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

Kenta Yamamoto,Yoshinori Fujiyoshi,Kazuhiro Abe,Hanayo Nakanishi,Himanshu Khandelia,Katsumasa Irie,Vikas Dubey

doi:10.7554/elife.47701

Kenta Yamamoto, Yoshinori Fujiyoshi + Show 5 more

Open Access

https://doi.org/10.7554/elife.47701

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Journal: eLife	Publication Date: Aug 22, 2019
Citations: 22	License type: CC BY 4.0

Affiliation: Nagoya University, University of Southern Denmark

Abstract

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 the H+,K+-ATPase, indicating an exchange of 1H+/1K+ per hydrolysis of one ATP molecule. This fulfills the energy requirement for the generation of a six pH unit gradient across the membrane. The structural basis of K+ recognition is resolved and supported by molecular dynamics simulations, establishing how the H+,K+-ATPase overcomes the energetic challenge to generate an H+ gradient of more than a million-fold-one of the highest cation gradients known in mammalian tissue-across the membrane.

Highlights

While the closely-related Na+,K+-ATPase mediates electrogenic transport of three Na+and two K+ ions coupled with the hydrolysis of one ATP molecule [1], the number of transported cations in the electroneutral operation [2,3] of the gastric H+,K+-ATPase remains unclear
The reported free energy derived from ATP hydrolysis in the parietal cell is about -13 kcal/mol [5]
The free energy derived from ATP hydrolysis, GATP, calculated from G’0 and the measured intracellular concentrations of ATP, ADP and Pi in the parietal cell is about -13 kcal/mol [5] as described in the previous report

Summary

Introduction

While the closely-related Na+,K+-ATPase mediates electrogenic transport of three Na+. The free energy derived from ATP hydrolysis, GATP, calculated from G’0 and the measured intracellular concentrations of ATP, ADP and Pi in the parietal cell is about -13 kcal/mol [5] as described in the previous report. MM K+ for intracellular conditions with the measured pH (1.0) and K+ concentration (10 mM) of the gastric juice gives concentration gradients across the parietal cell membrane of 106 and 12 times for H+ and K+, respectively. The ratio of H+ transported to ATP hydrolyzed must be approximately 1, and cannot be as large as 2, when gastric pH is around 1 This cannot be the case when luminal pH is neutral to weakly acidic, e.g., at pH 4. A different postulate is that two H+ are transported per ATP molecule hydrolyzed under these conditions [7], and that the number of transported H+, and K+ as well, changes from 2 to 1 as luminal pH decreases

Results

Discussion

Materials and Methods