Point mutations in SLC6 transporters cause misfolding, which can be remedied by pharmacochaperones. The serotonin transporter (SERT/SLC6A4) has a rich pharmacology including inhibitors, releasers (amphetamines, which promote the exchange mode), and more recently, discovered partial substrates. We hypothesized that partial substrates trapped the transporter in one or several states of the transport cycle. This conformational trapping may also be conducive to folding. We selected naphthylpropane-2-amines of the phenethylamine library (PAL) including the partial substrate PAL1045 and its congeners PAL287 and PAL1046. We analyzed their impact on the transport cycle of SERT by biochemical approaches and by electrophysiological recordings; substrate-induced peak currents and steady-state currents monitored the translocation of substrate and co-substrate Na⁺ across the lipid bilayer and the transport cycle, respectively. These experiments showed that PAL1045 and its congeners bound with different affinities (ranging from nm to μm) to various conformational intermediates of SERT during the transport cycle. Consistent with the working hypothesis, PAL1045 was the most efficacious compound in restoring surface expression and transport activity to the folding-deficient mutant SERT-⁶⁰¹PG⁶⁰²-AA. These experiments provide a proof-of-principle for a rational search for pharmacochaperones, which may be useful to restore function to clinically relevant folding-deficient transporter mutants.

The concentrative power of the transporters for dopamine (DAT), norepinephrine (NET), and serotonin (SERT) is thought to be fueled by the transmembrane Na⁺ gradient, but it is conceivable that they can also tap other energy sources, for example, membrane voltage and/or the transmembrane K⁺ gradient. We have addressed this by recording uptake of endogenous substrates or the fluorescent substrate APP⁺(4-(4-dimethylamino)phenyl-1-methylpyridinium) under voltage control in cells expressing DAT, NET, or SERT. We have shown that DAT and NET differ from SERT in intracellular handling of K⁺. In DAT and NET, substrate uptake was voltage-dependent due to the transient nature of intracellular K⁺ binding, which precluded K⁺ antiport. SERT, however, antiports K⁺ and achieves voltage-independent transport. Thus, there is a trade-off between maintaining constant uptake and harvesting membrane potential for concentrative power, which we conclude to occur due to subtle differences in the kinetics of co-substrate ion binding in closely related transporters.

All clinically approved drugs targeting the plasmalemmal transporters for dopamine, norepinephrine and serotonin act either as competitive uptake inhibitors or as amphetamine-like releasers. Recently, monoamine transporter (MAT) ligands were discovered that allosterically affect MAT-mediated substrate uptake, release, or both. Their modes of action have not been explained in a unified framework. Here, we go beyond competitive inhibitors and classical amphetamines and introduce concepts for partial efficacy at and allosteric modulation of MATs. After elaborating a kinetic account for amphetamine action, we provide an explanation for partial release, i.e. the observation that some amphetamines are less efficacious than others in inducing monoamine efflux. We then elucidate mechanisms of allosteric inhibition and stimulation of MATs, which can be functionally selective for either substrate uptake or amphetamine-induced release. These concepts are integrated into a parsimonious kinetic framework, which relies exclusively on physiological transport modes (without any deviation from an alternating access mechanism). The model posits cooperative substrate- and Na+-binding and functional selectivity by conformational selection, i.e. preference of the allosteric modulators for the substrate-loaded or substrate-free states of the transporter. Thus, the current knowledge about the kinetics of monoamine transport is sufficiently detailed to provide a quantitative description of the releasing action of amphetamines, of substrate uptake, and of selective modulation thereof by allosteric modulators.

Point mutations in the coding sequence for solute carrier 6 (SLC6) family members result in clinically relevant disorders, which are often accounted for by a loss-of-function phenotype. In many instances, the mutated transporter is not delivered to the cell surface because it is retained in the endoplasmic reticulum (ER). The underlying defect is improper folding of the transporter and is the case for many of the known dopamine transporter mutants. The monoamine transporters, i.e. the transporters for norepinephrine (NET/SLC6A2), dopamine (DAT/SLC6A3) and serotonin (SERT/SLC6A4), have a rich pharmacology; hence, their folding-deficient mutants lend themselves to explore the concept of pharmacological chaperoning. Pharmacochaperones are small molecules, which bind to folding intermediates with exquisite specificity and scaffold them to a folded state, which is exported from the ER and delivered to the cell surface. Pharmacochaperoning of mutant monoamine transporters, however, is not straightforward: ionic conditions within the ER are not conducive to binding of most typical monoamine transporter ligands. A collection of compounds exists, which are classified as atypical ligands because they trap monoamine transporters in unique conformational states. The atypical binding mode of some DAT inhibitors has been linked to their anti-addictive action. Here, we propose that atypical ligands and also compounds recently classified as partial releasers can serve as pharmacochaperones.

The plasmalemmal monoamine transporters for dopamine, norepinephrine, and serotonin (SERT) are targets for amphetamines. In vivo, amphetamines elicit most, if not all, of their actions by triggering monoamine efflux. This is thought to be accomplished by an amphetamine-induced switch from the forward-transport to the substrate-exchange mode. The mechanism underlying this switch has remained elusive; available kinetic models posit that substrates and cosubstrate Na⁺ ions bind either in a random or in a sequential order. Neither can account for all reported experimental observations. We used electrophysiological recordings to interrogate crucial conformational transitions associated with the binding of five different substrates (serotonin, para-chloroamphetamine, and the high-affinity naphthyl-propan-amines PAL-287, PAL-1045, and PAL-1046) to human SERT expressed in HEK293 cells; specifically, we determined the relaxation kinetics of SERT from a substrate-loaded to a substrate-free state at various intracellular and extracellular Na⁺ concentrations. These rates and their dependence on intracellular and extracellular Na⁺ concentrations differed considerably between substrates. We also examined the effect of K⁺ on substrate affinity and found that K⁺ enhanced substrate dissociation. A kinetic model was developed, which allowed for random, but cooperative, binding of substrate and Na⁺ (or K⁺). The synthetic data generated by this model recapitulated the experimental observations. More importantly, the cooperative binding model accounted for the releasing action of amphetamines without any digression from alternating access. To the best of our knowledge, this model is the first to provide a mechanistic framework for amphetamine-induced monoamine release and to account for the findings that some substrates are less efficacious than others in promoting the substrate-exchange mode.