Serotonin transporter function and pharmacology are sensitive to expression level: evidence for an endogenous regulatory factor.
We express mammalian serotonin transporters (SERTs) in Xenopus oocytes by cRNA injection and measure 5-hydroxytryptamine (5-HT) transport and 5-HT-induced current at varying expression levels. Transport and current both increase sigmoidally with the amount of cRNA injected, but current requires approximately 5-fold more cRNA to elicit a half-maximal response. Western blots of SERT protein demonstrate that current, but not transport, correlates linearly with the amount of SERT on the plasma membrane. In oocytes co-injected with wild-type SERT and an inactive SERT mutant, transport is similar to SERT alone, but current is attenuated. The charge/transport ratio reports the differential sensitivity of transport and current to increasing SERT cRNA injection and mutant co-expression. Manipulations that alter the charge/transport ratio also perturb substrate and inhibitor recognition. 5-HT, d-amphetamine, cocaine, and paroxetine inhibit transport more potently at lower expression levels; however, 5-HT potency for induction of current is similar at high and low expression. Moreover, the apparent potency of cRNA for transport depends on 5-HT concentration. We postulate that SERT interacts allosterically with an endogenous factor of limited abundance to alter substrate and inhibitor potency and the balance of 5-HT transport and channel-like activity.
Ramsey
I Scott
IS
Department of Pharmacology, Center for Molecular Neuroscience Vanderbilt University Medical Center, Nashville, Tennessee 37232-6600, USA.
DeFelice
Louis J
LJ
D000661:Amphetamine / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D002352:Carrier Proteins / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D003042:Cocaine / Q000494:pharmacology; D008562:Membrane Glycoproteins / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D017374:Paroxetine / Q000494:pharmacology; D018075:RNA, Complementary; D011994:Recombinant Proteins / Q000187:drug effects / Q000378:metabolism; D012701:Serotonin / Q000378:metabolism; D050486:Serotonin Plasma Membrane Transport Proteins; D014982:Xenopus laevis
D002352:Carrier Proteins; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D018075:RNA, Complementary; D011994:Recombinant Proteins; D050486:Serotonin Plasma Membrane Transport Proteins; D012701:Serotonin; D017374:Paroxetine; D000661:Amphetamine; D003042:Cocaine