How structural elements evolving from bacterial to human SLC6 transporters enabled new functional properties

Area of Science:

• Neuroscience
• Molecular Biology
• Evolutionary Biology

Background:

• Structure-function studies of SLC6A neurotransmitter transporters heavily rely on bacterial LeuT-fold homologs.
• Eukaryotic transporters possess unique N- and C-termini absent in bacterial counterparts, conferring expanded functional properties.
• Mechanistic understanding of how these eukaryotic-specific structural elements influence transporter function remains limited.

Purpose of the Study:

• To elucidate the evolutionary mechanisms by which extended termini in eukaryotic transporters enable novel functions.
• To investigate the role of the N- and C-termini of the human dopamine transporter (hDAT) in regulating transporter activity and mediating efflux.

Main Methods:

• Large-scale molecular dynamics simulations of wild-type and mutant hDAT.
• Comparative Markov state model analysis.
• Integration with experimentally determined transporter properties.

Main Results:

• Identified distinct roles for the proximal and distal segments of the hDAT N-terminus in regulating transporter function and mediating efflux.
• Demonstrated that N- and C-termini interactions contribute to phosphorylation-dependent regulation and reverse uptake (efflux).
• Provided quantitative insights into mechanisms underlying transporter regulation and novel phenotypes.

Conclusions:

• Eukaryotic transporter termini evolved to be larger, facilitating functions like efflux not present in bacterial homologs.
• Specific segments of the N-terminus play differential roles in membrane lipid adaptation and efflux modulation.
• These adaptations are critical for eukaryotic transporter function across diverse cellular membrane environments.