The gastric proton pump (H+,K+-ATPase), a P-type ATPase responsible for gastric acidification, mediates electro-neutral exchange of H+ and K+ coupled with ATP hydrolysis, but with an as yet undetermined transport stoichiometry. Here we show crystal structures at a resolution of 2.5 Å of the pump in the E2-P transition state, in which the counter-transporting cation is occluded. We found a single K+ bound to the cation-binding site of H+,K+-ATPase, indicating an exchange of 1H+/1K+ per hydrolysis of one ATP molecule. This fulfils the energy requirement for the generation of a six pH unit gradient across the membrane. The structural basis of K+ recognition is resolved, supported by molecular dynamics simulations, and this establishes how H+,K+-ATPase overcomes the energetic challenge to generate an H+ gradient of more than a million-fold - the highest cation gradient known in any mammalian tissue - across the membrane.

A single K+-binding site in the crystal structure of the gastric proton pump