Outward Facing Research Articles

On the Harm Inflicted by Urban Teacher Education Programs: Learning From the Historical Trauma Experienced by Teachers of Color

Throughout the country, urban teacher education programs ubiquitously employ the terms “diversity” and “social justice” on their websites. But—what do these terms mean and how do they influence (if at all) the programs represented by these terms according to the lived experiences of students of color in these programs? Through historical trauma theory, this study seeks to understand the experiences of teachers of color who graduated from urban teacher education programs that employed “diversity” and “social justice” on their websites. Through the analysis of interviews conducted with eight teachers of color, it unveils how teacher education programs dehumanize and exclude pre-service teachers of color, fostering psychological violence, promoting segregation and displacement, and cultivating economic deprivation. Findings show that the rhetorical and visual portraits of “diversity” and “social justice” on these programs’ websites are experienced by teachers of color as incongruent to their lived experiences within them. In particular, the outward facing diverse racial images do not reflect the populations these teacher education programs serve (mostly white) and the classroom and field experiences are dissonant from the commitments the programs espouse. Implications call for transforming teacher education from spaces where historical trauma is reproduced to spaces where racial healing and reconciliation can occur.

Transformative mindfulness: the role of mind-body practices in community-based activism

This article emerges from the simple observation that community-based social and environmental activists often engage with practices of mindfulness, either personally or collectively. It draws on two case studies, a UK-based Transition initiative and a community of social entrepreneurs in Germany. On the surface, social and environmental activists, committed to change in the ‘real world’, outward facing and public, jar with practices of ‘mindfulness’: personal and interior actions –‘private’. We argue that post-foundationalist understandings of community, particularly Nancy’s being-in-common – popularised within geography as ‘community economies’ – and the philosophical and spiritual roots of mindfulness are two lines of thought that provide clues to this co-occurrence. Going beyond understandings of collectivity that build on the coming together of preformed individuals or presuppose a common substance, we set the (Westernised) Buddhist influences on mindfulness, specifically the notion of interbeing, side by side with Nancy’s being-in-common. This article argues that both the political and spiritual aspects of activism are integral parts of social change. It concludes that post-foundational and Buddhist-inspired lines of thought cross-fertilise and chart a course towards transformative mindfulness.

Simulations of Active Transport Across the Blood-Brain Barrier

The blood-brain barrier (BBB) is responsible for maintaining homeostasis of the brain by regulating the chemical environment, immune cell transport, and the entry of toxins and pathogens, and misregulation of the BBB has been shown to be implicated in numerous CNS disorders. One of the challenges in studying the BBB include the bioavailability of therapies, as well as rising multidrug resistance of pathogens. A major contributor to drug resistance and lacking bioavailability of therapies is the over-expression of the broad-specificity P-glycoprotein (P-gp) efflux pump in the endothelial cells of the BBB. However, despite the role of P-gp in affecting the bioavailability, pharmacokinetics and efficacy of therapeutic drugs, the mechanism of the efflux of substrates via P-gp, as well as the inhibition of P-gp activity, are not well understood. To address the mechanisms of P-gp-mediated substrate efflux and P-gp inhibition, we performed multimicrosecond unbiased atomic detail molecular dynamics (MD) simulations of the P-glycoprotein from novel human cryo-EM models as well as X-ray crystal structures in the presence of substrates (doxorubicin, rhodamine-123) and an inhibitor (tariquidar). We compare the differential transport gradient of P-gp substrates and inhibitors between the inward-facing (IF) and outward-facing (OF) conformations of P-gp to propose a model of P-gp turnover. We compare this model of active transport to our model of passive transport across the BBB.

Microscopic View of Structural Transitions of Glucose Transporters

Glucose transporters (GLUTs) facilitate the diffusion of glucose across cellular membranes, thereby playing an essential role in cell metabolism. In this context, GLUT3 has been drawing special attention due to its reported over-expression in tumors (due to their increased demand for glucose) and, therefore, as a potential drug target in cancer therapy. The transport cycle of GLUT3 consists of large-scale structural changes between the outward-facing (OF) and inward-facing (IF) states. However, the molecular details of these structural changes are still unknown.

A Kinetic Description of Cytosolic K+ Binding to the Human Sertotonin Transporter Under Turnover Conditions

The human serotonin transporter (hSERT) belongs to the neurotransmitter:sodium symporter (NSS) family, which regulate neurotransmission by re-uptaking the released neurotransmitters into the presynaptic neuron. During the transport cycle, the transporter alternates between the outward-facing (OF) and inward-facing (IF) states to translocate the substrate across the membrane. Central to the cycle in hSERT is the OF-to-IF state transition, during which 5-HT (serotonin) is inwardly symported in a Na+- and Cl--dependent manner, followed by cytosolic-K+-binding-triggered returning to the OF state. K+ binding might act as a “kinetic decision point” by frustrating the outward transport of 5-HT, namely, the reverse of the physiological direction. However, the mechanism of such K+ regulation remains elusive. Moreover, the conventional transport stoichiometry (15-HTin:1Na+in:1Cl−in:1K+out) has been challenged by recent studies showing that Cl- might remain bound to hSERT during the entire cycle. To explore the role of cytosolic K+ binding to IF hSERT, we have constructed a Markov State Model from an extensive set of molecular dynamics simulations. Starting with the IF state, the system is modeled under turnover conditions, i.e., post-5-HT-release and exposed to cytosolic K+ concentration, resulting in 50 independent, 200-ns replicas. More than half of the trajectories capture spontaneous K+ binding to the Na2 site, a conserved Na+ binding site within NSSs deemed to control the transporter conformational changes and substrate release. Furthermore, consistent with recent experimental results, Cl- was found to remain bound in the majority of the ensembles, questioning the contribution of the Cl- gradient to the substrate transport. Together, these results characterize the previously unknown K+ site in hSERT and present a quantitative kinetic description of K+ binding, shedding light on the mechanism of the ion-dependence of the IF-to-OF transition that resets the transport cycle.

Interplay Between Membrane Curvature and Conformational States in ABC Transporters

ATP-Binding Cassette (ABC) transporters constitute one of the largest superfamily of transmembrane proteins, present in both prokaryotes and eukaryotes. ATP hydrolysis at the nucleotide binding domains (NBD) drives conformational changes in the transmembrane domains (TMD) allowing substrate translocation. During this process, the structure of the ABC transporter alternates between the inward-facing (IF) conformation and the outward-facing (OF) conformation. The most prevalent and best characterized ABC-member is the mammalian P-glycoprotein (Pgp).

Modelling of Ion and Water Permeation in the Outward Facing Conformation of Human Anion Exchanger 1

Modelling of Ion and Water Permeation in the Outward Facing Conformation of Human Anion Exchanger 1

Bile Acid Transport by the Symporter ASBTNM: Substrate Binding and Conformational Change

The apical sodium-dependent bile acid transporter (ASBT) allows reabsorption of bile acids from the intestine by coupling bile acid movement to the sodium gradient. Such transporters are attractive targets for drug delivery and in the treatment for hypercholesterolaemia. Several structures of bacterial homologues in both inward and outward facing conformations have been obtained experimentally, including a substrate-bound inward facing structure of the homologue from Neisseria meningitidis (ASBTNM). However, many details surrounding the binding of substrates and the conformational transition remain unclear. We have used experimental and computational methods to explore these details at an atomistic scale using the homologue ASBTNM and the bile acid taurocholate. Models of apo and substrate-bound ASBTNM in the outward-facing conformation were generated. Biased molecular dynamics simulations and free energy calculations were used to explore conformational changes and possible movement of taurocholate during the transition by an elevator mechanism. Probability distributions and the residues important for binding of sodium and taurocholate in wild type and mutant ASBTNM were investigated with unbiased simulations, revealing alternative sodium binding sites. These results further our understanding of the important molecular details of ASBT function.

Locking Two Rigid-body Bundles in an Outward-Facing Conformation: The Ion-coupling Mechanism in a LeuT-fold Transporter

Secondary active transporters use electrochemical gradient of ions to fuel the “uphill” translocation of the substrate following the alternating-access model. The coupling of ions to conformational dynamics of the protein remains one of the least characterized aspects of the transporter function. We employ extended molecular dynamics (MD) simulations to examine the Na+-binding effects on the structure and dynamics of a LeuT-fold, Na+-coupled secondary transporter (Mhp1) in its major conformational states, i.e., the outward-facing (OF) and inward-facing (IF) states, as well as on the OF ↔ IF state transition. Microsecond-long, unbiased MD simulations illustrate that Na+ stabilizes an OF conformation favorable for substrate association, by binding to a highly conserved site at the interface between the two helical bundles and restraining their relative position and motion. Furthermore, a special-protocol biased simulation for state transition suggests that Na+ binding hinders the OF ↔ IF transition. These synergistic Na+-binding effects allosterically couple the ion and substrate binding sites and modify the kinetics of state transition, collectively increasing the lifetime of an OF conformation with high substrate affinity, thereby facilitating substrate recruitment from a low-concentration environment. Based on the similarity between our findings for Mhp1 and experimental reports on LeuT, we propose that this model may represent a general Na+-coupling mechanism among LeuT-fold transporters.

ADVANCING OUR UNDERSTANDING OF ELDERHOOD: A NARRATIVE REVIEW

There are relatively few explorations of later life in the peer reviewed gerontological literature that holistically embrace the duality of potential and decline. This is in striking contrast to a growing body of non-scholarly literature, frequently authored by elders, displaying deep interest in the phenomenon of elderhood, i.e. the holistic, lived experience of later life. We conducted a narrative review with the aim of describing the state of the science with regard to the bio-psycho-social-spiritual experience of elderhood. Following a search of multiple databases for English language, peer reviewed articles published from 2000-2017, we identified 24 articles in the disciplines of gerontology, anthropology, psychology, the humanities, and spirituality studies, reflecting elderhood in Eastern and Western cultures. While the articles offered no shared operational definition of elderhood, nor applied any unifying conceptual or theoretical structures, several common themes emerged. These included the description of elderhood as both inward facing (inner development) and outward facing (social contributions of elders). Numerous articles also recognized that ageism socially mediates the experience of elderhood, resulting in a failure of social systems and structures to recognize or provide opportunities for lifelong growth in later years, including a lack of mentors and role models for individuals transitioning into elderhood. This review demonstrates that there is a compelling need for the discipline of gerontology to strengthen our understanding of the phenomenon of elderhood by leading on the development and implementation of theoretically driven empirical research into the subject of the holistic, lived experience of later life.

Exploring Conformational Transitions and Free-Energy Profiles of Proton-Coupled Oligopeptide Transporters.

Proteins involved in peptide uptake and transport belong to the proton-coupled oligopeptide transporter (POT) family. Crystal structures of POT family members reveal a common fold consisting of two domains of six transmembrane α helices that come together to form a "V" shaped transporter with a central substrate binding site. Proton-coupled oligopeptide transporters operate through an alternate access mechanism, where the membrane transporter undergoes global conformational changes, alternating between inward-facing (IF), outward-facing (OF), and occluded (OC) states. Conformational transitions are promoted by proton and ligand binding; however, due to the absence of crystallographic models of the outward-open state, the role of H+ and ligands is still not fully understood. To provide a comprehensive picture of the POT conformational equilibrium, conventional and enhanced sampling molecular dynamics simulations of PepTst in the presence or absence of ligand and protonation were performed. Free-energy profiles of the conformational variability of PepTst were obtained from microseconds of adaptive biasing force (ABF) simulations. Our results reveal that both proton and ligand significantly change the conformational free-energy landscape. In the absence of ligand and protonation, only transitions involving IF and OC states are allowed. After protonation of the residue Glu300, the wider free-energy well for Glu300 protonated PepTst indicates a greater conformational variability relative to the apo system, and OF conformations became accessible. For the Glu300 protonated Holo-PepTst, the presence of a second free-energy minimum suggests that OF conformations are not only accessible, but also stable. The differences in the free-energy profiles demonstrate that transitions toward outward-facing conformation occur only after protonation, which is likely the first step in the mechanism of peptide transport. Our extensive ABF simulations provide a fully atomic description of all states of the transport process, offering a model for the alternating access mechanism and how protonation and ligand control the conformational changes.

From Piggie Park v. Newman to Masterpiece Cakeshop v. Colorado Civil Rights Commission: Application of the Outward Facing Actions Test to LGBTQ Nondiscrimination Provisions

For over a century courts have embraced a framework distinguishing the behavior of religious organizations on the basis of whether the actions are inward or outward facing. This standard safeguards the rights of organizations to develop and enforce their religious tenets among their members and engage in self government without unconstitutional government intrusion. However, courts have also been clear that the rights of religious organizations to engage in actions that impact members of the general public and non members are not unlimited. As a federal court held in Newman v. Piggie Park Enterprises, the right to believe is not “absolute” and can not be practiced in “utter disregard of the clear constitutional rights of other citizens.” As the legal rights of LGBTQ people become more cemented, it is imperative that courts remain faithful to this well established standard and recognize the rights of LGBTQ people in a way that is consistent with this nation’s civil rights jurisprudence.

Effect of oxidative stress on cystine transportation by xC‾ antiporter

We performed computer simulations to investigate the effect of oxidation on the extracellular cystine (CYC) uptake by the xC− antiporter. The latter is important for killing of cancer cells. Specifically, applying molecular dynamics (MD) simulations we studied the transport of CYC across xCT, i.e., the light subunit of the xC− antiporter, in charge of bidirectional transport of CYC and glutamate. We considered the outward facing (OF) configuration of xCT, and to study the effect of oxidation, we modified the Cys327 residue, located in the vicinity of the extracellular milieu, to cysteic acid (CYO327).Our computational results showed that oxidation of Cys327 results in a free energy barrier for CYC translocation, thereby blocking the access of CYC to the substrate binding site of the OF system. The formation of the energy barrier was found to be due to the conformational changes in the channel. Analysis of the MD trajectories revealed that the reorganization of the side chains of the Tyr244 and CYO327 residues play a critical role in the OF channel blocking. Indeed, the calculated distance between Tyr244 and either Cys327 or CYO327 showed a narrowing of the channel after oxidation. The obtained free energy barrier for CYC translocation was found to be 33.9kJmol−1, indicating that oxidation of Cys327, by e.g., cold atmospheric plasma, is more effective in inhibiting the xC− antiporter than in the mutation of this amino acid to Ala (yielding a barrier of 32.4kJmol−1). The inhibition of the xC− antiporter may lead to Cys starvation in some cancer cells, eventually resulting in cancer cell death.

Conformation space of a heterodimeric ABC exporter under turnover conditions.

Cryo-electron microscopy (cryo-EM) has the capacity to capture molecular machines in action1–3. ATP-binding cassette (ABC) exporters are highly dynamic membrane proteins that extrude a wide range of substances from the cytosol4–6 and thereby contribute to essential cellular processes, adaptive immunity, and multidrug resistance7,8. Despite their vital importance, the coupling of nucleotide binding, hydrolysis, and release to the conformational dynamics remains poorly resolved, especially for heterodimeric/asymmetric ABC exporters that abound in humans. Here, we present eight high-resolution cryo-EM structures that delineate the full functional cycle of an asymmetric ABC exporter in lipid environment. Cryo-EM analysis under active turnover conditions reveals distinct inward-facing (IF) conformations, one of them with bound peptide substrate, and previously undescribed asymmetric post-hydrolysis states with dimerized nucleotide-binding domains (NBDs) and a closed extracellular gate. Capturing an outward-facing (OF) open conformation requires a slow-down in ATP hydrolysis, indicating the transient nature of this state vulnerable to substrate re-entry. ATP-bound pre-hydrolysis and vanadate-trapped states are conformationally equivalent and both comprise co-existing OF conformations with open and closed extracelluar gates. In contrast, the post-hydrolysis states from the turnover experiment exhibit asymmetric ADP/ATP occlusion after phosphate release from the canonical site and display a progressive separation of the nucleotide-binding domains and unlocking of the intracellular gate. Our findings reveal that phosphate release, not ATP hydrolysis, triggers the return of the exporter to the IF conformation. By mapping the conformational landscape during active turnover, aided by mutational and chemical modulation of kinetic rates to trap the key intermediates, we resolved fundamental and so-far hidden steps of the substrate translocation cycle of asymmetric ABC transporters.

ABCG36/PEN3/PDR8 Is an Exporter of the Auxin Precursor, Indole-3-Butyric Acid, and Involved in Auxin-Controlled Development.

The PDR-type ABCG transporter, ABCG36/PDR8/PEN3, is thought to be implicated in the export of a few structurally unrelated substrates, including the auxin precursor, indole-3-butyric acid (IBA), although a clear-cut proof of transport is lacking. An outward facing, lateral root (LR) location for ABCG36 fuelled speculations that it might secrete IBA into the rhizosphere. Here, we provide strong evidence that ABCG36 catalyzes the export of IBA – but not of indole-3-acetic acid – through the plasma membrane. ABCG36 seems to function redundantly with the closely related isoform ABCG37/PDR9/PIS1 in a negative control of rootward IBA transport in roots, which might be dampened by concerted, lateral IBA export. Analyses of single and double mutant phenotypes suggest that both ABCG36 and ABCG37 function cooperatively in auxin-controlled plant development. Both seem to possess a dual function in the control of auxin homeostasis in the root tip and long-range transport in the mature root correlating with non-polar and polar expression profiles in the LR cap and epidermis, respectively.

The heme-sensitive regulator SbnI has a bifunctional role in staphyloferrin B production by Staphylococcus aureus

Staphylococcus aureus infection relies on iron acquisition from its host. S. aureus takes up iron through heme uptake by the iron-responsive surface determinant (Isd) system and by the production of iron-scavenging siderophores. Staphyloferrin B (SB) is a siderophore produced by the 9-gene sbn gene cluster for SB biosynthesis and efflux. Recently, the ninth gene product, SbnI, was determined to be a free l-serine kinase that produces O-phospho-l-serine (OPS), a substrate for SB biosynthesis. Previous studies have also characterized SbnI as a DNA-binding regulatory protein that senses heme to control sbn gene expression for SB synthesis. Here, we present crystal structures at 1.9-2.1 Å resolution of a SbnI homolog from Staphylococcus pseudintermedius (SpSbnI) in both apo form and in complex with ADP, a product of the kinase reaction; the latter confirmed the active-site location. The structures revealed that SpSbnI forms a dimer through C-terminal domain swapping and a dimer of dimers through intermolecular disulfide formation. Heme binding had only a modest effect on SbnI enzymatic activity, suggesting that its two functions are independent and structurally distinct. We identified a heme-binding site and observed catalytic heme transfer between a heme-degrading protein of the Isd system, IsdI, and SbnI. These findings support the notion that SbnI has a bifunctional role contributing precursor OPS to SB synthesis and directly sensing heme to control expression of the sbn locus. We propose that heme transfer from IsdI to SbnI enables S. aureus to control iron source preference according to the sources available in the environment.

The Development of Autobiographical Memory, Autobiographical Narratives, and Autobiographical Consciousness.

In this article, we expand on aspects of autobiographical memory initially laid out in our earlier exposition of the sociocultural developmental model. We present a developmental account of the integration of an extended subjective perspective within an extended narrative framework both of which are mediated through language and shared cultural narratives that culminate in autobiographical consciousness. Autobiographical consciousness goes beyond simple memories of past events to create a sense of extended self through time that has experienced and reflexively evaluated events. We argue from philosophical, evolutionary, and developmental psychological perspectives that narratives are a critical form of human consciousness, and that this form is learned through everyday social interactions that are linguistically mediated. Language has "double-duality" in that it is both outward facing, allowing more explicit, organized and differentiated communication to and with others, and language is also inward facing, in that language provides tools for organizing and differentiating internal consciousness. Although consciousness itself is multifaceted, we argue that language is the mechanism without which this particular form of human autobiographical consciousness would not develop.

Molecular Basis of the Glucose Transport Mechanism in Plants.

The SWEET family belongs to a class of transporters in plants that undergoes large conformational changes to facilitate transport of sugar molecules across the cell membrane (SWEET, Sugars Will Eventually Be Exported Transporter). However, the structures of their functionally relevant conformational states in the transport cycle have not been reported. In this study, we have characterized the conformational dynamics and complete transport cycle of glucose in the OsSWEET2b transporter using extensive molecular dynamics simulations. Using Markov state models, we estimated the free energy barrier associated with different states as well as for the glucose transport mechanism. SWEETs undergo a structural transition to outward-facing (OF), occluded (OC), and inward-facing (IF) and strongly support an alternate access transport mechanism. The glucose diffuses freely from outside to inside the cell without causing major conformational changes which means that the conformations of glucose unbound and bound snapshots are exactly the same for OF, OC, and IF states. We identified a network of hydrophobic core residues at the center of the transporter that restricts the glucose entry to the cytoplasmic side and acts as an intracellular hydrophobic gate. The mechanistic predictions from molecular dynamics simulations are validated using site-directed mutagenesis experiments. Our simulation also revealed hourglass-like intermediate states making the pore radius narrower at the center. This work provides new fundamental insights into how substrate–transporter interactions actively change the free energy landscape of the transport cycle to facilitate enhanced transport activity.

Functionalizing Cell Membranes with DNA Origami for Multiplexed Biomolecular Sensing

The plasma membrane is the primary communicating interface between the cell and extracellular environment. Thus, the ability to engineer new functions into the cell membrane to externally monitor or manipulate cellular interactions is of great significance. Previous studies have focused on engineering cell surface proteins or incorporating synthetic protein constructs into the cell membrane, which are often highly challenging. Therefore, designing a robust approach to incorporate nanodevices with diverse structural and dynamic functions into the cell membrane can enable engineering of the cell surface as a functional material. DNA origami has enabled the fabrication of nanostructures with diverse biological applications such as molecular measurements, and biomolecular sensing1. Hence, to extend the functions of DNA nanostructures to the context of the cell plasma membrane, we developed a robust and controllable approach to incorporate DNA origami nanostructures into cell membranes2. Our approach enables the development of cell‐based multiplexed detection tools to examine and report the presence of various extracellular biomolecular and potentially biophysical cues.Our method implements a cholesterol conjugated DNA strand serving as the membrane incorporated oligo (MIO) to serve as an anchoring site for the binding of a DNA origami membrane bound breadboard (MBB). First, we confirmed the functionalization of the cell membrane with MIO by visualization of a complementary fluorescent oligo (Fig. 1A). Next, we utilized an intermediate oligo that binds to the MIO and extends a single‐stranded DNA binding site away from the surface for subsequent binding of the MBB (Fig. 1B). Using this functionalization scheme, we effectively coated the surface of five cell types with MBB structures (Fig. 1C, image shown for CH12.LX B Cells). In addition, confocal imaging techniques confirmed MBB presence around the cell periphery (Fig. 1D). Next, we leveraged this approach to facilitate controllable cell‐cell adhesion. We used oligos that connect two MBBs by binding to extended overhangs on the outward facing side. We used this approach to facilitate controllable binding between two heterotypic and homotypic cells functionalized with MBB structures (Fig. 2A). Finally, we used the overhangs on the outward facing side to incorporate aptamers that can detect the presence of extracellular single stranded DNA (Fig. 2B) and platelet‐derived growth factor (PDGF).We have developed a robust approach to functionalize the cell membrane with 3D DNA origami nanostructures. Our approach enables controlled cell‐cell attachment to construct higher order cellular assemblies. In addition, MBB can serve as a versatile and multiplexed cell‐based platform to monitor and report biomolecular or potentially biophysical cues in the cell surrounding environment.Support or Funding InformationFunding was provided by National Institute of Health grant R01HL14194.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Translating the atypical dopamine uptake inhibitor hypothesis toward therapeutics for treatment of psychostimulant use disorders.

Medication-assisted treatments are unavailable to patients with cocaine use disorders. Efforts to develop potential pharmacotherapies have led to the identification of a promising lead molecule, JJC8-091, that demonstrates a novel binding mode at the dopamine transporter (DAT). Here, JJC8-091 and a structural analogue, JJC8-088, were extensively and comparatively assessed to elucidate neurochemical correlates to their divergent behavioral profiles. Despite sharing significant structural similarity, JJC8-088 was more cocaine-like, increasing extracellular DA concentrations in the nucleus accumbens shell (NAS) efficaciously and more potently than JJC8-091. In contrast, JJC8-091 was not self-administered and was effective in blocking cocaine-induced reinstatement to drug seeking. Electrophysiology experiments confirmed that JJC8-091 was more effective than JJC8-088 at inhibiting cocaine-mediated enhancement of DA neurotransmission. Further, when VTA DA neurons in DAT-cre mice were optically stimulated, JJC8-088 produced a significant leftward shift in the stimulation-response curve, similar to cocaine, while JJC8-091 shifted the curve downward, suggesting attenuation of DA-mediated brain reward. Computational models predicted that JJC8-088 binds in an outward facing conformation of DAT, similar to cocaine. Conversely, JJC8-091 steers DAT towards a more occluded conformation. Collectively, these data reveal the underlying molecular mechanism at DAT that may be leveraged to rationally optimize leads for the treatment of cocaine use disorders, with JJC8-091 representing a compelling candidate for development.