Serotonin (5HT) 3 plays a role in the regulation of many behaviors (1, 2), and disturbances in the serotonergic system are implicated in a myriad of diseases (3-7). Following neurotransmission the serotonin transporter (SERT) clears 5HT from the synaptic cleft (8 -10). Numerous antidepressants such as fluoxetine or citalopram inhibit re-uptake of 5HT by SERT (11), and drugs of abuse, such as 3,4-methylenedioxymethamphetamine (MDMA) induce reverse 5HT transport through the transporter (12). SERT is a member of the solute carrier 6 gene family consisting of numerous ion-coupled co-transporters, such as the norepinephrine and dopamine transporters (13)(14)(15). These transporters use the Na ϩ ionic electrochemical potential to concentrate substrates against their concentration gradient (15-18). Traditionally, co-transport is described by the alternating access model where ions and substrate are transported with a fixed stoichiometry (19, 20); however, a competing model describes a channel within the transporter capable of conducing currents mediated by substrate and ions. These substrate-induced uncoupled currents are found in many neurotransmitter transporters (21-25). Additionally, in the absence of substrate, a constitutive leak current exists, which, for SERT, can be uncovered with inhibitors such as fluoxetine (25).Traditionally, monoamine transporter activity has been assessed by radiolabeled uptake assays; however, these biochemical assays have poor temporal resolution and do not yield spatial information of the transport process. Conversely, fluorescent substrates of monoamine transporters are advantageous tools to study mechanistic properties of SERT because they provide a continuous signal that can be measured with single live-cell imaging. In addition to the temporal and spatial advantages of fluorescent substrates, their use is amenable for high-throughput screening (27,28).Previously, we characterized 4-(4-(dimethylamino)styryl)-N-methylpyridinium (ASP ϩ ) as a fluorescent reporter for uptake activity for both the human norepinephrine transporter (hNET) and the human dopamine transporter (hDAT) and utilized it to study biophysical properties of hNET such as substrate-protein stoichiometry and substrate dwell time (29,30).

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The recent determination of high-resolution crystal structures of several transporters offers unprecedented insights into the structural mechanisms behind secondary transport. These proteins utilize the facilitated diffusion of the ions down their electrochemical gradients to transport the substrate against its concentration gradient. The structural studies revealed striking similarities in the structural organization of ion and solute binding sites and a well-conserved inverted-repeat topology between proteins from several gene families. In this paper we will overview recent atomistic simulations applied to study the mechanisms of selective binding of ion and substrate in LeuT, Glt, vSGLT and hSERT as well as its consequences for the transporter conformational dynamics. This article is part of a Special Issue entitled: Membrane protein structure and function.

The thermodynamics of ion solvation in non-aqueous solvents remains of great significance for understanding cellular transport and ion homeostasis for the design of novel ion-selective materials and applications in molecular pharmacology. Molecular simulations play pivotal roles in connecting experimental measurements to the microscopic structures of liquids. One of the most useful and versatile mimetic systems for understanding biological ion transport is N-methyl-acetamide (NMA). A plethora of theoretical studies for ion solvation in NMA have appeared recently, but further progress is limited by two factors. One is an apparent lack of experimental data on solubility and thermodynamics of solvation for a broad panel of 1 : 1 salts over an appropriate temperature and concentration range. The second concern is more substantial and has to do with the limitations hardwired in the additive (fixed charge) approximations used for most of the existing force-fields. In this submission, we report on the experimental evaluation of LiCl solvation in NMA over a broad range of concentrations and temperatures and compare the results with those of MD simulations with several additive and one polarizable force-field (Drude). By comparing our simulations and experimental results to density functional theory computations, we discuss the limiting factors in existing potential functions. To evaluate the possible implications of explicit and implicit polarizability treatments on ion permeation across biological channels, we performed potential of mean force (PMF) computations for Li(+) transport through a model narrow ion channel with additive and polarizable force-fields.