A lithium-induced conformational change in serotonin transporter alters cocaine binding, ion conductance, and reactivity of Cys-109.
Inactivation of serotonin transporter (SERT) expressed in HeLa cells by [2-(trimethylammonium)ethyl]methanethiosulfonate (MTSET) occurred much more readily when Na(+) in the reaction medium was replaced with Li(+). This did not result from a protective effect of Na(+) but rather from a Li(+)-specific increase in the reactivity of Cys-109 in the first external loop of the transporter. Li(+) alone of the alkali cations caused this increase in reactivity. Replacing Na(+) with N-methyl-d-glucamine (NMDG(+)) did not reduce the affinity of cocaine for SERT, as measured by displacement of a high affinity cocaine analog, but replacement of Na(+) with Li(+) led to a 2-fold increase in the K(D) for cocaine. The addition of either cocaine or serotonin (5-HT) protected SERT against MTSET inactivation. When SERT was expressed in Xenopus oocytes, inward currents were elicited by superfusing the cell with 5-HT (in the presence of Na(+)) or by replacing Na(+) with Li(+) but not NMDG(+). MTSET treatment of oocytes in Li(+) but not in Na(+) decreased both 5-HT and Li(+) induced currents, although 5-HT-induced currents were inhibited to a greater extent. Na(+) antagonized the effects of Li(+) on both inactivation and current. These results are consistent with Li(+) inducing a conformational change that exposes Cys-109, decreases cocaine affinity, and increases the uncoupled inward current.
Ni
Y G
YG
Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.
Chen
J G
JG
Androutsellis-Theotokis
A
A
Huang
C J
CJ
Moczydlowski
E
E
Rudnick
G
G
D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry*; D003042:Cocaine / Q000378:metabolism*; D003545:Cysteine; D005971:Glutamates / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D008094:Lithium / Q000494:pharmacology*; D008562:Membrane Glycoproteins / Q000737:chemistry*; D008564:Membrane Potentials / Q000187:drug effects; D026901:Membrane Transport Proteins; D008698:Mesylates / Q000494:pharmacology; D009419:Nerve Tissue Proteins; D011487:Protein Conformation; D050486:Serotonin Plasma Membrane Transport Proteins; D012964:Sodium / Q000494:pharmacology; D014981:Xenopus
D002352:Carrier Proteins; D005971:Glutamates; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D008698:Mesylates; D009419:Nerve Tissue Proteins; C493230:SLC6A4 protein, human; D050486:Serotonin Plasma Membrane Transport Proteins; C109154:(2-(trimethylammonium)ethyl)methanethiosulfonate; C014574:gamma-glutamylmethylamide; D008094:Lithium; D012964:Sodium; D003042:Cocaine; D003545:Cysteine