Parkinsonism and attention deficit hyperactivity disorder (ADHD) are widespread brain disorders that involve disturbances of dopaminergic signaling. The sodium-coupled dopamine transporter (DAT) controls dopamine homeostasis, but its contribution to disease remains poorly understood. Here, we analyzed a cohort of patients with atypical movement disorder and identified 2 DAT coding variants, DAT-Ile312Phe and a presumed de novo mutant DAT-Asp421Asn, in an adult male with early-onset parkinsonism and ADHD. According to DAT single-photon emission computed tomography (DAT-SPECT) scans and a fluoro-deoxy-glucose-PET/MRI (FDG-PET/MRI) scan, the patient suffered from progressive dopaminergic neurodegeneration. In heterologous cells, both DAT variants exhibited markedly reduced dopamine uptake capacity but preserved membrane targeting, consistent with impaired catalytic activity. Computational simulations and uptake experiments suggested that the disrupted function of the DAT-Asp421Asn mutant is the result of compromised sodium binding, in agreement with Asp421 coordinating sodium at the second sodium site. For DAT-Asp421Asn, substrate efflux experiments revealed a constitutive, anomalous efflux of dopamine, and electrophysiological analyses identified a large cation leak that might further perturb dopaminergic neurotransmission. Our results link specific DAT missense mutations to neurodegenerative early-onset parkinsonism. Moreover, the neuropsychiatric comorbidity provides additional support for the idea that DAT missense mutations are an ADHD risk factor and suggests that complex DAT genotype and phenotype correlations contribute to different dopaminergic pathologies.

BackgroundSyntaxin 1 (STX1) is a presynaptic plasma membrane protein that coordinates synaptic vesicle fusion. STX1 also regulates the function of neurotransmitter transporters, including the dopamine (DA) transporter (DAT). The DAT is a membrane protein that controls DA homeostasis through the high-affinity re-uptake of synaptically released DA.MethodsWe adopt newly developed animal models and state-of-the-art biophysical techniques to determine the contribution of the identified gene variants to impairments in DA neurotransmission observed in autism spectrum disorder (ASD).OutcomesHere, we characterize two independent autism-associated variants in the genes that encode STX1 and the DAT. We demonstrate that each variant dramatically alters DAT function. We identify molecular mechanisms that converge to inhibit reverse transport of DA and DA-associated behaviors. These mechanisms involve decreased phosphorylation of STX1 at Ser14 mediated by casein kinase 2 as well as a reduction in STX1/DAT interaction. These findings point to STX1/DAT interactions and STX1 phosphorylation as key regulators of DA homeostasis.InterpretationWe determine the molecular identity and the impact of these variants with the intent of defining DA dysfunction and associated behaviors as possible complications of ASD.

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 Zn 2+ exposure and a substituted cysteineaccessibility 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-offunction 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 Zn 2+ , 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.A substantial proportion of the disease etiology of common psychiatric disorders, such as attention deficit hyperactivity disorder (ADHD), autism spectrum disorders (ASD), bipolar disorder, and schizophrenia is attributed genetic components (1). The underlying neurobiological mechanisms are not clear, but dopamine disturbances are believed to constitute a central component (2-7). The allelic spectrum of these dopamine-related disorders is rapidly expanding and comprises both common variants and rare structural or exonic mutations, including de novo variants (1,(8)(9)(10)(11)(12). Moreover, an interesting overlap in the genetic architecture of psychiatric disorders has been observed, and this pleiotrophy is seen for both common and rare variants (13)(14)(15)(16)(17)(18). Despite progress, much of the genetic component remains unaccounted for, and we also have the challenge ahead of translating most of the comprehensive genetic information into an understanding of underlying biological processes, and subsequently into clinically applicable knowledge. Rare variants may provide a unique handle for obtaining new disease insights as they are expected to h...

Background:The relationship between ion binding and conductance states of the dopamine transporter is poorly understood. Results: The Li ϩ leak is mediated by the second sodium site (Na2) and is gated by Cl Ϫ .

Background:The presence of an extracellular gate in the dopamine transporter has been proposed. Results: We use rescue mutations, zinc site engineering, and cysteine-reactive chemistry to establish the presence of the gate. Conclusion: Arg-85 and Asp-476 constitute a functional thin gate in the dopamine transporter. Significance: This gains further insight into the molecular mechanisms behind substrate transport by mammalian neurotransmitter transporters.

The objective was to be prepared for a total and sudden loss scenario while drilling and coring a challenging well in the Barents sea. A dual-gradient Controlled Mud Level (CML) system with Controlled Mud Cap Drilling (CMCD) mode was installed on the rig to manage minor and/or total losses. Prior to spud of the section, an advanced dynamic simulator with the actual well configuration loaded was used to conduct offline training, and prepare the drilling team and involved service personnel for the operation. Experience from previous wells in the area identified the risk of drilling into karstified carbonate zones with the potential of leading to total and sudden losses. An advanced dynamic simulator was used to reflect the details of the CML system to be used. The rig crew together with the CML operator and other involved service personnel were trained on how to manage a total loss scenario by switching from CML to CMCD mode. All relevant operational procedures were used as a basis for creating training scenarios and operational preparations for the exercises. This paper will briefly present the simulator set-up, the operation/training procedures and results from the training. Feedback from the operation itself will also be described including lesson-learned from utilizing a full-scale dynamic simulator with the actual well loaded during preparation for operation.

Safety and efficiency have always been the primary performance measures in the coiled-tubing (CT) industry. This paper describes the feasibility study, development objectives, field test, and launch of a new-generation CT unit in the Norwegian sector of the North Sea. This automated, modular system with its distributed system architecture is designed to improve safety in CT operations, with 15% operational efficiency gain and 30% offshore personnel reduction targeted. A typical completion on Valhall is a horizontal well completed with 5 to 12 proppant-treated fractures per well in the soft chalk formations. The operations and logistics are complex and involve repeated sequences of perforating with CT, proppant fracture treatment with stimulation vessel, and a combined cleanout/perforating run with CT. On a 24-hour operation offshore, a 13-member crew operate the conventional CT spread comprising the unit, reel, and power stand; well-control equipment; two high-pressure, positive-displacement pumps; shakers; choke; and a CT tower with the injector head. The requirements and specifications of the new unit were finalized after a detailed study of operations, platform requirements, and local regulations. Efficiency gains were targeted in the rig-up and rig-down times, and in the overall zone cycle times (operational efficiency). Health, safety, and environmental improvements are achieved by reducing manual handling and lifting operations on board. Distinguishing equipment characteristics are as follows:Preassembly and integration of components on skids to enable quicker rig-up and reduce manual handling. For example, offshore rig-up typically involves 54 lifts; the new unit cuts this number to 36.Distributed-control architecture to reduce the number of rig-up connections required. For example, the system design allows a total rig-up with only 36 hydraulic connections instead of the usual 84.Ability to run larger and heavier pipe (6,000-m length, 27/8-in. diameter, 90,000-psi yield) at faster rates enables easier completion of difficult wells and reduces the overall zone-cycle time. It also eliminates handling of friction-reducing chemicals while maintaining cleanout efficiency.Planned reduction in crew through the utilization of automated process control, The benefits of this include the following:All controls for the choke, shakers, CT unit, and pumps are centralized in the CT unit's control cab. Many equipment controls-related tasks that conventionally were hands-on tasks are performed by the control system to allow the operator to focus on treatment efficiency.Automated safety features are incorporated into process-control software to reduce safety exposure, particularly in areas prone to human errors.Cab ergonomics allow the operator to oversee the entire operation and react to any situation quickly and efficiently. Introduction Valhall field completions have evolved over the years to the degree that, today, in a single well, completions with as many as 10 to 12 multiple-proppant fracture treatments are common. To save rig time, these operations are performed in a simultaneous operations (Sim-Ops) mode, in which the rig, after landing the completion, skids over to the next well. The well is then completed in a Sim-Ops mode with a stimulation vessel and large CT spread. The first run with CT is mud clean out/venturi followed by a tubing-conveyed perforating run. This is followed by proppant fracturing with the stimulation vessel. The purpose of the next run with CT is to clean out the well, leave a sand plug to isolate the fracture, and perforate the next zone. This combined functionality is achieved by running "pump-over-guns," a configuration that enables the CT to clean the well while running the guns. This cycle is continued until all zones are completed, and the final run with CT cleans out the entire well.