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Structure and function of H⁺/K⁺ pump mutants reveal Na⁺/K⁺ pump mechanisms.

ABSTRACT: Ion-transport mechanisms evolve by changing ion-selectivity, such as switching from Na⁺ to H⁺ selectivity in secondary-active transporters or P-type-ATPases. Here we study primary-active transport via P-type ATPases using functional and structural analyses to demonstrate that four simultaneous residue substitutions transform the non-gastric H⁺/K⁺ pump, a strict H⁺-dependent electroneutral P-type ATPase, into a bona fide Na⁺-dependent electrogenic Na⁺/K⁺ pump. Conversion of a H⁺-dependent primary-active transporter into a Na⁺-dependent one provides a prototype for similar studies of ion-transport proteins. Moreover, we solve the structures of the wild-type non-gastric H⁺/K⁺ pump, a suitable drug target to treat cystic fibrosis, and of its Na⁺/K⁺ pump-mimicking mutant in two major conformations, providing insight on how Na⁺ binding drives a concerted mechanism leading to Na⁺/K⁺ pump phosphorylation.

SUBMITTER: Young VC

PROVIDER: S-EPMC9463140 | biostudies-literature | 2022 Sep

REPOSITORIES: biostudies-literature

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Structure and function of H+/K+ pump mutants reveal Na+/K+ pump mechanisms.

Young Victoria C VC Nakanishi Hanayo H Meyer Dylan J DJ Nishizawa Tomohiro T Oshima Atsunori A Artigas Pablo P Abe Kazuhiro K

Nature communications 20220909 1

Ion-transport mechanisms evolve by changing ion-selectivity, such as switching from Na+ to H+ selectivity in secondary-active transporters or P-type-ATPases. Here we study primary-active transport via P-type ATPases using functional and structural analyses to demonstrate that four simultaneous residue substitutions transform the non-gastric H+/K+ pump, a strict H+-dependent electroneutral P-type ATPase, into a bona fide Na+-depend ...[more]

PMID: 36085139

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Project description:Na/K pumps build essential ion gradients across the plasmalemma of animal cells by coupling the extrusion of three Na+, with the import of two K+ and the hydrolysis of one ATP molecule. The mechanisms of selectivity and competition between Na+, K+, and inhibitory amines remain unclear. We measured the effects of external tetrapropylammonium (TPA+) and ethylenediamine (EDA2+) on three different Na/K pump transport modes in voltage-clamped Xenopus oocytes: 1) outward pump current (IP), 2) passive inward H+ current at negative voltages without Na+ or K+ (IH), and 3) transient charge movement reporting the voltage-dependent extracellular binding/release of Na+ (QNa). Both amines competed with K+ to inhibit IP. TPA+ inhibited IH without competing with H+, whereas EDA2+ did not alter IH at pH 7.6. TPA+ competed with Na+ in QNa measurements, reducing Na+-apparent affinity, evidenced by a ∼-75 mV shift in the charge-voltage curve (at 20 mM TPA+) without reduction of the total charge moved (Qtot). In contrast, EDA2+ and K+ did not compete with Na+ to inhibit QNa; both reduced Qtot without decreasing Na+-apparent affinity. EDA2+ (15 mM) right-shifted the charge-voltage curve by ∼+50 mV. Simultaneous occlusion of EDA2+ and Na+ by an E2P conformation unable to reach E1P was demonstrated by voltage-clamp fluorometry. Trypsinolysis experiments showed that EDA2+-bound pumps are much more proteolysis-resistant than Na+-, K+-, or TPA+-bound pumps, therefore uncovering unique EDA2+-bound conformations. K+ effects on QNa and IH were also evaluated in pumps inhibited with beryllium fluoride, a phosphate mimic. K+ reduced Qtot without shifting the charge-voltage curve, indicating noncompetitive effects, and partially inhibited IH to the same extent as TPA+ in non-beryllium-fluorinated pumps. These results demonstrate that K+ interacts with beryllium-fluorinated pumps inducing conformational changes that alter QNa and IH, suggesting that there are two external access pathways for proton transport by IH.

Dataset Information

Structure and function of H⁺/K⁺ pump mutants reveal Na⁺/K⁺ pump mechanisms.

Publications

Structure and function of H<sup>+</sup>/K<sup>+</sup> pump mutants reveal Na<sup>+</sup>/K<sup>+</sup> pump mechanisms.

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