Dopamine D2 receptors modulate the cholinergic pause and inhibitory learning.

Cholinergic interneurons (CINs) in the striatum respond to salient stimuli with a multiphasic response, including a pause, in neuronal activity. Slice-physiology experiments have shown the importance of dopamine D2 receptors (D2Rs) in regulating CIN pausing, yet the behavioral significance of the CIN pause and its regulation by dopamine in vivo is still unclear. Here, we show that D2R upregulation in CINs of the nucleus accumbens (NAc) lengthens the pause in CIN activity ex vivo and enlarges a stimulus-evoked decrease in acetylcholine (ACh) levels during behavior. This enhanced dip in ACh levels is associated with a selective deficit in the learning to inhibit responding in a Go/No-Go task. Our data demonstrate, therefore, the importance of CIN D2Rs in modulating the CIN response induced by salient stimuli and point to a role of this response in inhibitory learning. This work has important implications for brain disorders with altered striatal dopamine and ACh function, including schizophrenia and attention-deficit hyperactivity disorder (ADHD).

Dopamine D2 receptor overexpression in the nucleus accumbens core induces robust weight loss during scheduled fasting selectively in female mice.

Anorexia nervosa (AN) is an eating disorder observed predominantly in women and girls that is characterized by a low body-mass index, hypophagia, and hyperactivity. Activity-based anorexia (ABA), which refers to the weight loss, hypophagia, and hyperactivity exhibited by rodents exposed to both running wheels and scheduled fasting, provides a model for aspects of AN. Increased dopamine D2/D3 receptor binding in the anteroventral striatum has been reported in AN patients. We virally overexpressed D2Rs on nucleus accumbens core (D2R-OENAc) neurons that endogenously express D2Rs, and tested mice of both sexes in the open field test, ABA paradigm, and intraperitoneal glucose tolerance test (IGTT). D2R-OENAc did not alter baseline body weight, but increased locomotor activity in the open field across both sexes. During constant access to food and running wheels, D2R-OENAc mice of both sexes increased food intake and ran more than controls. However, when food was available only 7 h a day, only female D2R-OENAc mice rapidly lost 25% of their initial body weight, reduced food intake, and substantially increased wheel running. Surprisingly, female D2R-OENAc mice also rapidly lost 25% of their initial body weight during scheduled fasting without wheel access and showed no changes in food intake. In contrast, male D2R-OENAc mice maintained body weight during scheduled fasting. D2R-OENAc mice of both sexes also showed glucose intolerance in the IGTT. In conclusion, D2R-OENAc alters glucose metabolism in both sexes but drives robust weight loss only in females during scheduled fasting, implicating metabolic mechanisms in this sexually dimorphic effect.

Agonist-induced formation of unproductive receptor-G12 complexes.

G proteins are activated when they associate with G protein-coupled receptors (GPCRs), often in response to agonist-mediated receptor activation. It is generally thought that agonist-induced receptor-G protein association necessarily promotes G protein activation and, conversely, that activated GPCRs do not interact with G proteins that they do not activate. Here we show that GPCRs can form agonist-dependent complexes with G proteins that they do not activate. Using cell-based bioluminescence resonance energy transfer (BRET) and luminescence assays we find that vasopressin V2 receptors (V2R) associate with both Gs and G12 heterotrimers when stimulated with the agonist arginine vasopressin (AVP). However, unlike V2R-Gs complexes, V2R-G12 complexes are not destabilized by guanine nucleotides and do not promote G12 activation. Activating V2R does not lead to signaling responses downstream of G12 activation, but instead inhibits basal G12-mediated signaling, presumably by sequestering G12 heterotrimers. Overexpressing G12 inhibits G protein receptor kinase (GRK) and arrestin recruitment to V2R and receptor internalization. Formyl peptide (FPR1 and FPR2) and Smoothened (Smo) receptors also form complexes with G12 that are insensitive to nucleotides, suggesting that unproductive GPCR-G12 complexes are not unique to V2R. These results indicate that agonist-dependent receptor-G protein association does not always lead to G protein activation and may in fact inhibit G protein activation.

G12/13 is activated by acute tethered agonist exposure in the adhesion GPCR ADGRL3.

The adhesion G-protein-coupled receptor (GPCR) latrophilin 3 (ADGRL3) has been associated with increased risk of attention deficit hyperactivity disorder (ADHD) and substance use in human genetic studies. Knockdown in multiple species leads to hyperlocomotion and altered dopamine signaling. Thus, ADGRL3 is a potential target for treatment of neuropsychiatric disorders that involve dopamine dysfunction, but its basic signaling properties are poorly understood. Identification of adhesion GPCR signaling partners has been limited by a lack of tools to acutely activate these receptors in living cells. Here, we design a novel acute activation strategy to characterize ADGRL3 signaling by engineering a receptor construct in which we could trigger acute activation enzymatically. Using this assay, we found that ADGRL3 signals through G12/G13 and Gq, with G12/13 the most robustly activated. Gα12/13 is a new player in ADGRL3 biology, opening up unexplored roles for ADGRL3 in the brain. Our methodological advancements should be broadly useful in adhesion GPCR research.

Signalling profiles of a structurally diverse panel of synthetic cannabinoid receptor agonists.

Synthetic cannabinoid receptor agonists (SCRAs) represent the most rapidly proliferating class of "designer drugs" or "new psychoactive substances". SCRAs offer unregulated alternatives to cannabis that evade routine drug tests, but their use is increasingly associated with severe toxicity and death worldwide. Little is currently known about SCRA molecular pharmacology, or the mechanisms underpinning their toxicity, although the effects are believed to be primarily mediated by the type 1 cannabinoid receptor (CB1). In this study, we aimed to characterise the signalling profiles of a structurally diverse panel of novel SCRAs at CB1. We compare SCRAs to traditional reference cannabinoids CP55,940, WIN55,212-2, and THC. The activity of the SCRAs was assessed in key receptor signalling and regulatory pathways, including cAMP production, translocation of ß-arrestin 1 and 2, and receptor internalisation. The activity profiles of the ligands were also evaluated using operational analysis to identify ligand bias. Results revealed that SCRAs activities were relatively balanced in the pathways evaluated (compared to WIN55,212-2), although 5F-CUMYL-P7AICA and XLR-11 possessed partial efficacy in cAMP stimulation and ß-arrestin translocation. Notably, the SCRAs showed distinct potency and efficacy profiles compared to THC. In particular, while the majority of SCRAs demonstrated robust ß-arrestin translocation, cAMP stimulation, and internalisation, THC failed to elicit high efficacy responses in any of these assays. Further study is required to delineate if these pathways could contribute to SCRA toxicity in humans.

Detection of G Protein-Coupled Receptor Complexes in Postmortem Human Brain by Proximity Ligation Assay.

Combining immunological and molecular biological methods, the antibody-based proximity ligation assay (PLA) has been used for more than a decade to detect and quantify protein-protein interactions, protein modification, and protein expression in situ, including in brain tissue. However, the transfer of this technology to human brain samples requires a number of precautions due to the nature of the specimens and their specific processing. Here, we used the PLA brightfield detection technique to assess the expression of dopamine D2 receptor and adenosine A2A receptor and their proximity in human postmortem brains, and we developed a systematic random sampling method to help quantify the PLA signals. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Sample preparation and sectioning for PLA_BF Basic Protocol 2: PLA_BF staining of brain tissue Basic Protocol 3: Image acquisition and result analysis Support Protocol: Luxol fast blue/cresyl violet staining.

Detecting G protein-coupled receptor complexes in postmortem human brain with proximity ligation assay and a Bayesian classifier.

Despite the controversy regarding the existence and physiological relevance of class A G protein-coupled receptor dimerization, there is substantial evidence for functional interactions between the dopamine D2 receptor (D2R) and the adenosine A2A receptor (A2AR). A2AR-D2R complexes have been detected in rodent brains by proximity ligation assay; however, their existence in the human brain has not been demonstrated. In this study, we used Brightfield proximity ligation assay, combined with a systematic sampling and a parameter-free naive Bayesian classifier, and demonstrated proximity between the D2R and the A2AR in the adult human ventral striatum, consistent with their colocalization within complexes and the possible existence of D2R-A2AR heteromers. These methods are applicable to the relative quantification of proximity of two proteins, as well as the expression levels of individual proteins.

Do Toxic Synthetic Cannabinoid Receptor Agonists Have Signature in Vitro Activity Profiles? A Case Study of AMB-FUBINACA.

Recreational consumption of synthetic cannabinoid receptor agonists (SCRAs) is a growing crisis in public health in many parts of the world. AMB-FUBINACA is a member of this class of drugs and is responsible for a large proportion of SCRA-related toxicity both in New Zealand and internationally. Strikingly, little is currently known about the mechanisms by which SCRAs exert toxic effects or whether their activity through the CB1 cannabinoid receptor (the mediator of cannabinoid-related psychoactivity) is sufficient to explain clinical observations. The current study therefore set out to perform a basic molecular pharmacology characterization of AMB-FUBINACA (in comparison to traditional research cannabinoids CP55,940, WIN55,212-2, and Δ9-THC) in fundamental pathways of receptor activity, including cAMP inhibition, pERK activation, ability to drive CB1 internalization, and ability to induce translocation of ß-arrestins-1 and -2. Activity pathways were then compared by operational analysis to indicate whether AMB-FUBINACA may be a biased ligand. Results revealed that AMB-FUBINACA is highly efficacious and potent in all pathways assayed. However, surprisingly, bias analysis suggested that Δ9-THC, not AMB-FUBINACA, may be a biased ligand, with it being less active in both arrestin pathways than predicted by the activity of the other ligands tested. These data may help predict molecular characteristics of SCRAs. However, more research is required to determine whether these molecular effects manifest in toxicity at tissue/system level.

Gs- versus Golf-dependent functional selectivity mediated by the dopamine D1 receptor.

The two highly homologous subtypes of stimulatory G proteins Gαs (Gs) and Gαolf (Golf) display contrasting expression patterns in the brain. Golf is predominant in the striatum, while Gs is predominant in the cortex. Yet, little is known about their functional distinctions. The dopamine D1 receptor (D1R) couples to Gs/olf and is highly expressed in cortical and striatal areas, making it an important therapeutic target for neuropsychiatric disorders. Using novel drug screening methods that allow analysis of specific G-protein subtype coupling, we found that, relative to dopamine, dihydrexidine and N-propyl-apomorphine behave as full D1R agonists when coupled to Gs, but as partial D1R agonists when coupled to Golf. The Gs/Golf-dependent biased agonism by dihydrexidine was consistently observed at the levels of cellular signaling, neuronal function, and behavior. Our findings of Gs/Golf-dependent functional selectivity in D1R ligands open a new avenue for the treatment of cortex-specific or striatum-specific neuropsychiatric dysfunction.

Optical Control of Dopamine Receptors Using a Photoswitchable Tethered Inverse Agonist.

Family A G protein-coupled receptors (GPCRs) control diverse biological processes and are of great clinical relevance. Their archetype rhodopsin becomes naturally light sensitive by binding covalently to the photoswitchable tethered ligand (PTL) retinal. Other GPCRs, however, neither bind covalently to ligands nor are light sensitive. We sought to impart the logic of rhodopsin to light-insensitive Family A GPCRs in order to enable their remote control in a receptor-specific, cell-type-specific, and spatiotemporally precise manner. Dopamine receptors (DARs) are of particular interest for their roles in motor coordination, appetitive, and aversive behavior, as well as neuropsychiatric disorders such as Parkinson's disease, schizophrenia, mood disorders, and addiction. Using an azobenzene derivative of the well-known DAR ligand 2-(N-phenethyl-N-propyl)amino-5-hydroxytetralin (PPHT), we were able to rapidly, reversibly, and selectively block dopamine D1 and D2 receptors (D1R and D2R) when the PTL was conjugated to an engineered cysteine near the dopamine binding site. Depending on the site of tethering, the ligand behaved as either a photoswitchable tethered neutral antagonist or inverse agonist. Our results indicate that DARs can be chemically engineered for selective remote control by light and provide a template for precision control of Family A GPCRs.

Development of novel biosensors to study receptor-mediated activation of the G-protein α subunits Gs and Golf.

Gαs (Gs) and Gαolf (Golf) are highly homologous G-protein α subunits that activate adenylate cyclase, thereby serving as crucial mediators of intracellular signaling. Because of their dramatically different brain expression patterns, we studied similarities and differences between their activation processes with the aim of comparing their receptor coupling mechanisms. We engineered novel luciferase- and Venus-fused Gα constructs that can be used in bioluminescence resonance energy transfer assays. In conjunction with molecular simulations, these novel biosensors were used to determine receptor activation-induced changes in conformation. Relative movements in Gs were consistent with the crystal structure of ß2 adrenergic receptor in complex with Gs Conformational changes in Golf activation are shown to be similar to those in Gs Overall the current study reveals general similarities between Gs and Golf activation at the molecular level and provides a novel set of tools to search for Gs- and Golf-specific receptor pharmacology. In view of the wide functional and pharmacological roles of Gs- and Golf-coupled dopamine D1 receptor and adenosine A2A receptor in the brain and other organs, elucidating their differential structure-function relationships with Gs and Golf might provide new approaches for the treatment of a variety of neuropsychiatric disorders. In particular, these novel biosensors can be used to reveal potentially therapeutic dopamine D1 receptor and adenosine A2A receptor ligands with functionally selective properties between Gs and Golf signaling.

Dopamine receptor activation increases HIV entry into primary human macrophages.

Macrophages are the primary cell type infected with HIV in the central nervous system, and infection of these cells is a major component in the development of neuropathogenesis and HIV-associated neurocognitive disorders. Within the brains of drug abusers, macrophages are exposed to increased levels of dopamine, a neurotransmitter that mediates the addictive and reinforcing effects of drugs of abuse such as cocaine and methamphetamine. In this study we examined the effects of dopamine on HIV entry into primary human macrophages. Exposure to dopamine during infection increased the entry of R5 tropic HIV into macrophages, irrespective of the concentration of the viral inoculum. The entry pathway affected was CCR5 dependent, as antagonizing CCR5 with the small molecule inhibitor TAK779 completely blocked entry. The effect was dose-dependent and had a steep threshold, only occurring above 108 M dopamine. The dopamine-mediated increase in entry required dopamine receptor activation, as it was abrogated by the pan-dopamine receptor antagonist flupenthixol, and could be mediated through both subtypes of dopamine receptors. These findings indicate that the effects of dopamine on macrophages may have a significant impact on HIV pathogenesis. They also suggest that drug-induced increases in CNS dopamine may be a common mechanism by which drugs of abuse with distinct modes of action exacerbate neuroinflammation and contribute to HIV-associated neurocognitive disorders in infected drug abusers.

Discovery and characterization of a G protein-biased agonist that inhibits ß-arrestin recruitment to the D2 dopamine receptor.

A high-throughput screening campaign was conducted to interrogate a 380,000+ small-molecule library for novel D2 dopamine receptor modulators using a calcium mobilization assay. Active agonist compounds from the primary screen were examined for orthogonal D2 dopamine receptor signaling activities including cAMP modulation and ß-arrestin recruitment. Although the majority of the subsequently confirmed hits activated all signaling pathways tested, several compounds showed a diminished ability to stimulate ß-arrestin recruitment. One such compound (MLS1547; 5-chloro-7-[(4-pyridin-2-ylpiperazin-1-yl)methyl]quinolin-8-ol) is a highly efficacious agonist at D2 receptor-mediated G protein-linked signaling, but does not recruit ß-arrestin as demonstrated using two different assays. This compound does, however, antagonize dopamine-stimulated ß-arrestin recruitment to the D2 receptor. In an effort to investigate the chemical scaffold of MLS1547 further, we characterized a set of 24 analogs of MLS1547 with respect to their ability to inhibit cAMP accumulation or stimulate ß-arrestin recruitment. A number of the analogs were similar to MLS1547 in that they displayed agonist activity for inhibiting cAMP accumulation, but did not stimulate ß-arrestin recruitment (i.e., they were highly biased). In contrast, other analogs displayed various degrees of G protein signaling bias. These results provided the basis to use pharmacophore modeling and molecular docking analyses to build a preliminary structure-activity relationship of the functionally selective properties of this series of compounds. In summary, we have identified and characterized a novel G protein-biased agonist of the D2 dopamine receptor and identified structural features that may contribute to its biased signaling properties.

Conformational changes in dopamine transporter intracellular regions upon cocaine binding and dopamine translocation.

The dopamine transporter (DAT), a member of the neurotransmitter:sodium symporter family, mediates the reuptake of dopamine at the synaptic cleft. DAT is the primary target for psychostimulants such as cocaine and amphetamine. We previously demonstrated that cocaine binding and dopamine transport alter the accessibility of Cys342 in the third intracellular loop (IL3). To study the conformational changes associated with the functional mechanism of the transporter, we made cysteine substitution mutants, one at a time, from Phe332 to Ser351 in IL3 of the background DAT construct, X7C, in which 7 endogenous cysteines were mutated. The accessibility of the 20 engineered cysteines to polar charged sulfhydryl reagents was studied in the absence and presence of cocaine or dopamine. Of the 11 positions that reacted with methanethiosulfonate ethyl ammonium, as evidenced by inhibition of ligand binding, 5 were protected against this inhibition by cocaine and dopamine (S333C, S334C, N336C, M342C and T349C), indicating that reagent accessibility is affected by conformational changes associated with inhibitor and substrate binding. In some of the cysteine mutants, transport activity is disrupted, but can be rescued by the presence of zinc, most likely because the distribution between inward- and outward-facing conformations is restored by zinc binding. The experimental data were interpreted in the context of molecular models of DAT in both the inward- and outward-facing conformations. Differences in the solvent accessible surface area for individual IL3 residues calculated for these states correlate well with the experimental accessibility data, and suggest that protection by ligand binding results from the stabilization of the outward-facing configuration. Changes in the residue interaction networks observed from the molecular dynamics simulations also revealed the critical roles of several positions during the conformational transitions. We conclude that the IL3 region of DAT undergoes significant conformational changes in transitions necessary for both cocaine binding and substrate transport.

Discovery of a novel selective kappa-opioid receptor agonist using crystal structure-based virtual screening.

Kappa-opioid (KOP) receptor agonists exhibit analgesic effects without activating reward pathways. In the search for nonaddictive opioid therapeutics and novel chemical tools to study physiological functions regulated by the KOP receptor, we screened in silico its recently released inactive crystal structure. A selective novel KOP receptor agonist emerged as a notable result and is proposed as a new chemotype for the study of the KOP receptor in the etiology of drug addiction, depression, and/or pain.

Discovery of ß-arrestin-biased dopamine D2 ligands for probing signal transduction pathways essential for antipsychotic efficacy.

Elucidating the key signal transduction pathways essential for both antipsychotic efficacy and side-effect profiles is essential for developing safer and more effective therapies. Recent work has highlighted noncanonical modes of dopamine D(2) receptor (D(2)R) signaling via ß-arrestins as being important for the therapeutic actions of both antipsychotic and antimanic agents. We thus sought to create unique D(2)R agonists that display signaling bias via ß-arrestin-ergic signaling. Through a robust diversity-oriented modification of the scaffold represented by aripiprazole (1), we discovered UNC9975 (2), UNC0006 (3), and UNC9994 (4) as unprecedented ß-arrestin-biased D(2)R ligands. These compounds also represent unprecedented ß-arrestin-biased ligands for a G(i)-coupled G protein-coupled receptor (GPCR). Significantly, UNC9975, UNC0006, and UNC9994 are simultaneously antagonists of G(i)-regulated cAMP production and partial agonists for D(2)R/ß-arrestin-2 interactions. Importantly, UNC9975 displayed potent antipsychotic-like activity without inducing motoric side effects in inbred C57BL/6 mice in vivo. Genetic deletion of ß-arrestin-2 simultaneously attenuated the antipsychotic actions of UNC9975 and transformed it into a typical antipsychotic drug with a high propensity to induce catalepsy. Similarly, the antipsychotic-like activity displayed by UNC9994, an extremely ß-arrestin-biased D(2)R agonist, in wild-type mice was completely abolished in ß-arrestin-2 knockout mice. Taken together, our results suggest that ß-arrestin signaling and recruitment can be simultaneously a significant contributor to antipsychotic efficacy and protective against motoric side effects. These functionally selective, ß-arrestin-biased D(2)R ligands represent valuable chemical probes for further investigations of D(2)R signaling in health and disease.

Detection of antigen interactions ex vivo by proximity ligation assay: endogenous dopamine D2-adenosine A2A receptor complexes in the striatum.

The existence of G protein-coupled receptor (GPCR) dimers and/or oligomers has been demonstrated in heterologous systems using a variety of biochemical and biophysical assays. While these interactions are the subject of intense research because of their potential role in modulating signaling and altering pharmacology, evidence for the existence of receptor interactions in vivo is still elusive because of a lack of appropriate methods to detect them. Here, we adapted and optimized a proximity ligation assay (PLA) for the detection in brain slices of molecular proximity of two antigens located on either the same or two different GPCRs. Using this approach, we were able to confirm the existence of dopamine D2 and adenosine A2A receptor complexes in the striatum of mice ex vivo.

Making structural sense of dimerization interfaces of delta opioid receptor homodimers.

Opioid receptors, like other members of the G protein-coupled receptor (GPCR) family, have been shown to associate to form dimers and/or oligomers at the plasma membrane. Whether this association is stable or transient is not known. Recent compelling evidence suggests that at least some GPCRs rapidly associate and dissociate. We have recently calculated binding affinities from free energy estimates to predict transient association between mouse delta opioid receptor (DOR) protomers at a symmetric interface involving the fourth transmembrane (TM4) helix (herein termed "4" dimer). Here we present disulfide cross-linking experiments with DOR constructs with cysteines substituted at the extracellular ends of TM4 or TM5 that confirm the formation of DOR complexes involving these helices. Our results are consistent with the involvement of TM4 and/or TM5 at the DOR homodimer interface, but possibly with differing association propensities. Coarse-grained (CG) well-tempered metadynamics simulations of two different dimeric arrangements of DOR involving TM4 alone or with TM5 (herein termed "4/5" dimer) in an explicit lipid-water environment confirmed the presence of two structurally and energetically similar configurations of the 4 dimer, as previously assessed by umbrella sampling calculations, and revealed a single energetic minimum of the 4/5 dimer. Additional CG umbrella sampling simulations of the 4/5 dimer indicated that the strength of association between DOR protomers varies depending on the protein region at the interface, with the 4 dimer being more stable than the 4/5 dimer.

Exploring the binding site crevice of a family B G protein-coupled receptor, the type 1 corticotropin releasing factor receptor.

Family B of G protein-coupled receptors (GPCRs) is composed of receptors that bind peptides, such as secretin, glucagon, parathyroid hormone, and corticotropin releasing factor (CRF), which play critical physiological roles. These receptors, like all GPCRs, share a common structural motif of seven membrane-spanning segments, which have been proposed to bind small ligands, such as antalarmin, a nonpeptide antagonist of the type 1 receptor for CRF (CRF(1)). This leads to the hypothesis that as for family A GPCRs, the binding sites of small ligands for family B GPCRs are on the surface of a water-accessible crevice, the binding-site crevice, which is formed by the membrane-spanning segments and extends from the extracellular surface of the receptor into the plane of the membrane. To test this hypothesis we have begun to obtain structural information about family B GPCRs, using as a prototype the CRF(1), by determining the ability of sulfhydryl-specific methanethiosulfonate derivatives, such as the methanethiosulfonate-ethylammonium (MTSEA), to react with CRF(1) and thus irreversibly inhibit (125)I-Tyr(0)-sauvagine binding. We found that MTSEA inhibited (125)I-Tyr(0)-sauvagine binding to CRF(1) and that antalarmin protected against this irreversible inhibition. To identify the susceptible cysteine(s), we mutated, one at a time, four endogenous cysteines to serine. Mutation to serine of Cys211, Cys233, or Cys364 decreased the susceptibility of sauvagine binding to irreversible inhibition by MTSEA. Thus, Cys211, Cys233, and Cys364 at the cytoplasmic ends of the third, fourth, and seventh membrane-spanning segments, respectively, are exposed in the binding site crevice of CRF(1).

Single-molecule dynamics of gating in a neurotransmitter transporter homologue.

Neurotransmitter:Na(+) symporters (NSS) remove neurotransmitters from the synapse in a reuptake process that is driven by the Na(+) gradient. Drugs that interfere with this reuptake mechanism, such as cocaine and antidepressants, profoundly influence behaviour and mood. To probe the nature of the conformational changes that are associated with substrate binding and transport, we have developed a single-molecule fluorescence imaging assay and combined it with functional and computational studies of the prokaryotic NSS homologue LeuT. Here we show molecular details of the modulation of intracellular gating of LeuT by substrates and inhibitors, as well as by mutations that alter binding, transport or both. Our direct observations of single-molecule transitions, reflecting structural dynamics of the intracellular region of the transporter that might be masked by ensemble averaging or suppressed under crystallographic conditions, are interpreted in the context of an allosteric mechanism that couples ion and substrate binding to transport.