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Mutation of an asparagine in the first transmembrane alters ion-coupled serotonin transport and cation selectivity in the human serotonin transporter.

The serotonin neurotransmitter transporter (SERT) like the norepinephrine (NET) and dopamine (DAT) transporters is a member of the [Na.sup.+]- and [Cl.sup.-]-dependent SLC6 transporter family and is the target of several drugs of abuse as well as clinically important antidepressants. Serotonin (5-HT) transport via SERT functions to terminate synaptic serotonergic transmission and is energetically coupled to the inwardly-directed electrochemical gradients of [Na.sup.+] and [Cl.sup.-]. However, the process by which 5-HT movement is coupled to the [Na.sup.+] and [Cl.sup.-] chemiosmotic gradients is poorly understood at the molecular level. Previously, we identified an asparagine residue (N101) in transmembrane domain 1 of SERT that upon replacement with the smaller amino acids, alanine and cysteine, renders the transporter Cl-independent. The study presented here suggests that in contrast to native SERT, N101 mutants lack the strict requirement for Na+ to drive 5-HT translocation. Interestingly, transport of 5-HT by wildtype SERT does not occur in the absence of [Na.sup.+] even at very high 5-HT concentrations. However, SERT N101 mutants exhibit a dose-dependent increase in 5-HT translocation in the absence of [Na.sup.+] suggesting at least a partial mechanistic uncoupling between substrate movement and the requirement for [Na.sup.+]. Remarkably, N101 mutants exhibit an increase in basal activity of SERT about 20% above native SERT when [Na.sup.+] is removed. Full cation replacement studies suggest this enhanced basal SERT activity is not mediated by cations. To further characterize the altered cation selectivity of the N101 mutants, we are investigating the ability of other cations (e.g., [K.sup.+], [Ba.sup.2+], [Mg.sup.2+], [Li.sup.+], [Tris.sup.+], choline, [NH.sub.4.sup.+]) to functionally replace [Na.sup.+] in the N101 mutants. Preliminary results suggest enhanced transport of 5-HT in the presence of [Ca.sup.2+] when compared to [Na.sup.+]-free conditions in the N101 mutants. Finally, this study supports the critical role N101 plays in the coordination of ion binding to substrate translocation in SERT and by understanding N101 we hope to gain valuable insight into the transport mechanism.

Nathan Burbach [1] *, Kristin Pavlish [1] and L. Keith Henry [1]

[1] Department of Pharmacology, Physiology and Therapeutics University of North Dakota School of Medicine and Health Sciences, Grand Forks, North Dakota 58201
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Title Annotation:Undergraduate Communication in the A. Roger Denison competition
Author:Burbach, Nathan; Pavlish, Kristin; Henry, L. Keith
Publication:Proceedings of the North Dakota Academy of Science
Date:Apr 1, 2010
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