Interrogating the Role of Extracellular Loops 2 and 4 (EL2 and EL4) of the Serotonin Transporter with Antibody Fragments
Authors: Esendir Eray , Burtscher Verena , Sandtner Walter , Freissmuth Michael 
Published: April 2020

The serotonin transporter (SERT) mediates its eponymous action, i.e. the uptake of serotonin (5‐HT), by relying on the electrochemical gradient of sodium as the driving force. By retrieving 5‐HT from the synaptic cleft, SERT regulates serotonergic signaling in relay with the vesicular monoamine transporters, which results in refilling of presynaptic 5‐HT stores. SERT is an integral membrane protein comprising 12 transmembrane segments linked by 6 extracellular (EL) and 5 intracellular loops (IL). Crystallography studies employing antigen binding fragments (Fabs) directed against extracellular loop 2 (EL2) and extracellular loop 4 (EL4) of SERT indicate that EL4 may act as a lid which occludes the extracellular vestibule and limits the access to the substrate binding site (S1‐site). Our working hypothesis posited that restricting the mobility of EL4 (but not of EL2) by the antibody or the F(ab) impeded the conformational rearrangement required for the forward transport mode. The conformational switch associated with substrate translocation and the cycling of the transporter can be monitored by recording 5‐HT‐induced currents through human SERT expressed in HEK293 cells in the patch‐clamp, whole‐cell configuration. Accordingly, we explored, if 5‐HT‐induced currents were reversibly inhibited by an antibody and F(abs) against EL4 and/or EL2 of SERT. In addition, binding of F(ab)s and 5‐HT to SERT were assessed by capacitance measurements. Association and dissociation rate constants (kon and koff) were calculated for the antibody and for the F(ab) against EL4 as 3.1x107 M−1*s−1 and 1.4 s−1 and 2.0x106 M−1*s−1 and 0.02 s−1, respectively. Consistent with the working hypothesis, occupancy of EL4 by an antibody or a F(ab) fragment reduced the peak current associated with initial substrate translocation by 60%. Sole binding of EL2‐Fab and EL4‐Fab had no effect and a modest effect (inhibition by 16%) of substrate transport, respectively. In contrast, simultaneous binding of Fabs to EL2 and EL4 inhibited 5‐HT transport by 64%. Because restriction of EL4 movement impeded entry of into the forward cycling mode, we conclude that EL4 plays a role in the transition of SERT from the outward open to the occluded state during transport. In addition, because inhibition of transport was more pronounced upon concomitant binding of both Fabs, we surmise that EL2 also undergoes a conformational rearrangement during 5‐HT translocation presumably by interacting with EL4 in the occluded state. The selectivity of inhibitors for SERT over DAT is known to be specified by their association rates. This implies a selectivity filter in SERT. Our current observations are consistent with a model, where EL4 participates in limiting the access pathway, because the affinity of [3H]imipramine is increased by EL4‐Fab. Hence, EL4 may contribute to the formation of the selectivity filter.Support or Funding InformationThis study is supported by the doctoral program CCHD jointly funded by the Austrian Science Fund FWF and the Medical University of Vienna.