Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

J Vis Exp. 2016 Nov 27:(117):54792. doi: 10.3791/54792.

Abstract

The serotonin transporter is a sodium and chloride-coupled transporter that "pumps" extracellular serotonin into cells. S-citalopram is a drug used to treat depression and anxiety by binding to the serotonin transporter with high-affinity, blocking serotonin reuptake. Here we report an efficient procedure and a set of tools to stabilize, express, purify, and crystallize serotonin transporter-antibody complexes bound to S-citalopram and other antidepressants. Mutations which stabilize the serotonin transporter were identified using an S-citalopram binding assay. Serotonin transporter expressed in baculovirus-transduced HEK293S GnTI- cells, was reconstituted into proteoliposomes and used to raise high-affinity antibodies. We have developed a strategy to discover antibodies that are useful for structural studies. A straightforward approach for the expression of antibody fragments in Sf9 cells has also been established. Transporter-antibody complexes purified using this procedure are well-behaved and readily crystallize, producing complexes with S-citalopram that diffract X-rays to 3-4 Å resolution. The strategies developed here can be utilized to determine the structure of other challenging membrane proteins.

Publication types

  • Video-Audio Media

MeSH terms

  • Citalopram / metabolism*
  • Crystallization
  • Humans
  • Protein Binding
  • Selective Serotonin Reuptake Inhibitors
  • Serotonin
  • Serotonin Plasma Membrane Transport Proteins* / chemistry
  • Serotonin Plasma Membrane Transport Proteins* / isolation & purification
  • Serotonin Plasma Membrane Transport Proteins* / metabolism

Substances

  • SLC6A4 protein, human
  • Serotonin Plasma Membrane Transport Proteins
  • Serotonin Uptake Inhibitors
  • Citalopram
  • Serotonin