Abstract
Neurotransmitter:sodium symporters are highly expressed in the human brain and catalyze the uptake of substrate through the plasma membrane by using the electrochemical gradient of sodium as the energy source. The bacterial homolog LeuT, a small amino acid transporter isolated from the bacteria Aquifex aeolicus, is the founding member of the family and has been crystallized in three conformations. The N-terminus is structurally well defined and strongly interacts with the transporter core in the outward-facing conformations. However, it could not be resolved in the inward-facing conformation, which indicates enhanced mobility. Here we investigate conformations and dynamics of the N-terminus, by combining molecular dynamics simulations with experimental verification using distance measurements and accessibility studies. We found strongly increased dynamics of the N-terminus, but also that helix TM1A is subject to enhanced mobility. TM1A moves towards the transporter core in the membrane environment, reaching a conformation that is closer to the structure of LeuT with wild type sequence, indicating that the mutation introduced to create the inward-facing structure might have altered the position of helix TM1A. The mobile N-terminus avoids entering the open vestibule of the inward-facing state, as accessibility studies do not show any reduction of quenching by iodide of a fluorophore attached to the N-terminus.
Highlights
Neurotransmitter:sodium symporters (NSS) are secondary active transporters that belong to the solute carrier family 6 (SLC6), and include transporters for the neurotransmitters serotonin (SERT), dopamine (DAT), norepinephrine (NET) and γ-aminobutyric acid (GAT)
We found that the fluorescence signal of tetramethylrhodamine maleimide (TMR) conjugated to the LeuT mutants was quenched at the tested positions and comparable to quenching measured with the control residue R86C, which is known to be water exposed and insensitive to conformational changes (Fig. 6)
Substrates bind with high affinity to the central substrate binding site site 1 (S1), which is reachable through the open outer vestibule
Summary
Neurotransmitter:sodium symporters (NSS) are secondary active transporters that belong to the solute carrier family 6 (SLC6), and include transporters for the neurotransmitters serotonin (SERT), dopamine (DAT), norepinephrine (NET) and γ-aminobutyric acid (GAT). The small amino acid transporter LeuT from Aquifex aeolicus is a bacterial homolog of NSS transporters that has been crystalized in three different conformations [2,3,4]. The SLC6 transporters operate according to the alternating access model [8], which states that the substrate binding site within the transporter can be accessed from both sides of the membrane, but at any given time the path to the substrate binding site is only accessible from one side of the membrane This avoids the formation of a channel-like mode and imposes directionality of transport, in the presence of pertinent ion gradients
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
