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J. Biol. Chem., Vol. 277, Issue 17, 14475-14482, April 26, 2002
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From the Department of Pharmacology, Center for Molecular
Neuroscience Vanderbilt University Medical Center,
Nashville, Tennessee 37232-6600
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
~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.
Serotonin Transporter Function and Pharmacology Are Sensitive to
Expression Level
EVIDENCE FOR AN ENDOGENOUS REGULATORY FACTOR*
and
*
This work was supported by National Institutes of Health
Grant NS-34075 (to L. J. D.) and National Institutes of
Health Fellowship MH-12393 (to I. S. R.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Present address: Dept. of Cardiology, Children's Hospital,
Harvard Medical School, Enders 1309, Boston, MA 02115.
§
To whom correspondence should be addressed: Dept. of Pharmacology,
Vanderbilt University Medical Center, Nashville, TN 37232-6600. Tel.: 615-343-6278; Fax: 615-343-1679; E-mail:
lou.defelice@mcmail.vanderbilt.edu.
Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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