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The intracellular lipid-binding domain of human Na⁺/H⁺ exchanger 1 forms a lipid-protein co-structure essential for activity.

ABSTRACT: Dynamic interactions of proteins with lipid membranes are essential regulatory events in biology, but remain rudimentarily understood and particularly overlooked in membrane proteins. The ubiquitously expressed membrane protein Na⁺/H⁺-exchanger 1 (NHE1) regulates intracellular pH (pH_i) with dysregulation linked to e.g. cancer and cardiovascular diseases. NHE1 has a long, regulatory cytosolic domain carrying a membrane-proximal region described as a lipid-interacting domain (LID), yet, the LID structure and underlying molecular mechanisms are unknown. Here we decompose these, combining structural and biophysical methods, molecular dynamics simulations, cellular biotinylation- and immunofluorescence analysis and exchanger activity assays. We find that the NHE1-LID is intrinsically disordered and, in presence of membrane mimetics, forms a helical ??-hairpin co-structure with the membrane, anchoring the regulatory domain vis-a-vis the transport domain. This co-structure is fundamental for NHE1 activity, as its disintegration reduced steady-state pH_i and the rate of pH_i recovery after acid loading. We propose that regulatory lipid-protein co-structures may play equally important roles in other membrane proteins.

SUBMITTER: Hendus-Altenburger R

PROVIDER: S-EPMC7713384 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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The intracellular lipid-binding domain of human Na+/H+ exchanger 1 forms a lipid-protein co-structure essential for activity.

Hendus-Altenburger Ruth R Vogensen Jens J Pedersen Emilie Skotte ES Luchini Alessandra A Araya-Secchi Raul R Bendsoe Anne H AH Prasad Nanditha Shyam NS Prestel Andreas A Cardenas Marité M Pedraz-Cuesta Elena E Arleth Lise L Pedersen Stine F SF Kragelund Birthe B BB

Communications biology 20201203 1

Dynamic interactions of proteins with lipid membranes are essential regulatory events in biology, but remain rudimentarily understood and particularly overlooked in membrane proteins. The ubiquitously expressed membrane protein Na+/H+-exchanger 1 (NHE1) regulates intracellular pH (pHi) with dysregulation linked to e.g. cancer and cardiovascular diseases. NHE1 has a long, regulatory cytosolic domain carrying a membrane-proximal region described as a lipid-interact ...[more]

PMID: 33273619

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Project description:Fully-activated Na+/H+ exchanger-1 (NHE1) generates the cardiomyocyte's largest trans-membrane extrusion of H+ ions for an equimolar influx of Na+ ions. This has the desirable effect of clearing excess intracellular acidity, but comes at a large energetic premium because the exchanged Na+ ions must ultimately be extruded by the sodium pump, a process that consumes the majority of the heart's non-contractile ATP. We hypothesize that the state of NHE1 activation depends on metabolic resources, which become limiting in periods of myocardial hypoxia. To test this functionally, NHE1 activity was measured in response to in vitro and in vivo hypoxic treatments. NHE1 flux was interrogated as a function of intracellular pH by fluorescence imaging of rodent ventricular myocytes loaded with pH-sensitive dyes BCECF or cSNARF1. Anoxic superfusates promptly inhibited NHE1, tracking the time-course of mitochondrial depolarization. Mass spectrometry of NHE1 immuno-precipitated from Langendorff-perfused anoxic hearts identified Tyr-581 dephosphorylation and Tyr-561 phosphorylation. The latter residue is part of the domain that interacts with phosphatidylinositol 4,5-bisphosphate (PIP2), a membrane lipid that becomes depleted under metabolic inhibition. Tyr-561 phosphorylation is expected to electrostatically weaken this activatory interaction. To test if a period of hypoxia produces a persistent inhibition of NHE1, measurements under normoxia were performed on myocytes that had been incubated in 2% O2 for 4 h. NHE1 activity remained inhibited, but the effect was ablated in the presence of Dasatinib, an inhibitor of Abl/Src-family tyrosine kinases. Chronic tissue hypoxia in vivo, attained in a mouse model of anemic hypoxia, also resulted in persistently slower NHE1. In summary, we show that NHE1 responds to oxygen, a physiologically-relevant metabolic regulator, ostensibly to divert ATP for contraction. We describe a novel mechanism of NHE1 inhibition that may be relevant in cardiac disorders featuring altered oxygen metabolism, such as myocardial ischemia and reperfusion injury.

Dataset Information

The intracellular lipid-binding domain of human Na⁺/H⁺ exchanger 1 forms a lipid-protein co-structure essential for activity.

Publications

The intracellular lipid-binding domain of human Na<sup>+</sup>/H<sup>+</sup> exchanger 1 forms a lipid-protein co-structure essential for activity.

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