Dokument: Role of extracellular gate in cation coupling in the glutamate transporter family

Titel:	Role of extracellular gate in cation coupling in the glutamate transporter family
URL für Lesezeichen:	https://docserv.uni-duesseldorf.de/servlets/DocumentServlet?id=54165
URN (NBN):	urn:nbn:de:hbz:061-20210915-094839-1
Kollektion:	Dissertationen
Sprache:	Englisch
Dokumententyp:	Wissenschaftliche Abschlussarbeiten » Dissertation
Medientyp:	Text
Autor:	Alleva, Claudia [Autor]
Dateien:	[Dateien anzeigen] Adobe PDF [Details] 11,76 MB in einer Datei [ZIP-Datei erzeugen] Dateien vom 09.09.2020 / geändert 09.09.2020
Beitragende:	Prof. Dr. Fahlke, Christoph [Gutachter] Prof. Dr. Gohlke, Holger [Gutachter]
Dewey Dezimal-Klassifikation:	500 Naturwissenschaften und Mathematik » 570 Biowissenschaften; Biologie
Beschreibung:	Excitatory aminoacid transporters(EAATs)terminate synaptic transmission by taking up glutamate from the synaptic cleft and moving it back into nearby neurons and glial cells.Thereby, EAATs ensure low extracellular glutamate concentrations in the brain and permit reliable high frequency signal transduction in excitatory synapses. Under pathological conditions, e.g. ischemia, impaired EAATs function leads to accumulation of glutamate in the synaptic cleft, causing glutamate excitotoxic effects. Concentrative glutamate uptake is achieved through a secondary active transport cycle, which couples the uptake of one glutamate molecule to the co-transport of three Na+, and one H+, with the counter-transport of one K+. This coupling stoichiometry permits glutamate accumulation up to 106-fold gradients. X-ray crystal structures of the prokaryotic EAAT homologs GltPh and GltTk, and the human glutamate transporter EAAT1 have revealed the position of the substrate- and Na+-binding sites, and defned two hairpin-like structures that serve as gates to regulate access to the substrate-bindingpocket. Glutamate transport involves at least two major conformational changes: extracellular gate opening and closing, and transmembrane translocation of the transport domain harboring thesubstrate and ion-binding sites. It has recently been established that substrate binding causes subsequent extracellular gateclosure, and this induced-fit binding mechanism has been shown toconfer substrate selectivity. In contrast, the coupling between cation binding and conformational changes is still unclear. Here, we perform extensive all-atom molecular dynamics simulations to study the coupling mechanisms between Na+ , K+, and H+ binding and extracellular gate dynamics. Our molecular dynamics simulations suggest that the binding of two Na+ prior to substrate binding induces gate opening via a conformational selection-like mechanism, and we define the allosteric interaction network underlying this Na+-gate coupling. We confirmed our results through stopped flow fluorescence spectroscopy experiments, and we identified two point mutations, M311A and R397A in GltPh that uncouple the Na+ binding from gate dynamics by selectively altering Na+ binding or the Na+-induced gate opening. The coupling mechanism involves Na+ binding to the so-called Na1 and Na3 sites: Na+ binding at Na1 is preceded by a binding-pocket rearrangement, while Na3 binding involves passage through the Na1 site. We show that only occupation of both sites ensures complete gate opening to permit subsequent substrate binding. Moreover, we show that K+ binding is also coupled to extracellular gate dynamics, but exerting an opposite effect to Na+ binding and causing hairpin closure in EAAT1. Similarly to Na+, the coupling to K+ is driven by allosteric interactions, which we demonstrate by disrupting it with mutations that destabilize the open state (E386Q, E386D, and R459A EAAT1) or increase the flexibility of the hairpin (L428A EAAT1). Finally, we show the physiological role of the putative H+-acceptor site, demonstrating that protonating it stabilizes the closed state in the extracellular gate. In summary, Na+ binding causes extracellular gate opening via a conformational selection-like mechanism, which permits induced-fit substrate binding causing gate re-closure, which finally permits transmembrane translocation. Our analysis reveals how the energetic coupling of the extracellular gate to ion and substrate binding forms the molecular basis for secondary active glutamate transport.
Lizenz:	Urheberrechtsschutz
Fachbereich / Einrichtung:	Mathematisch- Naturwissenschaftliche Fakultät
Dokument erstellt am:	15.09.2021
Dateien geändert am:	15.09.2021
Promotionsantrag am:	30.08.2018
Datum der Promotion:	06.09.2019