Cytosolic K⁺ Binding to the Human Serotonin Transporter

Area of Science:

• Neuroscience
• Structural Biology
• Biochemistry

Background:

• The human serotonin transporter (hSERT) is crucial for serotonin reuptake, operating via alternating outward-facing (OF) and inward-facing (IF) states.
• Cytosolic potassium (K+) binding is proposed to facilitate the hSERT's return to the OF state, but its mechanism and binding site remain unclear.
• Recent studies challenge conventional transport stoichiometry, suggesting chloride (Cl-) may remain bound throughout the cycle.

Purpose of the Study:

• To investigate the role and mechanism of cytosolic K+ binding to the inward-facing (IF) state of hSERT.
• To identify the specific binding site and kinetics of K+ interaction with hSERT.
• To explore the implications of K+ binding for the transporter's conformational transitions and transport cycle.

Main Methods:

• Extensive molecular dynamics (MD) simulations of hSERT in the presence of cytosolic K+.
• Generation of 50 independent 200 ns trajectories starting from the post-release IF conformation.
• Markov state model (MSM) analysis of ion dynamics to quantify binding kinetics and identify dominant states.

Main Results:

• Spontaneous K+ binding was observed at the Na2 site in over half of the simulations.
• MSM analysis identified K+ occupancy of the Na2 site, with Na+ retained at Na1, as the dominant post-release state.
• Chloride (Cl-) remained bound to hSERT in most simulations, suggesting limited Cl- release during this phase.

Conclusions:

• Cytosolic K+ binding to the Na2 site is a key event in the hSERT transport cycle.
• This K+ binding likely facilitates the transition from the IF to the OF state, resetting the transporter.
• The findings provide a mechanistic framework for K+ regulation of hSERT function and challenge previous transport models.