On the Na+/H+ Selectivity of Membrane Transporters and Enzymes: Experimental and Theoretical Studies of an ATP-Synthase Rotor Ring

Electrochemical gradients of sodium and protons are the primary driving forces for a wide array of cellular processes mediated by membrane proteins, such as energy conversion, solute uptake and multi-drug extrusion. The factors that confer ion specificity to these systems are poorly understood. Sodium and proton-driven systems of different functionality are often found in the same organism, and membrane proteins within the same functional family frequently feature distinct specificities despite a high similarity in their structures. Therefore, it appears that the specificity of H+/Na+-coupled systems is largely determined by localized variations in their amino-acid sequence, rather than by architectural constraints, function type or the organism's environment.Here, we use experimental and computational methods to quantify and rationalize the ion specificity of a rotor ring from an F-type ATP synthase. Specifically we employ Isothermal Titration Calorimetry to experimentally determine the H+/Na+-selectivity of the rotor ring from Ilyobacter tartaricus, and find this ring to be selective for H+ over Na+ by three orders of magnitude. This result explains the observation that this ATP synthase is functionally Na+-specific, owing to the much larger concentration of Na+ over H+ under physiological conditions. Using free-energy molecular simulations, we then compare the I. tartaricus ring with that of the Enterococcus hirae V-type ATPase, which is known to be essentially non-selective (and thus also coupled to Na+), as well as with that of Spirulina platensis, whose H+ selectivity is at least a billion-fold. Taken together with previous theoretical studies, this analysis establishes the general principle underlying the broad spectrum of H+/Na+ selectivities in the rotary ATPase family, which might also be applicable to other membrane transport systems.