Behavioral encoding across timescales by region-specific dopamine dynamics.

The dorsal (DS) and ventral striatum (VS) receive dopaminergic projections that control motor functions and reward-related behavior. It remains poorly understood how dopamine release dynamics across different temporal scales in these regions are coupled to behavioral outcomes. Here, we employ the dopamine sensor dLight1.3b together with multiregion fiber photometry and machine learning-based analysis to decode dopamine dynamics across the striatum during self-paced exploratory behavior in mice. Our data show a striking coordination of rapidly fluctuating signal in the DS, carrying information across dopamine levels, with a slower signal in the VS, consisting mainly of slow-paced transients. Importantly, these release dynamics correlated with discrete behavioral motifs, such as turns, running, and grooming on a subsecond-to-minute time scale. Disruption of dopamine dynamics with cocaine caused randomization of action selection sequencing and disturbance of DS-VS coordination. The data suggest that distinct dopamine dynamics of DS and VS jointly encode behavioral sequences during unconstrained activity with DS modulating the stringing together of actions and VS the signal to initiate and sustain the selected action.

Amphetamine-induced reverse transport of dopamine does not require cytosolic Ca2.

Amphetamines (AMPHs) are substrates of the dopamine transporter (DAT) and reverse the direction of dopamine (DA) transport. This has been suggested to depend on activation of Ca2+-dependent pathways, but the mechanism underlying reverse transport via endogenously expressed DAT is still unclear. Here, to enable concurrent visualization by live imaging of extracellular DA dynamics and cytosolic Ca2+ levels, we employ the fluorescent Ca2+ sensor jRGECO1a expressed in cultured dopaminergic neurons together with the fluorescent DA sensor GRABDA1H expressed in cocultured "sniffer" cells. In the presence of the Na+-channel blocker tetrodotoxin to prevent exocytotic DA release, AMPH induced in the cultured neurons a profound dose-dependent efflux of DA that was blocked both by inhibition of DAT with cocaine and by inhibition of the vesicular monoamine transporter-2 with Ro-4-1284 or reserpine. However, the AMPH-induced DA efflux was not accompanied by an increase in cytosolic Ca2+ and was unaffected by blockade of voltage-gated calcium channels or chelation of cytosolic Ca2+. The independence of cytosolic Ca2+ was further supported by activation of N-methyl-D-aspartate-type ionotropic glutamate receptors leading to a marked increase in cytosolic Ca2+ without affecting AMPH-induced DA efflux. Curiously, AMPH elicited spontaneous Ca2+ spikes upon blockade of the D2 receptor, suggesting that AMPH can regulate intracellular Ca2+ in an autoreceptor-dependent manner regardless of the apparent independence of Ca2+ for AMPH-induced efflux. We conclude that AMPH-induced DA efflux in dopaminergic neurons does not require cytosolic Ca2+ but is strictly dependent on the concerted action of AMPH on both vesicular monoamine transporter-2 and DAT.

Role of dopamine neurons in familiarity.

Dopamine neurons signal the salience of environmental stimuli and influence learning, although it is less clear if these neurons also determine the salience of memories. Ventral tegmental area (VTA) dopamine neurons increase their firing in the presence of new objects and reduce it upon repeated, inconsequential exposures, marking the shift from novelty to familiarity. This study investigates how dopamine neuron activity during repeated familiar object exposure affects an animal's preference for new objects in a subsequent novel object recognition (NOR) test. We hypothesize that a single familiarization session will not sufficiently lower dopamine activity, such that the memory of a familiar object remains salient, leading to equal exploration of familiar and novel objects and weaker NOR discrimination. In contrast, multiple familiarization sessions likely suppress dopamine activity more effectively, reducing the salience of the familiar object and enhancing subsequent novelty discrimination. Our experiments in mice indicated that multiple familiarization sessions reduce VTA dopamine neuron activation, as measured by c-Fos expression, and enhance novelty discrimination compared with a single familiarization session. Dopamine neurons that show responsiveness to novelty were primarily located in the paranigral nucleus of the VTA and expressed vesicular glutamate transporter 2 transcripts, marking them as dopamine-glutamate neurons. Chemogenetic inhibition of dopamine neurons during a single session paralleled the effects of multiple sessions, improving NOR. These findings suggest that a critical role of dopamine neurons during the transition from novelty to familiarity is to modulate the salience of an object's memory.

It takes two-or more-to tango: Revisiting the role of dopamine transporter oligomerization.

The dopamine transporter utilizes the transmembrane sodium gradient to mediate reuptake of dopamine from the extracellular space. The dopamine transporter can form dimers and possibly also higher order structures in the plasma membrane, and this oligomerization has been implicated in both trafficking and transport. However, we still do not fully understand its biological importance. A study by Sorkina et al. now describes a series of small molecules that link transporter conformation to oligomerization and endocytosis, providing an interesting step forward in an intricate dance.

Disruption of the PDZ domain-binding motif of the dopamine transporter uniquely alters nanoscale distribution, dopamine homeostasis, and reward motivation.

The dopamine (DA) transporter (DAT) is part of a presynaptic multiprotein network involving interactions with scaffold proteins via its C-terminal PDZ domain-binding sequence. Using a mouse model expressing DAT with mutated PDZ-binding sequence (DAT-AAA), we previously demonstrated the importance of this binding sequence for striatal expression of DAT. Here, we show by application of direct stochastic reconstruction microscopy not only that the striatal level of transporter is reduced in DAT-AAA mice but also that the nanoscale distribution of this transporter is altered with a higher propensity of DAT-AAA to localize to irregular nanodomains in dopaminergic terminals. In parallel, we observe mesostriatal DA adaptations and changes in DA-related behaviors distinct from those seen in other genetic DAT mouse models. DA levels in the striatum are reduced to ∼45% of that of WT, accompanied by elevated DA turnover. Nonetheless, fast-scan cyclic voltammetry recordings on striatal slices reveal a larger amplitude and prolonged clearance rate of evoked DA release in DAT-AAA mice compared with WT mice. Autoradiography and radioligand binding show reduced DA D2 receptor levels, whereas immunohistochemistry and autoradiography show unchanged DA D1 receptor levels. In behavioral experiments, we observe enhanced self-administration of liquid food under both a fixed ratio of one and progressive ratio schedule of reinforcement but a reduction compared with WT when using cocaine as reinforcer. In summary, our data demonstrate how disruption of PDZ domain interactions causes changes in DAT expression and its nanoscopic distribution that in turn alter DA clearance dynamics and related behaviors.

Dopaminergic neurons establish a distinctive axonal arbor with a majority of non-synaptic terminals.

Chemical neurotransmission typically occurs through synapses. Previous ultrastructural examinations of monoamine neuron axon terminals often failed to identify a pre- and postsynaptic coupling, leading to the concept of "volume" transmission. Whether this results from intrinsic properties of these neurons remains undefined. We find that dopaminergic neurons in vitro establish a distinctive axonal arbor compared to glutamatergic or GABAergic neurons in both size and propensity of terminals to avoid direct contact with target neurons. While most dopaminergic varicosities are active and contain exocytosis proteins like synaptotagmin 1, only ~20% of these are synaptic. The active zone protein bassoon was found to be enriched in dopaminergic terminals that are in proximity to a target cell. Finally, we found that the proteins neurexin-1αSS4- and neuroligin-1A+B play a critical role in the formation of synapses by dopamine (DA) neurons. Our findings suggest that DA neurons are endowed with a distinctive developmental connectivity program.

Elucidating the Mechanism Behind Sodium-Coupled Neurotransmitter Transporters by Reconstitution.

Sodium-coupled neurotransmitter transporters play a fundamental role in the termination of synaptic neurotransmission, which makes them a major drug target. The reconstitution of these secondary active transporters into liposomes has shed light on their molecular transport mechanisms. From the earliest days of the reconstitution technique up to today's single-molecule studies, insights from live functioning transporters have been indispensable for our understanding of their physiological impact. The two classes of sodium-coupled neurotransmitter transporters, the neurotransmitter: sodium symporters and the excitatory amino acid transporters, have vastly different molecular structures, but complementary proteoliposome studies have sought to unravel their ion-dependence and transport kinetics. Furthermore, reconstitution experiments have been used on both protein classes to investigate the role of e.g. the lipid environment, of posttranslational modifications, and of specific amino acid residues in transport. Techniques that allow the detection of transport at a single-vesicle resolution have been developed, and single-molecule studies have started to reveal single transporter kinetics, which will expand our understanding of how transport across the membrane is facilitated at protein level. Here, we review a selection of the results and applications where the reconstitution of the two classes of neurotransmitter transporters has been instrumental.

Increased vulnerability of nigral dopamine neurons after expansion of their axonal arborization size through D2 dopamine receptor conditional knockout.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the loss of dopamine (DA) neurons in the substantia nigra pars compacta (SNc). Rare genetic mutations in genes such as Parkin, Pink1, DJ-1, α-synuclein, LRRK2 and GBA are found to be responsible for the disease in about 15% of the cases. A key unanswered question in PD pathophysiology is why would these mutations, impacting basic cellular processes such as mitochondrial function and neurotransmission, lead to selective degeneration of SNc DA neurons? We previously showed in vitro that SNc DA neurons have an extremely high rate of mitochondrial oxidative phosphorylation and ATP production, characteristics that appear to be the result of their highly complex axonal arborization. To test the hypothesis in vivo that axon arborization size is a key determinant of vulnerability, we selectively labeled SNc or VTA DA neurons using floxed YFP viral injections in DAT-cre mice and showed that SNc DA neurons have a much more arborized axon than those of the VTA. To further enhance this difference, which may represent a limiting factor in the basal vulnerability of these neurons, we selectively deleted in mice the DA D2 receptor (D2-cKO), a key negative regulator of the axonal arbour of DA neurons. In these mice, SNc DA neurons have a 2-fold larger axonal arborization, release less DA and are more vulnerable to a 6-OHDA lesion, but not to α-synuclein overexpression when compared to control SNc DA neurons. This work adds to the accumulating evidence that the axonal arborization size of SNc DA neurons plays a key role in their vulnerability in the context of PD.

Modulating Dopamine Signaling and Behavior with Chemogenetics: Concepts, Progress, and Challenges.

For more than 60 years, dopamine (DA) has been known as a critical modulatory neurotransmitter regulating locomotion, reward-based motivation, and endocrine functions. Disturbances in DA signaling have been linked to an array of different neurologic and psychiatric disorders, including Parkinson's disease, schizophrenia, and addiction, but the underlying pathologic mechanisms have never been fully elucidated. One major obstacle limiting interpretation of standard pharmacological and transgenic interventions is the complexity of the DA system, which only appears to widen as research progresses. Nonetheless, development of new genetic tools, such as chemogenetics, has led to an entirely new era for functional studies of neuronal signaling. By exploiting receptors that are engineered to respond selectively to an otherwise inert ligand, so-called Designer Receptors Exclusively Activated by Designer Drugs (DREADDs), chemogenetics enables pharmacological remote control of neuronal activity. Here we review the recent, extensive application of this technique to the DA field and how its use has advanced the study of the DA system and contributed to our general understanding of DA signaling and related behaviors. Moreover, we discuss the challenges and pitfalls associated with the chemogenetic technology, such as the metabolism of the DREADD ligand clozapine N-oxide (CNO) to the D2 receptor antagonist clozapine. We conclude that despite the recent concerns regarding CNO, the chemogenetic toolbox provides an exceptional approach to study neuronal function. The huge potential should promote continued investigations and additional refinements to further expound key mechanisms of DA signaling and circuitries in normal as well as maladaptive behaviors.

Neuropsychiatric disease-associated genetic variants of the dopamine transporter display heterogeneous molecular phenotypes.

Genetic factors are known to significantly contribute to the etiology of psychiatric diseases such as attention deficit hyperactivity disorder (ADHD) and autism spectrum and bipolar disorders, but the underlying molecular processes remain largely elusive. The dopamine transporter (DAT) has received continuous attention as a potential risk factor for psychiatric disease, as it is critical for dopamine homeostasis and serves as principal target for ADHD medications. Constrain metrics for the DAT-encoding gene, solute carrier family 6 member 3 (SLC6A3), indicate that missense mutations are under strong negative selection, pointing to pathophysiological outcomes when DAT function is compromised. Here, we systematically characterized six rare genetic variants of DAT (I312F, T356M, D421N, A559V, E602G, and R615C) identified in patients with neuropsychiatric disorders. We evaluated dopamine uptake and ligand interactions, along with ion coordination and electrophysiological properties, to elucidate functional phenotypes, and applied Zn2+ exposure and a substituted cysteine-accessibility approach to identify shared structural changes. Three variants (I312F, T356M, and D421N) exhibited impaired dopamine uptake associated with changes in ligand binding, ion coordination, and distinct conformational disturbances. Remarkably, we found that all three variants displayed gain-of-function electrophysiological phenotypes. I312F mediated an increased uncoupled anion conductance previously suggested to modulate neuronal excitability. T356M and D421N both mediated a cocaine-sensitive leakage of cations, which for T356M was potentiated by Zn2+, concurrent with partial functional rescue. Collectively, our findings support that gain of disruptive functions due to missense mutations in SLC6A3 may be key to understanding how dopaminergic dyshomeostasis arises in heterozygous carriers.

Initial rewarding effects of cocaine and amphetamine assessed in a day using the single-exposure place preference protocol.

In rodents, only a single dose of cocaine or amphetamine is required to cause a marked increase in extracellular dopamine, induce hyperlocomotion and cause persistent plasticity changes within dopaminergic neurons of the ventral tegmental area (VTA). The initial drug experience is suggested to predict vulnerability of developing addiction, but only few studies have assessed the perception of reward accompanying this initial exposure. We recently presented an approach to assess the initial rewarding effects of cocaine in mice with a single-exposure place preference (sePP) protocol, avoiding repeated drug injections. Here, we demonstrate a condensed version of the sePP, allowing assessment of initial subjective reward-perception within a day. By using this protocol, we demonstrate that a single exposure to both cocaine and amphetamine is sufficient to induce place preference. Furthermore, we use chemogenetics ( Designer Receptors Exclusively Activated by Designer Drugs [DREADD]) to show that both inhibitory and stimulatory modulation of VTA DA signalling disrupts cocaine-induced place preference in the condensed sePP. Our findings support the presence of initial reward-perception of both cocaine and amphetamine, and the formation of drug-context association. In addition, our data support that VTA DA signalling prior to drug exposure affects either reward-perception or the time during which associations are formed, thereby preventing induction of cocaine-induced place preference in the sePP. The easy and timesaving sePP protocol should form a critical basis for further deciphering the complex mechanisms underlying the progression from the initial drug experience to escalating drug intake and addiction.

The GPRC6A receptor displays constitutive internalization and sorting to the slow recycling pathway.

The class C G protein-coupled receptor GPRC6A is a putative nutrient-sensing receptor and represents a possible new drug target in metabolic disorders. However, the specific physiological role of this receptor has yet to be identified, and the mechanisms regulating its activity and cell surface availability also remain enigmatic. In the present study, we investigated the trafficking properties of GPRC6A by use of both a classical antibody feeding internalization assay in which cells were visualized using confocal microscopy and a novel internalization assay that is based on real-time measurements of fluorescence resonance energy transfer. Both assays revealed that GPRC6A predominantly undergoes constitutive internalization, whereas the agonist-induced effects were imperceptible. Moreover, postendocytic sorting was investigated by assessing the co-localization of internalized GPRC6A with selected Rab protein markers. Internalized GPRC6A was mainly co-localized with the early endosome marker Rab5 and the long loop recycling endosome marker Rab11 and to a much lesser extent with the late endosome marker Rab7. This suggests that upon agonist-independent internalization, GPRC6A is recycled via the Rab11-positive slow recycling pathway, which may be responsible for ensuring a persistent pool of GPRC6A receptors at the cell surface despite chronic agonist exposure. Distinct trafficking pathways have been reported for several of the class C receptors, and our results thus substantiate that non-canonical trafficking mechanisms are a common feature for the nutrient-sensing class C family that ensure functional receptors in the cell membrane despite prolonged agonist exposure.

Differential Internalization Rates and Postendocytic Sorting of the Norepinephrine and Dopamine Transporters Are Controlled by Structural Elements in the N Termini.

The norepinephrine transporter (NET) mediates reuptake of synaptically released norepinephrine in central and peripheral noradrenergic neurons. The molecular processes governing availability of NET in the plasma membrane are poorly understood. Here we use the fluorescent cocaine analogue JHC 1-64, as well as several other approaches, to investigate the trafficking itinerary of NET in live noradrenergic neurons. Confocal imaging revealed extensive constitutive internalization of JHC 1-64-labeled NET in the neuronal somata, proximal extensions and presynaptic boutons. Phorbol 12-myristate 13-acetate increased intracellular accumulation of JHC 1-64-labeled NET and caused a parallel reduction in uptake capacity. Internalized NET strongly colocalized with the "long loop" recycling marker Rab11, whereas less overlap was seen with the "short loop" recycling marker Rab4 and the late endosomal marker Rab7. Moreover, mitigating Rab11 function by overexpression of dominant negative Rab11 impaired NET function. Sorting of NET to the Rab11 recycling compartment was further supported by confocal imaging and reversible biotinylation experiments in transfected differentiated CATH.a cells. In contrast to NET, the dopamine transporter displayed markedly less constitutive internalization and limited sorting to the Rab11 recycling compartment in the differentiated CATH.a cells. Exchange of domains between the two homologous transporters revealed that this difference was determined by non-conserved structural elements in the intracellular N terminus. We conclude that NET displays a distinct trafficking itinerary characterized by continuous shuffling between the plasma membrane and the Rab11 recycling compartment and that the functional integrity of the Rab11 compartment is critical for maintaining proper presynaptic NET function.

Conformational Dynamics on the Extracellular Side of LeuT Controlled by Na+ and K+ Ions and the Protonation State of Glu290.

Ions play key mechanistic roles in the gating dynamics of neurotransmitter:sodium symporters (NSSs). In recent microsecond scale molecular dynamics simulations of a complete model of the dopamine transporter, a NSS protein, we observed a partitioning of K(+) ions from the intracellular side toward the unoccupied Na2 site of dopamine transporter following the release of the Na2-bound Na(+) Here we evaluate with computational simulations and experimental measurements of ion affinities under corresponding conditions, the consequences of K(+) binding in the Na2 site of LeuT, a bacterial homolog of NSS, when both Na(+) ions and substrate have left, and the transporter prepares for a new cycle. We compare the results with the consequences of binding Na(+) in the same apo system. Analysis of >50-µs atomistic molecular dynamics and enhanced sampling trajectories of constructs with Glu(290), either charged or neutral, point to the Glu(290) protonation state as a main determinant in the structural reconfiguration of the extracellular vestibule of LeuT in which a "water gate" opens through coordinated motions of residues Leu(25), Tyr(108), and Phe(253) The resulting water channel enables the binding/dissociation of the Na(+) and K(+) ions that are prevalent, respectively, in the extracellular and intracellular environments.

Preserved dopaminergic homeostasis and dopamine-related behaviour in hemizygous TH-Cre mice.

Cre-driver mouse lines have been extensively used as genetic tools to target and manipulate genetically defined neuronal populations by expression of Cre recombinase under selected gene promoters. This approach has greatly advanced neuroscience but interpretations are hampered by the fact that most Cre-driver lines have not been thoroughly characterized. Thus, a phenotypic characterization is of major importance to reveal potential aberrant phenotypes prior to implementation and usage to selectively inactivate or induce transgene expression. Here, we present a biochemical and behavioural assessment of the dopaminergic system in hemizygous tyrosine hydroxylase (TH)-Cre mice in comparison to wild-type (WT) controls. Our data show that TH-Cre mice display preserved dopaminergic homeostasis with unaltered levels of TH and dopamine as well as unaffected dopamine turnover in striatum. TH-Cre mice also show preserved dopamine transporter expression and function supporting sustained dopaminergic transmission. In addition, TH-Cre mice demonstrate normal responses in basic behavioural paradigms related to dopaminergic signalling including locomotor activity, reward preference and anxiolytic behaviour. Our results suggest that TH-Cre mice represent a valid tool to study the dopamine system, though careful characterization must always be performed to prevent false interpretations following Cre-dependent transgene expression and manipulation of selected neuronal pathways.

The Environment Shapes the Inner Vestibule of LeuT.

Human neurotransmitter transporters are found in the nervous system terminating synaptic signals by rapid removal of neurotransmitter molecules from the synaptic cleft. The homologous transporter LeuT, found in Aquifex aeolicus, was crystallized in different conformations. Here, we investigated the inward-open state of LeuT. We compared LeuT in membranes and micelles using molecular dynamics simulations and lanthanide-based resonance energy transfer (LRET). Simulations of micelle-solubilized LeuT revealed a stable and widely open inward-facing conformation. However, this conformation was unstable in a membrane environment. The helix dipole and the charged amino acid of the first transmembrane helix (TM1A) partitioned out of the hydrophobic membrane core. Free energy calculations showed that movement of TM1A by 0.30 nm was driven by a free energy difference of ~15 kJ/mol. Distance measurements by LRET showed TM1A movements, consistent with the simulations, confirming a substantially different inward-open conformation in lipid bilayer from that inferred from the crystal structure.

Amphetamine action at the cocaine- and antidepressant-sensitive serotonin transporter is modulated by αCaMKII.

Serotonergic neurotransmission is terminated by reuptake of extracellular serotonin (5-HT) by the high-affinity serotonin transporter (SERT). Selective 5-HT reuptake inhibitors (SSRIs) such as fluoxetine or escitalopram inhibit SERT and are currently the principal treatment for depression and anxiety disorders. In addition, SERT is a major molecular target for psychostimulants such as cocaine and amphetamines. Amphetamine-induced transport reversal at the closely related dopamine transporter (DAT) has been shown previously to be contingent upon modulation by calmodulin kinase IIα (αCaMKII). Here, we show that not only DAT, but also SERT, is regulated by αCaMKII. Inhibition of αCaMKII activity markedly decreased amphetamine-triggered SERT-mediated substrate efflux in both cells coexpressing SERT and αCaMKII and brain tissue preparations. The interaction between SERT and αCaMKII was verified using biochemical assays and FRET analysis and colocalization of the two molecules was confirmed in primary serotonergic neurons in culture. Moreover, we found that genetic deletion of αCaMKII impaired the locomotor response of mice to 3,4-methylenedioxymethamphetamine (also known as "ecstasy") and blunted d-fenfluramine-induced prolactin release, substantiating the importance of αCaMKII modulation for amphetamine action at SERT in vivo as well. SERT-mediated substrate uptake was neither affected by inhibition of nor genetic deficiency in αCaMKII. This finding supports the concept that uptake and efflux at monoamine transporters are asymmetric processes that can be targeted separately. Ultimately, this may provide a molecular mechanism for putative drug developments to treat amphetamine addiction.

Mechanism of the Association between Na+ Binding and Conformations at the Intracellular Gate in Neurotransmitter:Sodium Symporters.

Neurotransmitter:sodium symporters (NSSs) terminate neurotransmission by Na(+)-dependent reuptake of released neurotransmitters. Previous studies suggested that Na(+)-binding reconfigures dynamically coupled structural elements in an allosteric interaction network (AIN) responsible for function-related conformational changes, but the intramolecular pathway of this mechanism has remained uncharted. We describe a new approach for the modeling and analysis of intramolecular dynamics in the bacterial NSS homolog LeuT. From microsecond-scale molecular dynamics simulations and cognate experimental verifications in both LeuT and human dopamine transporter (hDAT), we apply the novel method to identify the composition and the dynamic properties of their conserved AIN. In LeuT, two different perturbations disrupting Na(+) binding and transport (i.e. replacing Na(+) with Li(+) or the Y268A mutation at the intracellular gate) affect the AIN in strikingly similar ways. In contrast, other mutations that affect the intracellular gate (i.e. R5A and D369A) do not significantly impair Na(+) cooperativity and transport. Our analysis shows these perturbations to have much lesser effects on the AIN, underscoring the sensitivity of this novel method to the mechanistic nature of the perturbation. Notably, this set of observations holds as well for hDAT, where the aligned Y335A, R60A, and D436A mutations also produce different impacts on Na(+) dependence. Thus, the detailed AIN generated from our method is shown to connect Na(+) binding with global conformational changes that are critical for the transport mechanism. That the AIN between the Na(+) binding sites and the intracellular gate in bacterial LeuT resembles that in eukaryotic hDAT highlights the conservation of allosteric pathways underlying NSS function.

Substrate-induced unlocking of the inner gate determines the catalytic efficiency of a neurotransmitter:sodium symporter.

Neurotransmitter:sodium symporters (NSSs) mediate reuptake of neurotransmitters from the synaptic cleft and are targets for several therapeutics and psychostimulants. The prokaryotic NSS homologue, LeuT, represents a principal structural model for Na(+)-coupled transport catalyzed by these proteins. Here, we used site-directed fluorescence quenching spectroscopy to identify in LeuT a substrate-induced conformational rearrangement at the inner gate conceivably leading to formation of a structural intermediate preceding transition to the inward-open conformation. The substrate-induced, Na(+)-dependent change required an intact primary substrate-binding site and involved increased water exposure of the cytoplasmic end of transmembrane segment 5. The findings were supported by simulations predicting disruption of an intracellular interaction network leading to a discrete rotation of transmembrane segment 5 and the adjacent intracellular loop 2. The magnitude of the spectroscopic response correlated inversely with the transport rate for different substrates, suggesting that stability of the intermediate represents an unrecognized rate-limiting barrier in the NSS transport mechanism.