Unraveling the mechanism of glutamate transport

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Glutamate concentration in the synaptic cleft is tightly regulated by a family of secondary active transporters called excitatory amino acid transporters (EAAT1-5) that are able to symport three Na+ ions and one H+ and counter-transport one K+ ion, with uncoupled chloride conductance. In this thesis, the studies have been conducted on the archeal homologue, GltTk.
During the initial steps of binding, the Na+ ions entering the binding pocket from the bulk most likely follow a series of transient bindings with other amino acid residues before reaching their final states. This was revealed by combination of atomistic-scale MD simulations including the application of Markov state model and functional studies.
I present the attempts of creating larger nanodiscs for structural purposes using three different approaches: two by extracting the protein directly from the membranes with the diisobutylene/maleic acid copolymer, called DIBMA, and one by changing the reconstitution ratios of the protein with the protein belt MSP2N2 and the lipids.
During the transport cycle, an aqueous channel between the scaffold and the transport domain forms and it allows the thermodynamically uncoupled anionic flux. This state is called the chloride-conductive state (ClCS). I present the characterization of the chloride channel in GltTk using solid-supported membrane electrophysiology (SSM). This same electrophysiological method was used to study the implications of the single point mutation of the only proline present in TM 5 to arginine, that in humans is responsible for the neurological disorder called episodic ataxia 6.
Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • University of Groningen
  • Guskov, Albert, Supervisor
  • Slotboom, Dirk, Supervisor
Award date6-Feb-2023
Place of Publication[Groningen]
Publication statusPublished - 2023

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