Download Electrophysiological characterization of Na transporter

Electrophysiological characterization of Na+-site mutants of the human serotonin
transporter
Pella Söderhielm
1,
2
1
2
, Pernille Noer , Kristian Strømgaard , Steffen Sinning , Anders S. Kristensen
1
1
University of Copenhagen, Department of Medicinal Chemistry, Universitetsparken 2, 2100 Copenhagen,
2
Denmark,
Center for Psychiatric Research, Aarhus University Hospital, 8240 Risskov, Denmark.

[email protected]
The serotonin transporter (SERT) mediates reuptake of serotonin (5-HT) from the synaptic cleft and is a
molecular target for drugs used to treat affective disorders such as depression. SERT belongs to the family
+
-
of Na /Cl coupled neurotransmitter transporters which transports Na
+
exchange for a K .
+
-
and Cl together with 5-HT in
Although the transport process overall is thought to be electroneutral, several
macroscopic membrane currents have been found to be associated with mammalian and insect SERT
1-3
activity . Specifically, studies using two-electrode voltage clamp electrophysiology to measure macroscopic
membrane currents in X. laevis oocytes expressing SERT have reported three distinct SERT-associated
1,2
currents . i) a 5-HT-induced transport-associated inward current, ii) a constitutive inward leak current that is
detected as an outward deflection in the base-line membrane current when applying SERT inhibitors, and iii)
a transient current can be induced by voltage jumps to high negative potentials. The transient current have
been suggested to represent that SERT can adopt ion-channel states consistent with the finding of single4
channel activity in membrane patches from SERT-expressing oocytes .
Recently, the structural understanding of SERT has been advanced by X-ray crystal structures of the
5
bacterial SERT-homolog LeuT , which have allowed construction of homology models of SERT; showing two
sodium binding sites (Na1 and Na2) to exist within the central substrate binding pocket. In the present study,
we study the influence of the Na1 and Na2 sites for the conducting states of human SERT. We find that
+
+
disruption of the Na -sites by mutation of key residues involved in Na coordination in each site changes the
+
macroscopic currents observed in oocytes. Our results offer novel insight into the significance of Na binding
to Na1 and Na2 for the conducting states of SERT.
1
Mager, S., Min, C., Henry, DJ., Chavkin, C., Hoffman, BJ. et al. (1994). Conducting states of a mammalian serotonin transporter.
Neuron, 12, 845-859.
Adams SV, DeFelice LJ (2003) Ionic currents in the human serotonin transporter reveal inconsistencies in the alternating access
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hypothesis. Biophys J. 85(3):1548-59.
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Petersen CI, DeFelice LJ (1999) Ionic interactions in the Drosophila serotonin transporter identify it as a serotonin channel. Nat
Neurosci. 2(7):605-10.
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Lin F, Lester HA, Mager S (1996) Single-channel currents produced by the serotonin transporter and analysis of a mutation affecting
ion permeation. Biophys J. 71(6):3126-35.
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Yamashita, A., Singh, S.K., Kawate, T., Jin, Y. & Gouaux, E. (2005). Crystal structure of a bacterial homologue of Na+/Cl--dependent
neurotransmitter transporters. Nature, 437, 215-23.