Substrate and Inhibitor–Specific Conformational Changes in the Human Serotonin Transporter Revealed by Voltage-Clamp Fluorometry

Abstract

The serotonin transporter (SERT) regulates neurotransmission by the biogenic monoamine neurotransmitter serotonin (5-HT, 5-hydroxytryptamine) in the central nervous system, and drugs inhibiting SERT are widely used for the treatment of a variety of central nervous system diseases. The conformational dynamics of SERT transport function and inhibition is currently poorly understood. We used voltage-clamp fluorometry to study conformational changes in human SERT (hSERT) during 5-HT transport and inhibitor binding. Cys residues were introduced at 12 positions in hSERT to enable covalent attachment of a rhodamine-based fluorophore. Transport-associated changes in fluorescence from fluorophore-labeled hSERT expressed in Xenopus oocytes could be robustly detected at four positions in hSERT: endogenous Cys109 in the top of transmembrane domain (TM) 1b, Cys substituted for Thr323 in the top of TM6, Ala419 in the interface between TM8 and extracellular loop (EL) 4, and Leu481 in EL5. The reporter positions were used for timeresolved measurement of conformational changes during 5-HT transport and binding of cocaine and the selective serotonin reuptake inhibitors fluoxetine and escitalopram. At all reporter positions, fluorescence changes observed upon substrate application were distinctly different from those observed upon inhibitor application, with respect to relative amplitude or direction. Furthermore, escitalopram, fluoxetine, and cocaine induced a very similar pattern of fluorescent changes overall, which included movements within or around TM1b, EL4, and EL5. Taken together, our data lead us to suggest that competitive inhibitors stabilize hSERT in a state that is different from the apo outward-open conformation as well as inward-facing conformations.

Deep Mutagenesis of a Transporter for Uptake of a Non-Native Substrate Identifies Conformationally Dynamic Regions

“…Na1 site, followed by a second Na + occupying the Na2 site. APP+ then enters the transporter through the extracellular vestibule and moves to the orthosteric site, with localized dynamics around extracellular loop 4 (EL4) and the extracellular gate, consistent with static structures (18,19), HDX data (42,43), and voltage-clamp fluorometry (84). Unlike the endogenous substrate 5-HT, the increased structural rigidity of APP+ (Figure S1) results in its translocation 'down' the extracellular vestibule to be more constrained, with decreased rotational freedom.…”

Conformational Changes in the 5-HT3A Receptor Extracellular Domain Measured by Voltage-Clamp Fluorometry

“…Electrophysiology and Voltage-Clamp Fluorometry. VCF recordings were performed essentially as described previously (Söderhielm et al, 2015). A glass bottom perfusion chamber was mounted on an inverted epifluorescence microscope (Diaphot 300; Nikon, Japan) equipped with a 20Â air objective and a G-1B filter set (Chroma 11002v2, excitation filter 541-551 nm, dichroic mirror .565 nm, emission .590 nm).…”

A Novel Bromine-Containing Paroxetine Analogue Provides Mechanistic Clues for Binding Ambiguity at the Central Primary Binding Site of the Serotonin Transporter

“…Human SERT (hSERT) is the primary target for the selective serotonin reuptake inhibitors (SSRIs), such as the U.S. Food and Drug Administration (FDA)-approved and widely prescribed escitalopram and paroxetine, both of which competitively inhibit 5-HT transport by stabilizing hSERT in an outward-open conformation . Of all the SSRIs, paroxetine binds with the highest affinity to hSERT ( K i < 1 nM) and is highly selective. , It is one of the most therapeutically useful SSRIs, prescribed for the treatment of a wide array of neuropsychiatric disorders such as depression, panic disorder, and obsessive-compulsive disorder .…”