Deceleration of the E1P-E2P Transition and Ion Transport by Mutation of Potentially Salt Bridge-forming Residues Lys-791 and Glu-820 in Gastric H+/K+-ATPase

Katharina L Dürr,Ina Seuffert,Thomas Friedrich

doi:10.1074/jbc.m110.133470

Katharina L Dürr, Ina Seuffert + Show 1 more

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https://doi.org/10.1074/jbc.m110.133470

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Abstract

A lysine residue within the highly conserved center of the fifth transmembrane segment in P(IIC)-type ATPase α-subunits is uniquely found in H,K-ATPases instead of a serine in all Na,K-ATPase isoforms. Because previous studies suggested a prominent role of this residue in determining the electrogenicity of non-gastric H,K-ATPase and in pK(a) modulation of the proton-translocating residues in the gastric H,K-ATPases as well, we investigated its functional significance for ion transport by expressing several Lys-791 variants of the gastric H,K-ATPase in Xenopus oocytes. Although the mutant proteins were all detected at the cell surface, none of the investigated mutants displayed any measurable K(+)-induced stationary currents. In Rb(+) uptake measurements, replacement of Lys-791 by Arg, Ala, Ser, and Glu substantially impaired transport activity and reduced the sensitivity toward the E(2)-specific inhibitor SCH28080. Furthermore, voltage clamp fluorometry using a reporter site in the TM5/TM6 loop for labeling with tetra-methylrhodamine-6-maleimide revealed markedly changed fluorescence signals. All four investigated mutants exhibited a strong shift toward the E(1)P state, in agreement with their reduced SCH28080 sensitivity, and an about 5-10-fold decreased forward rate constant of the E(1)P ↔ E(2)P conformational transition, thus explaining the E(1)P shift and the reduced Rb(+) transport activity. When Glu-820 in TM6 adjacent to Lys-791 was replaced by non-charged or positively charged amino acids, severe effects on fluorescence signals and Rb(+) transport were also observed, whereas substitution by aspartate was less disturbing. These results suggest that formation of an E(2)P-stabilizing interhelical salt bridge is essential to prevent futile proton exchange cycles of H(+) pumping P-type ATPases.

Highlights

740 and Cluster of Excellence Unifying Concepts in Catalysis). □S The on-line version of this article contains supplemental Appendices A and B. 1 Present address: Vollum Institute, Oregon Health and Science University
Rbϩ Uptake and Cell Surface Expression of Lys-791 Mutants—Because mutation of Lys-791 in the non-gastric H,K-ATPase produced interesting effects on pump stoichiometry resulting in electrogenic pump activity, we first examined whether the corresponding amino acid replacements in rat gastric H,K-ATPase would give rise to Kϩ-stimulated pump currents upon expression in Xenopus oocytes
We have demonstrated recently that the increased Rbϩ uptake activity observed for the gastric H,K-ATPase at pHex ϭ 5.5 is caused by a slight intracellular acidification (ϳ0.5 pH units) as a consequence of the acidic extracellular pH, because a similar pH change of the cell interior, which can be achieved at pHex ϭ 7.4 by adding 40 mM butyrate to the extracellular solution, results in very similar Rbϩ uptake in the case of the wild-type protein

Summary

Introduction

740 and Cluster of Excellence Unifying Concepts in Catalysis). □S The on-line version of this article (available at http://www.jbc.org) contains supplemental Appendices A and B. 1 Present address: Vollum Institute, Oregon Health and Science University,. Electrophysiological experiments using purified H,K-ATPasecontaining membrane fragments on planar lipid bilayers [5, 6] have shown that the proton branch of the cycle, which involves the E1P-E2P conformational change investigated in the current study, includes an electrogenic step As it is the case for the electrogenic Na,K-ATPase, the E1P-E2P distribution might be driven by the redistribution of cations within intraor extracellularly oriented high-field access channels to the transport sites. Mutagenesis studies have shown that Na,KATPase and H,K-ATPase extrude Naϩ and Hϩ (or H3Oϩ), generally utilizing the same conserved carboxylic acids of their respective cation binding sites in the transmembrane domains TM42 to TM6 (see Fig. 1) This raises the important question of how the different stoichiometries are achieved on a molecular level and why these carboxyls with an expected pK around 3–5 can release protons at a lumenal pH of ϳ1 in the case of the gastric H,K-ATPase. Lys-791/Glu-820 Mutations Slow Turnover of Gastric H,K-ATPase [17,18,19,20] under sufficiently steep ion gradients and high ADP/ ATP ratios in presence of Pi

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