Proton exclusion by an aquaglyceroprotein: a voltage clamp study

Background information. In silico both orthodox aquaporins and aquaglyceroporins are shown to exclude protons. Supporting experimental evidence is available only for orthodox aquaporins. In contrast, the subset of the aquaporin water channel family that is permeable to glycerol and certain small, uncharged solutes has not yet been shown to exclude protons. Moreover, different aquaglyceroporins have been reported to conduct ions when reconstituted in planar bilayers. Results. To clarify these discrepancies, we have measured proton permeability through the purified Escherichia coli glycerol facilitator (GlpF). Functional reconstitution into planar lipid bilayers was demonstrated by imposing an osmotic gradient across the membrane and detecting the resulting small changes in ionic concentration close to the membrane surface. The osmotic water flow corresponds to a GlpF single channel water permeability of 0.7×10^-14 cm^3·subunit^-1·s^-1. Proton conductivity measurements carried out in the presence of a pH gradient (1 unit) revealed an upper limit of the H+ (OH-) to H2O molecules transport stoichiometry of 2×10^-9. A significant GlpFmediated ion conductivity was also not detectable. Conclusions. The lack of a physiologically relevant GlpF-mediated proton conductivity agrees well with predictions made by molecular dynamics simulations.