Latch and trigger role for R445 in DAT transport explains molecular basis of DTDS

Area of Science:

• Neuroscience
• Molecular Biology
• Biochemistry

Background:

• Dopamine transporter (DAT) deficiency syndrome (DTDS) is linked to various mutations.
• The R445C mutation's mechanism causing DAT deficiency was unclear, as it's located away from known substrate binding sites.

Purpose of the Study:

• To elucidate the molecular mechanism by which the R445C mutation leads to DAT transport deficiency.
• To investigate the role of a putative salt bridge between R445 and E428 in DAT function.

Main Methods:

• Generation of DAT mutants: R445E, E428R, and the double mutant E428R/R445E.
• Assessment of substrate ([3H]DA) and inhibitor ([3H]CFT) binding and transport properties at the cell surface for wild-type and mutant DAT.
• Structure-based analysis to model the role of R445 in DAT function.

Main Results:

• Single mutants R445E and E428R showed a loss of substrate and inhibitor binding and transport.
• The double mutant E428R/R445E, while nonfunctional, restored binding affinity to wild-type levels.
• R445 appears to play a dual role: forming a salt bridge with E428 to stabilize the inward-closed state and acting as a trigger to open the transporter.

Conclusions:

• The R445C mutation likely impairs DAT function by disrupting the R445-E428 salt bridge, affecting transporter conformation and substrate binding.
• This salt bridge is critical for maintaining the DAT's inward-closed state and facilitating substrate transport.
• The study provides a structural model explaining how R445C causes DAT deficiency, impacting substrate binding and transport.