Influence of the Selectivity Filter Properties on Proton Selectivity in the Influenza A M2 Channel

The homotetrameric M2 proton channel of influenza A plays a crucial role in the viral life cycle and is thus an important therapeutic target. It selectively conducts protons against a background of other competing cations whose concentrations are up to a million times greater than the proton concentration. Its selectivity is largely determined by a constricted region of its open pore known as the selectivity filter, which is lined by four absolutely conserved histidines. While the mechanism of proton transport through the channel has been studied, the physical principles underlying the selectivity for protons over other cations in the channel's His₄ selectivity filter remain elusive. Furthermore, it is not known if proton selectivity absolutely requires all four histidines with two of the four histidines protonated and if other titratable amino acid residues in lieu of the histidines could bind protons and how they affect proton selectivity. Here, we elucidate how the competition between protons and rival cations such as Na⁺ depends on the selectivity filter's (1) histidine protonation state, (2) solvent exposure, (3) oligomeric state (the number of protein chains and thus the number of His ligands), and (4) ligand composition by evaluating the free energies for replacing monovalent Na⁺ with H₃O⁺ in various model selectivity filters. We show that tetrameric His₄ filters are more proton-selective than their trimeric His₃ counterparts, and a dicationic His₄ filter where two of the four histidines are protonated is more proton-selective than tetrameric filters with other charge states/composition (different combinations of His protonation states or different metal-ligating ligands). The [His₄]²⁺ filter achieves proton selectivity by providing suboptimal binding conditions for rival cations such as Na⁺, which prefers a neutral or negatively charged filter instead of a dicationic one, and three rather than four ligands with oxygen-ligating atoms.