Structure and function of Serotonin Transporter

1. Structure and function of Serotonin Transporter Erice, April 9, 2002 Gary Rudnick Department of Pharmacology Yale University

2. + K Cl- Na+ + - H+ NT NT Na+ H+ ATP Na+ K+

3. Stoichiometry Bioenergetics of ion coupling Drug interactions Structure Binding site Conformational changes Regulation

5. Na+ SCN- Cl- 5-HT KPi

6. Curt Keller Hannah Glomska Jie-Guang Chen Susan Liu-Chen Yan Ni Fusun Kilic Andreas Androutsellis-Theotokis Yuan-Wei Zhang Pam Nelson Jane Talvenheimo Susan Keyes Steve Wall Howard Gu Megan Stephan Marisa Chen Yoel Smicun Scott Campbell Dennis Murphy

7. Stoichiometry and ion coupling

8. NaClout KPiin gramicidin No gradient (NaCl in=out)

9. For A-B symport (co-transport): at equilibrium and

14. + + + - - V + - - + - + - - + - + D H+ TPMP+ K+

18. + GABA, Proline NE+ 5-HT + 2 Na + + Na Na - - - Cl Cl Cl + K DA, Betaine, Taurine... Glutamic acid Glycine + 2 or 3 Na+ ? Na - + - 3 Na Cl Cl + K + ? K

19. Drug Interactions

20. C H 3 N H 2 N H C l C H 3 N C l A m p h e t a m i n e S e r t r a l i n e N H I m i p r a m i n e 2 N H C H C H 3 3 C H N H O 3 F C O 3 M D M A O Fluoxetine N H 2 H O C H 3 C H 3 N O N N O O C H 3 O C H 3 S e r o t o n i n ( 5 - H T ) O - C I T b O C o c a i n e I

22. Dilution Imipramine control 5-HT + Imip. 5-HT in 5-HT Time

23. C H 3 N H 2 N H C l C H 3 N C l A m p h e t a m i n e S e r t r a l i n e N H I m i p r a m i n e 2 N H C H C H 3 3 C H N H O 3 F C O 3 M D M A O Fluoxetine N H 2 H O C H 3 C H 3 N O N O N O C H 3 O C H 3 S e r o t o n i n ( 5 - H T ) O - C I T b O C o c a i n e I

24. +PCA +PCA

25. NaCl 5-HT+ To NaCl 5-HT+ Ti NaCl 5-HT+ NaCl 5-HT+ Ti To K+ K+ Ti To K+ K+

26. NaCl MDMA+ SERT MDMA+ ADP ATP K+ H+ H+ H+ MDMA+ MDMA MDMA MDMA

27. in in in K+ 5-HT+ - Cl - Na+ Cl- Cl- (or H+) Na+ out out out in Mechanism of serotonin in 5-HT+ transport. Coupling to Cl- K+ Na+ sodium, chloride and (or H+) out out potassium gradients in in in 5-HT+ Cl- Cl- out out out

28. Structure

29. N T W T T G I N W L D Q N A F S H Y N N C T T S C E x t e r i o r T S R H L V D Y A K T S L F A G P K E V N L P W Q P E Y A E W F I S P V S Q H L L V V K L Q R G D L F S Q D F L R Y E Q C I Y L T K I N E T L R N G N Y W L S Q G D T Y L S L P A Y R I L L E A L G G G T Q G M F E Y P E V G T V V Y Y E P Y P R A H G A L I L G A P I A S T S A W S A W W A M W A T V T M F W A N Q Y G S L G D L I L M I L G I R T P A F N L P I T L V V L A A S G P A T L V L L L V A C L Y S N C T L G S Q L I D T T Y I F Y V G T I I V I F Y V I C S L F V F A M F S L V L I V F A L Y A A A F I L G V C S I L G I I M F G I T L I F F I I Y T S Y S I E G L V G F F V V S P V A P G P S F A I F V F V G V S L I L I Y S T I I F A I P L M I F T C A L G V A F L L C S M I T V V S V I I I C V P L F W W T A W N F W T W F L C L V Y Y V Y A Y I G M V I F G R D L S I A I W M Y T S G F K E R K Y V K L G D T E I W W V L R Q N I A K G G S T L L R F G S K K T V T G E G P A K C G P F K F Q F S R T A T T T I A T D A L S V S A N E E P I P R G Q A Y Q L R I E K W N I D F Y C I R N C K H G E I V I D S G S D R L H K K H T L P K I T S M E E C T E A E S T A N P G E D P S M R P C I E G Q G F V W C I P I T V E S E I D E A T L A V C T S E A R P D N G S S A P Q V T A I N D M S T R I Y L N G L G P N V V V S G L Q K Q K C y t o p l a s m

30. Exterior C109 Cytoplasm

31. O N H H N O O S C C H H C N S C H 2 ( C H ) S 3 2 H 2 4 O N - B i o t i n y l a m i n o m e t h y l m e t h a n e t h i o s u l f o n a t e ( M T S E A - B i o t i n ) O O N a O S N H H N 3 O O ( C H ) ( C H ) ( C H ) C N O S S N C 2 4 2 2 2 2 S O N H S - S S - B i o t i n

32. K399 Exterior K314 L406 Q111 R194 L564 V310 C109 K319 K243 T490 V489 H571 Cytoplasm

33. Exterior Cytoplasm

34. Res-FLAG Sens-myc C109A I172C Anti-FLAG 64 kD Anti-myc F L A G -1 mg Activity 16 +MTSEA -1 14 +cocaine 12 10 C 8 5-HT Influx, pmol min 6 4 2 0 c-myc pRSTag C109A I172C res-FLAG sens-myc construct

35. anti-myc IP myc IP total + - + + + res-FLAG + + + VSV-G sens-myc sens-myc - + + + + VSV-G SERT anti-FLAG anti-VSV-G Streptavidin extract Depleted res-FLAG + + + + sens-myc + - + - 68 kD

36. X X X Sens-myc Res-FLAG + Sens-myc Res-FLAG + Sens-myc + Cocaine Res-FLAG n o D N A 0 . 0 0 . 2 0 . 4 0 . 6 0 . 8 1 . 0 1 2 5 b [ I ] - C I T b o u n d , f m o l / m g

37. 10 8 6 Activity, pmol min-1 4 2 80 100 40 60 0 20 percentsensitive

38. 1.4 1.2 1.0 0.8 Remaining Activity 0.6 0.4 0.2 0.0 0 20 40 60 80 100 Percent Sensitive

39. Where is the substrate binding site?

40. 4 2 3 5 6 7 8 9 1 0 1 1 1 2 1 W T B i n d i n g T r a n s p o r t E L 2 b + c o n s e r v e d + ’ + v a r i a n t E L 2 b E L 2 a

42. 1.4 G484C E494C V488C L491C T497C 1.2 1.0 0.8 0.6 0.4 0.2 0.0 .001 .01 0.1 1 10 100 1.4 A486C L492C V489C Y495C 1.2 1.0 Activity, fraction remaining 0.8 0.6 0.4 0.2 0.0 1.4 Y487C T490C E493C A496C 1.2 1.0 0.8 0.6 0.4 0.2 0.0 1E-4 0.01 0.1 1 10 100 .001 .01 0.1 1 10 100 1E-4 0.01 0.1 1 10 100 .001 .01 0.1 1 10 100 [MTSET], mM

43. Activity, % WT Remaining Activity, % Inactivation Rate 0 40 80 120 0 20 40 60 80 100 0.01 0.1 1 10 100 T480C L481C T482C S483C G484C G485C A486C Y487C V488C V489C T490C L491C L492C E493C E494C Y495C A496C T497C G498C P499C A500C V501C L502C

45. L A W A I I T N Y Y S A I Y F A I I C I A “Gate” residue Binding site residues

46. Not protected. Modification does not block b-CIT binding. Accessibility sensitive to ion and ligand binding. Occluded in NET upon NE translocation. L A W A I Functional, inactivated by MTS reagents. Protected against inactivation by 5-HT and cocaine. Modification prevents b-CIT binding. I T N Y Conserved, required for transport. Cys supports low affinity binding. Y S A I Y F A Labeled from outside, reduced from cytoplasm. I I C I A

47. L A W A I I T N Y Y S A I Y F A I I C I A “Gate” residue Binding site residues

48. C109 outside W103 hSERT N101 D98 Y95 inside I172, Y176, I179 N C Residue MTS sensitivity Ligand selectivity Trp-103 -EA, -ET, -ES Asn-101 -EA, -ET Asp-98 -EA, -ET, -ES Gramine-5HT Tyr-95 Mazindol-Citalopram Tryptamine deravitives

49. Conformational changes