the oligomeric interface, but their mechanisms of assembly into functional, thermodynamically stable units within the membrane are largely unknown. Here we characterize the assembly of the inverted-topology, homodimeric fluoride channel Fluc, leveraging a known mutation, N43S, that weakens Na+ binding to the dimer interface, thereby unlocking the complex. While single-channel recordings show Na+ is required for activation, single-molecule photobleaching and bulk Förster Resonance Energy Transfer experiments in lipid bilayers demonstrate that N43S Fluc monomers and dimers exist in dynamic equilibrium, even without Na+. Molecular dynamics simulations indicate this equilibrium is dominated by a differential in the lipid-solvation energetics of monomer and dimer, which stems from hydrophobic exposure of the polar ion pathway in the monomer. These results suggest a model wherein membrane-associated forces induce channel assembly while subsequent factors, in this case Na+ binding, result in channel activation.

Molecular mechanisms of Na+-driven bile acid transport in human NTCP

“…Na + can play diverse roles in membrane transport. For example, the importance of the sodium ion was found to be structural for maintaining the homodimer assembly of Fluc 35,42 , a sodium-dependent uoride channel, although a recent study challenged this idea and suggested that Na + binding directly activates the F − transport 43 . In the case of LeuT-fold transporters, sodium ions at two distinct binding sites are thought to play different roles, with one Na + directly coordinating with the substrate and the other primarily coordinating with backbone and side chain atoms on the TM helices to regulate gating 32 .…”

Molecular Mechanisms of Na+-driven Bile Acid Transport in Human NTCP

“…Na + can play diverse roles in membrane transport. For example, the importance of the sodium ion was found to be structural for maintaining the homodimer assembly of Fluc 46, 71 , a sodium-dependent fluoride channel, although a recent study challenged this idea and suggested that Na + binding directly activates the F − transport 72 . In the case of LeuT-fold transporters, sodium ions at two distinct binding sites are thought to play different roles, with one Na + directly coordinating with the substrate and the other primarily coordinating with backbone and side chain atoms on the TM helices to regulate gating 32 .…”

Mitigation of membrane morphology defects explain stability and orientational specificity of CLC dimers

“…These studies have been applied to several systems, including the homodimeric chloride/proton antiporter CLC-ec1 ( Fig. 1A ) from E. coli (Chadda et al 2016)and the dual-topology homodimeric fluoride ion channel Fluc (Ernst et al 2023). But CLC-ec1 remains a particularly interesting system in that it forms dimers via a membrane embedded, hydrophobic interface, achieving strong stability in biologically relevant membranes, with ΔG° 2:1POPE/POPG = -10.9 kcal/mole (Chadda et al 2018) and ΔG° EPL = -12.7 kcal/mole (Chadda et al 2023), relative to the 1 subunit/lipid standard state.…”