De novo genetic variation is an important class of risk factors for autism spectrum disorder (ASD). Recently, whole exome sequencing of ASD families has identified a novel de novo missense mutation in the human dopamine (DA) transporter (hDAT) gene, which results in a Thr to Met substitution at site 356 (hDAT T356M). The dopamine transporter (DAT) is a presynaptic membrane protein that regulates dopaminergic tone in the central nervous system by mediating the high-affinity re-uptake of synaptically released DA, making it a crucial regulator of DA homeostasis. Here, we report the first functional, structural, and behavioral characterization of an ASD-associated de novo mutation in the hDAT. We demonstrate that the hDAT T356M displays anomalous function, characterized as a persistent reverse transport of DA (substrate efflux). Importantly, in the bacterial homolog leucine transporter, substitution of A289 (the homologous site to T356) with a Met promotes an outward-facing conformation upon substrate binding. In the substrate-bound state, an outward-facing transporter conformation is a required for substrate efflux. In Drosophila melanogaster, expression of hDAT T356M in DA neurons lacking Drosophila DAT leads to hyperlocomotion, a trait associated with DA dysfunction and ASD. Taken together, our findings demonstrate that alterations in DA homeostasis, mediated by aberrant DAT function, may confer risk for ASD and related neuropsychiatric conditions.
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.
We present the dynamic mechanism of concerted motions in a full-length molecular model of the human dopamine transporter (hDAT), a member of the neurotransmitter/sodium symporter (NSS) family, involved in state-to-state transitions underlying function. The findings result from an analysis of unbiased atomistic molecular dynamics simulation trajectories (totaling >14 μs) of the hDAT molecule immersed in lipid membrane environments with or without phosphatidylinositol 4,5-biphosphate (PIP2) lipids. The N-terminal region of hDAT (N-term) is shown to have an essential mechanistic role in correlated rearrangements of specific structural motifs relevant to state-to-state transitions in the hDAT. The mechanism involves PIP2-mediated electrostatic interactions between the N-term and the intracellular loops of the transporter molecule. Quantitative analyses of collective motions in the trajectories reveal that these interactions correlate with the inward-opening dynamics of hDAT and are allosterically coupled to the known functional sites of the transporter. The observed large-scale motions are enabled by specific reconfiguration of the network of ionic interactions at the intracellular end of the protein. The isomerization to the inward-facing state in hDAT is accompanied by concomitant movements in the extracellular vestibule and results in the release of an Na+ ion from the Na2 site and destabilization of the substrate dopamine in the primary substrate binding S1 site. The dynamic mechanism emerging from the findings highlights the involvement of the PIP2-regulated interactions between the N-term and the intracellular loop 4 in the functionally relevant conformational transitions that are also similar to those found to underlie state-to-state transitions in the leucine transporter (LeuT), a prototypical bacterial homologue of the NSS.
The dopamine transporter (DAT) is a transmembrane protein belonging to the family of Neurotransmitter:Sodium Symporters (NSS). Members of the NSS are responsible for the clearance of neurotransmitters from the synaptic cleft, and for their translocation back into the presynaptic nerve terminal. The DAT contains long intracellular N- and C-terminal domains that are strongly implicated in the transporter function. The N-terminus (N-term), in particular, regulates the reverse transport (efflux) of the substrate through DAT. Currently, the molecular mechanisms of the efflux remain elusive in large part due to lack of structural information on the N-terminal segment. Here we report a computational model of the N-term of the human DAT (hDAT), obtained through an ab initio structure prediction, in combination with extensive atomistic molecular dynamics (MD) simulations in the context of a lipid membrane. Our analysis reveals that whereas the N-term is a highly dynamic domain, it contains secondary structure elements that remain stable in the long MD trajectories of interactions with the bilayer (totaling >2.2 µs). Combining MD simulations with continuum mean-field modeling we found that the N-term engages with lipid membranes through electrostatic interactions with the charged lipids PIP2 (phosphatidylinositol 4,5-Biphosphate) or PS (phosphatidylserine) that are present in these bilayers. We identify specific motifs along the N-term implicated in such interactions and show that differential modes of N-term/membrane association result in differential positioning of the structured segments on the membrane surface. These results will inform future structure-based studies that will elucidate the mechanistic role of the N-term in DAT function.
ABSTRACT:With the construction and implementation of a logical and standardized numbering of atomic nuclei, to define mono-, di-, and oligo-peptide systems, automation of input file generation and data extraction could greatly improve the efficiency of the search for the structural energy minima on the potential energy hypersurface of these systems. The internal hierarchy of the database covering constitutional structures, protective groups, levels of theory, and basis sets used, as well as the variety of possible conformations, is also discussed. © 2002 Wiley Periodicals, Inc. Int J Quantum Chem 90: 933-968, 2002 Key words: peptide computations; atomic numbering; protein folding; internal coordinates; database internal hierarchy; ab initio DedicationT his article is written in the "teaching spirit" of our immortal teacher Professor P. O. Löwdin. We hope that our article and the methodology and ideals within will improve the understanding of young researchers in the whole field of molecular science, from physics and mathematics through chemistry and biology, all the way to medicine and even computational sciences, specifically for those who wish to enter the field of study comprising peptide and protein folding. In this spirit, we wish to dedicate this article to the legacy and living memory of Professor P. O. Löwdin. PreambleWe are now in a new age where we are able to produce vast amounts of data but do not capitalize on their value. We cannot thoroughly analyze all of the data, due to limitations in extraction of relevant variables and values, coupled with a serious lack of cross-referencing/pattern-matching. Consequently, one must develop "data-engines" and "data-mechanics" for multivariable analyses of systems commonly having hundreds or thousands of degrees of freedom. The process is particularly difficult when intersystem assemblies are made of known intrasystem building blocks, such as with modularly constructed oligopeptides and glycopeptides interacting with substrate molecules. In fact, the direction of approach, environmental conditions, and many other interactive relations become very important, especially in the development of kinetic models. Although intersystem dynamics and mechanics are very complex and yield a wealth of understanding, they are more of a holistic approach [1] to modern challenges in theoretical areas. The internal thermodynamics of oligopeptidic secondary and tertiary structural elements is perhaps one of the most challenging aspects of chemistry as a whole, in all of its related subdisciplines.Using this reductionist approach [1] and breaking the problem of secondary and tertiary peptide/protein structure up into its constituent elements, one is able to isolate each, creating and refining them, until they are understood in a form that allows them to be reassembled into a more accurate and holistic picture [2]. This would include the formulation of a set of analytic rules to account for the coupling behavior of all parameters in the system. However, at this point there is litt...
Novel psychoactive substances (NPS) may have unsuspected addiction potential through possessing stimulant properties. Stimulants normally act at the dopamine transporter (DAT) and thus increase dopamine (DA) availability in the brain, including nucleus accumbens, within the reward and addiction pathway. This paper aims to assess DAT responses to dissociative diarylethylamine NPS by means of in vitro and in silico approaches. We compared diphenidine (DPH) and 2-methoxydiphenidine (methoxphenidine, 2-MXP/MXP) for their binding to rat DAT, using autoradiography assessment of [125I]RTI-121 displacement in rat striatal sections. The drugs’ effects on electrically-evoked DA efflux were measured by means of fast cyclic voltammetry in rat accumbens slices. Computational modeling, molecular dynamics and alchemical free energy simulations were used to analyse the atomistic changes within DAT in response to each of the five dissociatives: DPH, 2-MXP, 3-MXP, 4-MXP and 2-Cl-DPH, and to calculate their relative binding free energy. DPH increased DA efflux as a result of its binding to DAT, whereas MXP had no significant effect on either DAT binding or evoked DA efflux. Our computational findings corroborate the above and explain the conformational responses and atomistic processes within DAT during its interactions with the dissociative NPS. We suggest DPH can have addictive liability, unlike MXP, despite the chemical similarities of these two NPS.
Valvular interstitial cells (VICs) play a critical role in the pathophysiology of heart valves. The objective of this study was to investigate the roles of different subcellular structures of the VICs, especially a-SMA stress fibers, to the VIC mechanical responses under different mechanical loading conditions and activation states. We modeled the VIC as a continuum with two distinct domains: the cell nucleus and cytoplasm. The nucleus was modeled as an incompressible neo-Hookean material while the cytoplasm was modeled as a mixture of two solid phases: the basal, isotropic cytoskeleton phase and a-SMA stress fiber phase, which exhibit some orientations at each point described by an orientation density function. The a-SMA stress fibers also exhibit passive elastic and active contractile responses in the direction of the orientations. We developed VIC mechanical model, which integrated the data from two experiments: micropipette aspiration (MA) and atomic force microscopy (AFM) of the aortic VIC (AVIC) and pulmonary VIC (PVIC) that each exhibits different expression levels of the a-SMA and contraction strength. In the MA experiment, VICs are in inactivated states while in AFM experiment, VICs are in activated states, exhibiting higher level of a-SMA expression and contraction. Thus, using our model in conjunction with the experimental data, we investigated how the expression level and active contraction of the a-SMA fibers affect the mechanical responses of the VICs. We implemented our model on the finite element method. We determined a~10-fold difference in the contractions strength between the AVICs and PVICs, implying that not only the expression level of the a-SMA fibers but also the contraction level increases from PVICs compared to AVICs. The higher expression of the a-SMA fibers may facilitate more efficient contraction and increased ability to synthesize collagen and other ECM components.
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