Fluorescent non-canonical amino acid provides insight into the human serotonin transporter.

Publisher: Nature Pub. Group Country of Publication: England NLM ID: 101528555 Publication Model: Electronic Cited Medium: Internet ISSN: 2041-1723 (Electronic) Linking ISSN: 20411723 NLM ISO Abbreviation: Nat Commun Subsets: MEDLINE

Original Publication: [London] : Nature Pub. Group

Serotonin Plasma Membrane Transport Proteins*/metabolism ; Serotonin Plasma Membrane Transport Proteins*/genetics ; Serotonin Plasma Membrane Transport Proteins*/chemistry ; Serotonin*/metabolism ; Amino Acids*/metabolism; Humans ; Sodium/metabolism ; Protein Binding ; Protein Conformation ; Fluorescent Dyes/chemistry ; Fluorescent Dyes/metabolism ; Binding Sites ; HEK293 Cells ; Allosteric Regulation

The serotonin transporter (SERT), responsible for the reuptake of released serotonin, serves as a major target for antidepressants and psychostimulants. Nevertheless, refining the mechanistic models for SERT remains challenging. Here, we expand the molecular understanding of the binding of ions, substrates, and inhibitors to SERT by incorporating the fluorescent non-canonical amino acid Anap through genetic code expansion. We elucidate steady-state changes in conformational dynamics of purified SERT with Anap inserted at intracellular- or extracellular sites. This uncovers the competitive mechanisms underlying cation binding and assigns distinct binding- and allosteric coupling patterns for several inhibitors and substrates. Finally, we track in real-time conformational transitions in response to the interaction with Na ⁺ or serotonin. In this work, we present a methodological platform reporting on SERT conformational dynamics, which together with other approaches will deepen our insights into the molecular mechanisms of SERT.
(© 2024. The Author(s).)

Biochemistry. 1997 Feb 11;36(6):1479-86. (PMID: 9063896)
J Biol Chem. 2016 May 6;291(19):9939-47. (PMID: 26969165)
Nucleic Acids Res. 2022 Jul 5;50(W1):W276-W279. (PMID: 35412617)
Neuron. 2024 Jun 5;112(11):1815-1831.e4. (PMID: 38492574)
Proc Natl Acad Sci U S A. 2013 May 14;110(20):8272-7. (PMID: 23630265)
Nat Commun. 2022 May 4;13(1):2446. (PMID: 35508541)
Elife. 2024 Jan 25;12:. (PMID: 38271216)
Nat Commun. 2019 Apr 11;10(1):1687. (PMID: 30976000)
Chembiochem. 2016 Apr 1;17(7):529-53. (PMID: 26789551)
Nat Struct Mol Biol. 2019 Aug;26(8):686-694. (PMID: 31285608)
Nature. 2024 Aug;632(8026):930-937. (PMID: 39085602)
Nature. 2019 May;569(7754):141-145. (PMID: 31019304)
J Biol Chem. 2014 Jan 17;289(3):1825-40. (PMID: 24293367)
J Phys Chem B. 2014 Aug 28;118(34):10085-91. (PMID: 25079965)
Nature. 2005 Sep 8;437(7056):215-23. (PMID: 16041361)
Proc Natl Acad Sci U S A. 2023 Jul 18;120(29):e2304602120. (PMID: 37436958)
J Biol Chem. 2001 Aug 17;276(33):30942-7. (PMID: 11408487)
Chembiochem. 2017 Mar 16;18(6):511-514. (PMID: 28093840)
ACS Chem Biol. 2017 Mar 17;12(3):805-813. (PMID: 28121133)
Biophys J. 1996 Dec;71(6):3126-35. (PMID: 8968583)
ACS Chem Neurosci. 2017 May 17;8(5):1011-1018. (PMID: 27959497)
J Biol Chem. 1979 Oct 25;254(20):10084-9. (PMID: 489585)
Biochemistry. 1978 Oct 31;17(22):4739-42. (PMID: 728383)
Nature. 2017 Oct 4;550(7674):53-60. (PMID: 28980641)
Nat Struct Mol Biol. 2019 Jul;26(7):545-556. (PMID: 31270469)
Sci Adv. 2021 Dec 03;7(49):eabl3857. (PMID: 34851672)
Nat Protoc. 2012 Sep;7(9):1569-78. (PMID: 22864198)
Sci Adv. 2018 May 11;4(5):eaar6179. (PMID: 29756037)
J Biol Chem. 2006 Jan 27;281(4):2012-23. (PMID: 16272152)
Assay Drug Dev Technol. 2022 Feb-Mar;20(2):83-94. (PMID: 35171002)
Sci Rep. 2015 Jul 08;5:11866. (PMID: 26153354)
J Am Chem Soc. 2014 Nov 5;136(44):15577-83. (PMID: 25350841)
Proc Natl Acad Sci U S A. 1992 Mar 1;89(5):1817-21. (PMID: 1347426)
Nature. 2016 Apr 21;532(7599):334-9. (PMID: 27049939)
Int J Mol Sci. 2019 Oct 28;20(21):. (PMID: 31661895)
J Biol Chem. 2010 Dec 10;285(50):39201-10. (PMID: 20889976)
Front Neurosci. 2019 Feb 27;13:156. (PMID: 30872996)
RNA Biol. 2015;12(9):950-8. (PMID: 26176195)
J Am Chem Soc. 2013 Aug 28;135(34):12540-3. (PMID: 23924161)
J Gen Physiol. 2016 Feb;147(2):201-16. (PMID: 26755770)
Methods Enzymol. 2021;654:49-84. (PMID: 34120725)
Nature. 2024 Aug;632(8025):678-685. (PMID: 39112703)
Nat Methods. 2007 Mar;4(3):239-44. (PMID: 17322890)
J Am Chem Soc. 2009 Sep 16;131(36):12921-3. (PMID: 19702307)
J Gen Physiol. 2019 Nov 4;151(11):1248-1264. (PMID: 31570504)
J Biol Chem. 2007 Oct 5;282(40):29441-7. (PMID: 17698848)
J Biol Chem. 2014 Sep 12;289(37):25764-73. (PMID: 25063810)
Science. 1989 Apr 14;244(4901):182-8. (PMID: 2649980)
Nucleic Acids Res. 2021 Jun 21;49(11):e62. (PMID: 33684219)
Neuron. 1994 Apr;12(4):845-59. (PMID: 8161456)
J Biol Chem. 2006 Aug 4;281(31):22092-22099. (PMID: 16757479)
Front Cell Neurosci. 2021 May 10;15:673782. (PMID: 34040506)
J Neurosci. 2011 Apr 27;31(17):6605-15. (PMID: 21525301)
Elife. 2021 Jun 01;10:. (PMID: 34061030)
J Biol Chem. 2012 Jan 2;287(1):438-445. (PMID: 22072712)
Trends Pharmacol Sci. 2000 Feb;21(2):61-4. (PMID: 10664610)
Pharmacol Rev. 2011 Sep;63(3):585-640. (PMID: 21752877)
Mol Pharmacol. 2003 Aug;64(2):440-6. (PMID: 12869649)
J Biol Chem. 2016 Jan 15;291(3):1456-71. (PMID: 26582198)
Nature. 2024 Aug;632(8026):921-929. (PMID: 39048818)
Mol Cell. 2008 Jun 20;30(6):667-77. (PMID: 18570870)
Nat Commun. 2016 Sep 28;7:12755. (PMID: 27678200)
Nucleic Acids Res. 2014 Jul;42(Web Server issue):W320-4. (PMID: 24753421)

NNF22OC0079091 Novo Nordisk Fonden (Novo Nordisk Foundation); 1030-00036B Sundhed og Sygdom, Det Frie Forskningsråd (Medical Sciences, Danish Council for Independent Research); CF20-0345 Carlsbergfondet (Carlsberg Foundation)

0 (Serotonin Plasma Membrane Transport Proteins)
333DO1RDJY (Serotonin)
0 (Amino Acids)
0 (SLC6A4 protein, human)
9NEZ333N27 (Sodium)
0 (Fluorescent Dyes)

Date Created: 20241028 Date Completed: 20241028 Latest Revision: 20241030

20241031

PMC11514162

10.1038/s41467-024-53584-9

39463388