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Dissecting the proton transport pathway in electrogenic Na+/H+ antiporters
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
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Number of Authors: 72017 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 114, no 7, p. E1101-E1110Article in journal (Refereed) Published
Abstract [en]

Sodium/proton exchangers of the SLC9 family mediate the transport of protons in exchange for sodium to help regulate intracellular pH, sodium levels, and cell volume. In electrogenic Na+/H+ antiporters, it has been assumed that two ion-binding aspartate residues transport the two protons that are later exchanged for one sodium ion. However, here we show that we can switch the antiport activity of the bacterial Na+/H+ antiporter NapA from being electrogenic to electroneutral by the mutation of a single lysine residue (K305). Electroneutral lysine mutants show similar ion affinities when driven by Delta pH, but no longer respond to either an electrochemical potential (psi) or could generate one when driven by ion gradients. We further show that the exchange activity of the human Na+/H+ exchanger NHA2 (SLC9B2) is electroneutral, despite harboring the two conserved aspartic acid residues found in NapA and other bacterial homologues. Consistently, the equivalent residue to K305 in human NHA2 has been replaced with arginine, which is a mutation that makes NapA electroneutral. We conclude that a transmembrane embedded lysine residue is essential for electrogenic transport in Na+/H+ antiporters.

Place, publisher, year, edition, pages
2017. Vol. 114, no 7, p. E1101-E1110
Keywords [en]
secondary active transporters, proton transport, membrane protein, Na+/H+ exchangers, energetics
National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-141418DOI: 10.1073/pnas.1614521114ISI: 000393989300010PubMedID: 28154142OAI: oai:DiVA.org:su-141418DiVA, id: diva2:1089122
Available from: 2017-04-18 Created: 2017-04-18 Last updated: 2019-08-14Bibliographically approved
In thesis
1. Establishing the molecular mechanism of sodium/proton exchangers
Open this publication in new window or tab >>Establishing the molecular mechanism of sodium/proton exchangers
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Sodium/proton exchangers are ubiquitous secondary active transporters that can be found in all kingdoms of life. These proteins facilitate the transport of protons in exchange for sodium ions to help regulate internal pH, sodium levels, and cell volume. Na+/H+ exchangers belong to the SLC9 family and are involved in many physiological processes including cell proliferation, cell migration and vesicle trafficking. Dysfunction of these proteins has been linked to physiological disorders, such as hypertension, heart failure, epilepsy and diabetes.

The goal of my thesis is to establish the molecular basis of ion exchange in Na+/H+ exchangers. By establishing how they bind and catalyse the movement of ions across the membrane, we hope we can better understand their role in human physiology.

In my thesis, I will first present an overview of Na+/H+ exchangers and their molecular mechanism of ion translocation as was currently understood by structural and functional studies when I started my PhD studies. I will outline our important contributions to this field, which were to (i) obtain the first atomic structures of the same Na+/H+ exchanger (NapA) in two major alternating conformations, (ii) show how a transmembrane embedded lysine residue is essential for carrying out electrogenic transport, and (iii) isolate and recorde the first kinetic data of a mammalian Na+/H+ exchanger (NHA2) in an isolated liposome reconstitution system.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2017. p. 47
Keywords
membrane protein, secondary active transporters, sodium/proton exchangers, proton transport, structure, energetics
National Category
Biochemistry and Molecular Biology Structural Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-147333 (URN)978-91-7649-964-1 (ISBN)978-91-7649-965-8 (ISBN)
Public defence
2017-11-14, William-Olssonsalen, Geovetenskapens hus, Svante Arrhenius väg 14, Stockholm, 10:00 (English)
Opponent
Supervisors
Available from: 2017-10-20 Created: 2017-09-22 Last updated: 2017-10-20Bibliographically approved
2. Structure and Biogenesis of Membrane Proteins
Open this publication in new window or tab >>Structure and Biogenesis of Membrane Proteins
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Membrane proteins make up about one-third of the cellular proteome. The diverse roles that membrane proteins have in cells include major life-sustaining processes, making them major drug targets. The respiratory chain comprises a series of complexes of membrane proteins residing in the inner mitochondrial membrane, which serve as major drivers of ATP synthesis. Assembly of the respiratory chain complexes (RCC) requires coordinated synthesis of nuclear and mitochondrial subunits. Cbp3-Cbp6 complex binds to the mitoribosome as translational activator for cytochrome b synthesis and binds the nascent polypeptide to facilitate its hemylation. Cbp3 consists of an N-terminal domain specific to mitochondrial homologues and a conserved C-terminal ubiquinol-cytochrome c chaperone domain. In this thesis I present the first crystal structure of the C-terminal domain from a bacterial homologue that has enabled us to identify the interaction sites of yeast Cbp3 with Cbp6 and cytochrome b using site-specific photo-crosslinking. Our finding suggests that Cbp3 contacts the mitoribosome via the N-terminal domain in a manner that positions the substrate binding site close to the tunnel exit. In the second project, we have analyzed the effects of disease causing cytochrome b mutations, on bc1 complex assembly. We found that complex III assembly is blocked at either intermediate 0 or I due to impaired insertion of bL or bH heme respectively, which indicates that assembly processes are involved in disease development. We then focused on NADH; a product of alpha-ketoglutarate dehydrogenase complex (KGDH) catalyzed citric acid cycle reaction and one of the substrates that supply electron to the respiratory chain. Kgd4 is a novel subunit of this enzyme complex and two functional variants (Kgd4S and Kgd4L) of unknown origins exist in yeast. We report in our work that Kgd4L originates from a UUG alternative start site, 90 nucleotides upstream and in frame of the annotated start codon. The sequence context upstream of UUG determines the efficiency of recognition of this alternative start codon. Finally, Na+/H+ antiporters are present in all species and are involved in regulation of intracellular pH, cell volume and sodium concentration. ATP formed during oxidative phosphorylation serves as energy source for Na+/K+ ATPase to generate Na+ gradient across the inner mitochondrial membrane, which drives local Na+/H+ antiporters. We show that K305 is involved in proton transport and responsible for the electrogenicity of NapA, while human NHA2 shows electroneutral antiporter activity.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2019. p. 53
Keywords
Cbp3, cytochrome b, respiratory complex III, alternative translation initiation and sodium/proton exchange
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-171519 (URN)978-91-7797-749-0 (ISBN)978-91-7797-750-6 (ISBN)
Public defence
2019-09-26, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 13:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 1: Manuscript. Paper 2: Manuscript.

Available from: 2019-09-03 Created: 2019-08-13 Last updated: 2019-08-26Bibliographically approved

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