(581a) Elucidating Mechanisms of Substrate Transport in Neurotransmitter Transporters

The serotonin transporter, SERT, catalyzes serotonin reuptake at the synapse to terminate neurotransmission via an alternating access mechanism, and SERT inhibitors are the most widely prescribed antidepressants. Comprehensive simulations of the entire ion-coupled import process have illuminated protein dynamics and binding events at atomic resolution, yet experimental validation remains incomplete. Here, deep mutagenesis is used to determine the effects of nearly all amino acid substitutions on human SERT surface expression and transport of the fluorescent substrate analogue APP+. Simulations show that while the native substrate, serotonin promotes SERT dynamics, the conformational free energy landscape in the presence of APP+ instead resembles Na⁺ bound-SERT, with higher free energy barriers for isomerization to an inward-facing state. The deep mutational scan for SERT-catalyzed import of APP+ identifies mutations that impose dynamic changes that would otherwise be facilitated by the native substrate. Indeed, hundreds of gain-of-function mutations for APP+ import are found along the permeation pathway, most notably mutations that favor opening of a solvent-exposed intracellular vestibule. The mutagenesis data support the simulated mechanism in which metal ions bind to two sites, with the neurotransmitter and a symported sodium sharing a common cytosolic exit pathway to achieve coupling. Furthermore, the mutational landscape for SERT surface trafficking, which likely filters out misfolded sequences, reveals that residues along the permeation pathway are mutationally tolerant, providing plausible evolutionary pathways for changes in transporter properties while maintaining folded structure.