PMID- 20159976
OWN - NLM
STAT- MEDLINE
DCOM- 20100504
LR  - 20240419
IS  - 1083-351X (Electronic)
IS  - 0021-9258 (Print)
IS  - 0021-9258 (Linking)
VI  - 285
IP  - 15
DP  - 2010 Apr 9
TI  - Transmembrane domain 6 of the human serotonin transporter contributes to an 
      aqueously accessible binding pocket for serotonin and the psychostimulant 
      3,4-methylene dioxymethamphetamine.
PG  - 11270-80
LID - 10.1074/jbc.M109.093658 [doi]
AB  - The plasma membrane serotonin (5-HT) transporter (SERT, SLC6A4) clears 5-HT after 
      release at nerve termini and is targeted by both antidepressant medications and 
      psychostimulants (e.g. MDMA, cocaine). Homology modeling of human SERT (hSERT), 
      based on high resolution structures of the microbial SLC6 family member LeuT(Aa), 
      along with biochemical studies of wild type and mutant transporters, predicts 
      transmembrane (TM) domains 1, 3, 6, and 8 comprise the 5-HT-binding pocket. We 
      utilized the substituted cysteine accessibility method along with surface and 
      site-specific biotinylation to probe TM6 for aqueous accessibility and 
      differential interactions with 5-HT and psychostimulants. Our results are 
      consistent with TM6 being composed of an aqueous-accessible, alpha-helical 
      extracellular domain (TM6a) that is separated by a central, unwound section from 
      a cytoplasmically localized domain (TM6b) with limited aqueous accessibility. The 
      substitution G338C appears to lock hSERT in an outward-facing conformation that, 
      although accessible to aminoethylmethanethiosulfonate-biotin, 5-HT, and 
      citalopram, is incapable of inward 5-HT transport. Transport of 5-HT by G338C can 
      be partially restored by the TM1 mutation Y95F. With regard to 
      methanethiosulfonate (MTS) inactivation of uptake, TM6a Cys mutants demonstrate 
      Na(+)-dependent [2-(trimethylammonium)ethyl]-MTS sensitivity. Studies with the 
      centrally located substitution S336C reveal features of a common binding pocket 
      for 5-HT and 3,4-methylenedioxymethamphetamine (MDMA). Interestingly, the 
      substitution I333C reveals an MDMA-induced conformation not observed with 5-HT. 
      In the context of prior studies on TM1, our findings document shared and unique 
      features of TM6 contributing to hSERT aqueous accessibility, ligand recognition, 
      and conformational dynamics.
FAU - Field, Julie R
AU  - Field JR
AD  - Department of Pharmacology, Vanderbilt University Medical Center, Nashville, 
      Tennessee 37232-8548, USA.
FAU - Henry, L Keith
AU  - Henry LK
FAU - Blakely, Randy D
AU  - Blakely RD
LA  - eng
GR  - R01 DA007390/DA/NIDA NIH HHS/United States
GR  - K01 DA022378/DA/NIDA NIH HHS/United States
GR  - DA07390/DA/NIDA NIH HHS/United States
GR  - DA022378/DA/NIDA NIH HHS/United States
GR  - F31 DA023337/DA/NIDA NIH HHS/United States
GR  - DA23337/DA/NIDA NIH HHS/United States
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
DEP - 20100216
PL  - United States
TA  - J Biol Chem
JT  - The Journal of biological chemistry
JID - 2985121R
RN  - 0 (SLC6A4 protein, human)
RN  - 0 (Serotonin Agents)
RN  - 0 (Serotonin Plasma Membrane Transport Proteins)
RN  - 059QF0KO0R (Water)
RN  - 333DO1RDJY (Serotonin)
RN  - KE1SEN21RM (N-Methyl-3,4-methylenedioxyamphetamine)
SB  - IM
MH  - Biological Transport
MH  - Cytoplasm/metabolism
MH  - Humans
MH  - Kinetics
MH  - Models, Biological
MH  - Mutagenesis, Site-Directed
MH  - Mutation
MH  - N-Methyl-3,4-methylenedioxyamphetamine/pharmacology
MH  - Protein Binding
MH  - Protein Conformation
MH  - Protein Structure, Tertiary
MH  - Serotonin/*chemistry
MH  - Serotonin Agents/pharmacology
MH  - Serotonin Plasma Membrane Transport Proteins/*chemistry/*metabolism
MH  - Water/chemistry
PMC - PMC2857005
EDAT- 2010/02/18 06:00
MHDA- 2010/05/05 06:00
PMCR- 2011/04/09
CRDT- 2010/02/18 06:00
PHST- 2010/02/18 06:00 [entrez]
PHST- 2010/02/18 06:00 [pubmed]
PHST- 2010/05/05 06:00 [medline]
PHST- 2011/04/09 00:00 [pmc-release]
AID - S0021-9258(19)40577-2 [pii]
AID - M109.093658 [pii]
AID - 10.1074/jbc.M109.093658 [doi]
PST - ppublish
SO  - J Biol Chem. 2010 Apr 9;285(15):11270-80. doi: 10.1074/jbc.M109.093658. Epub 2010 
      Feb 16.