Selective Serotonin Reuptake Inhibitors Within Cells: Temporal Resolution in Cytoplasm, Endoplasmic Reticulum, and Membrane

bioRxiv (Cold Spring Harbor Laboratory)

Abstract

ABSTRACT Selective serotonin reuptake inhibitors (SSRIs) are the most prescribed treatment for individuals experiencing major depressive disorder (MDD). The therapeutic mechanisms that take place before, during, or after SSRIs bind the serotonin transporter (SERT) are poorly understood, partially because no studies exist of the cellular and subcellular pharmacokinetic properties of SSRIs in living cells. We studied escitalopram and fluoxetine using new intensity- based drug-sensing fluorescent reporters (“iDrugSnFRs”) targeted to the plasma membrane (PM), cytoplasm, or endoplasmic reticulum (ER) of cultured neurons and mammalian cell lines. We also employed chemical detection of drug within cells and phospholipid membranes. The drugs attain equilibrium in neuronal cytoplasm and ER, at approximately the same concentration as the externally applied solution, with time constants of a few s (escitalopram) or 200-300 s (fluoxetine). Simultaneously, the drugs accumulate within lipid membranes by ≥ 18-fold (escitalopram) or 180-fold (fluoxetine), and possibly by much larger factors. Both drugs leave cytoplasm, lumen, and membranes just as quickly during washout. We synthesized membrane-impermeant quaternary amine derivatives of the two SSRIs. The quaternary derivatives are substantially excluded from membrane, cytoplasm, and ER for > 2.4 h. They inhibit SERT transport-associated currents 6- or 11-fold less potently than the SSRIs (escitalopram or fluoxetine derivative, respectively), providing useful probes for distinguishing compartmentalized SSRI effects. Although our measurements are orders of magnitude faster than the “therapeutic lag” of SSRIs, these data suggest that SSRI-SERT interactions within organelles or membranes may play roles during either the therapeutic effects or the “antidepressant discontinuation syndrome”. SIGNIFICANCE STATEMENT Selective serotonin reuptake inhibitors stabilize mood in several disorders. In general, these drugs bind to the serotonin (5-hydroxytryptamine) transporter (SERT), which clears serotonin from CNS and peripheral tissues. SERT ligands are effective and relatively safe; primary care practitioners often prescribe them. However, they have several side effects and require 2 to 6 weeks of continuous administration until they act effectively. How they work remains perplexing, contrasting with earlier assumptions that the therapeutic mechanism involves SERT inhibition followed by increased extracellular serotonin levels. This study establishes that two SERT ligands, fluoxetine and escitalopram, enter neurons within minutes, while simultaneously accumulating in many membranes. Such knowledge will motivate future research, hopefully revealing where and how SERT ligands “engage” their therapeutic target(s).

Authors 15

1. Aaron L. Nichols

   California Institute of Technology

   Affiliation as printed

   Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91106

   Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 906

2. Zack Blumenfeld

   University of Southern California · California Institute of Technology

   Affiliation as printed

   Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91106

   Keck School of Medicine, University of Southern California, Los Angeles, CA 90007

   Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 906

3. Laura Luebbert Aachen Institute of Biology, Leiden University

   Leiden University · California Institute of Technology

   Affiliation as printed

   Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91106

   Institute of Biology, Leiden University, Leiden, The Netherlands

   Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 906

4. Hailey J. Knox

   California Institute of Technology

   Affiliation as printed

   Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91106

5. Anand K. Muthusamy

   California Institute of Technology

   Affiliation as printed

   Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91106

6. Jonathan S. Marvin

   Howard Hughes Medical Institute · Janelia Research Campus

   Affiliation as printed

   Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147

7. Charlene H. Kim

   California Institute of Technology

   Affiliation as printed

   Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91106

   Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 906

8. Stephen N. Grant

   California Institute of Technology

   Affiliation as printed

   Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91106

9. David P. Walton

   California Institute of Technology

   Affiliation as printed

   Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91106

10. Bruce N. Cohen

    California Institute of Technology

    Affiliation as printed

    Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91106

    Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 906

11. Rebekkah Hammar

    Uppsala University

    Affiliation as printed

    Department of Pharmacy, Uppsala University, Uppsala, Sweden

12. Loren L. Looger

    Howard Hughes Medical Institute · Janelia Research Campus

    Affiliation as printed

    Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147

13. Per Artursson

    Uppsala University · Science for Life Laboratory

    Affiliation as printed

    Department of Pharmacy, Uppsala University, Uppsala, Sweden

    Science for Life Laboratory Drug Discovery and Development platform (SciLifelab DDD-P), and Uppsala University Drug Optimization and Pharmaceutical Profiling Platform (UDOPP), Uppsala University, Uppsala, Sweden

14. Dennis A. Dougherty

    California Institute of Technology

    Affiliation as printed

    Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91106

15. Henry A. Lester corresponding

    California Institute of Technology

    Affiliation as printed

    Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91106

    Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 906