Persistent binding at dopamine transporters determines sustained psychostimulant effects.

Psychostimulants interacting with the dopamine transporter (DAT) can be used illicitly or for the treatment of specific neuropsychiatric disorders. However, they can also produce severe and persistent adverse events. Often, their pharmacological properties in vitro do not fully correlate to their pharmacological profile in vivo. Here, we investigated the pharmacological effects of enantiomers of pyrovalerone, α-pyrrolidinovalerophenone, and 3,4-methylenedioxypyrovalerone as compared to the traditional psychostimulants cocaine and methylphenidate, using a variety of in vitro, computational, and in vivo approaches. We found that in vitro drug-binding kinetics at DAT correlate with the time-course of in vivo psychostimulant action in mice. In particular, a slow dissociation (i.e., slow koff) of S-enantiomers of pyrovalerone analogs from DAT predicts their more persistent in vivo effects when compared to cocaine and methylphenidate. Overall, our findings highlight the critical importance of drug-binding kinetics at DAT for determining the in vivo profile of effects produced by psychostimulant drugs.

Unveiling the crucial role of betaine: modulation of GABA homeostasis via SLC6A1 transporter (GAT1).

Betaine is an endogenous osmolyte that exhibits therapeutic potential by mitigating various neurological disorders. However, the underlying cellular and molecular mechanisms responsible for its neuroprotective effects remain puzzling.In this study, we describe a possible mechanism behind the positive impact of betaine in preserving neurons from excitotoxicity. Here we demonstrate that betaine at low concentration modulates the GABA uptake by GAT1 (slc6a1), the predominant GABA transporter in the central nervous system. This modulation occurs through the temporal inhibition of the transporter, wherein prolonged occupancy by betaine impedes the swift transition of the transporter to the inward conformation. Importantly, the modulatory effect of betaine on GAT1 is reversible, as the blocking of GAT1 disappears with increased extracellular GABA. Using electrophysiology, mass spectroscopy, radiolabelled cellular assay, and molecular dynamics simulation we demonstrate that betaine has a dual role in GAT1: at mM concentration acts as a slow substrate, and at µM as a temporal blocker of GABA, when it is below its K0.5. Given this unique modulatory characteristic and lack of any harmful side effects, betaine emerges as a promising neuromodulator of the inhibitory pathways improving GABA homeostasis via GAT1, thereby conferring neuroprotection against excitotoxicity.

Free energy profile of the substrate-induced occlusion of the human serotonin transporter.

The serotonin transporter (SERT) is a member of the Solute Carrier 6 (SLC6) family and is responsible for maintaining the appropriate level of serotonin in the brain. Dysfunction of SERT has been linked to several neuropsychiatric disorders, including depression, anxiety and obsessive-compulsive disorder. Therefore, an in-depth understanding of the mechanism on an atomistic level, coupled with a quantification of transporter dynamics and the associated free energies is required. Here, we constructed Markov state models (MSMs) from extensive unbiased molecular dynamics simulations to quantify the free energy profile of serotonin (5HT) triggered SERT occlusion and explored the driving forces of the mechanism of occlusion. Our results reveal that SERT occludes via multiple intermediate conformations and show that the motion of occlusion is energetically downhill for the 5HT-bound transporter. Force distribution analyses show that the interactions of 5HT with the bundle domain are crucial. During occlusion, attractive forces steadily increase and pull on the bundle domain, which leads to SERT occlusion. Some interactions become repulsive upon full occlusion, suggesting that SERT creates pressure on 5HT to promote its movement towards the cytosol.

Bioisosteric analogs of MDMA: Improving the pharmacological profile?

3,4-Methylenedioxymethamphetamine (MDMA, 'ecstasy') is re-emerging in clinical settings as a candidate for the treatment of specific neuropsychiatric disorders (e.g. post-traumatic stress disorder) in combination with psychotherapy. MDMA is a psychoactive drug, typically regarded as an empathogen or entactogen, which leads to transporter-mediated monoamine release. Despite its therapeutic potential, MDMA can induce dose-, individual-, and context-dependent untoward effects outside safe settings. In this study, we investigated whether three new methylenedioxy bioisosteres of MDMA improve its off-target profile. In vitro methods included radiotracer assays, transporter electrophysiology, bioluminescence resonance energy transfer and fluorescence-based assays, pooled human liver microsome/S9 fraction incubations, metabolic stability studies, isozyme mapping, and liquid chromatography coupled to high-resolution mass spectrometry. In silico methods included molecular docking. Compared with MDMA, all three MDMA bioisosteres (ODMA, TDMA, and SeDMA) showed similar pharmacological activity at human serotonin, dopamine, and norepinephrine transporters (hSERT, hDAT, and hNET, respectively) but decreased agonist activity at 5-HT2A/2B/2C receptors. Regarding their hepatic metabolism, they differed from MDMA, with N-demethylation being the only metabolic route shared, and without forming phase II metabolites. In addition, TDMA showed an enhanced intrinsic clearance in comparison to its congeners. Additional screening for their interaction with human organic cation transporters (hOCTs) and plasma membrane monoamine transporter (hPMAT) revealed a weaker interaction of the MDMA analogs with hOCT1, hOCT2, and hPMAT. Our findings suggest that these new MDMA bioisosteres might constitute appealing therapeutic alternatives to MDMA, sparing the primary pharmacological activity at hSERT, hDAT, and hNET, but displaying a reduced activity at 5-HT2A/2B/2C receptors and alternative hepatic metabolism. Whether these MDMA bioisosteres may pose lower risk alternatives to the clinically re-emerging MDMA warrants further studies.

Diacylglycerols interact with the L2 lipidation site in TRPC3 to induce a sensitized channel state.

Coordination of lipids within transient receptor potential canonical channels (TRPCs) is essential for their Ca2+ signaling function. Single particle cryo-EM studies identified two lipid interaction sites, designated L1 and L2, which are proposed to accommodate diacylglycerols (DAGs). To explore the role of L1 and L2 in TRPC3 function, we combined structure-guided mutagenesis and electrophysiological recording with molecular dynamics (MD) simulations. MD simulations indicate rapid DAG accumulation within both L1 and L2 upon its availability within the plasma membrane. Electrophysiological experiments using a photoswitchable DAG-probe reveal potentiation of TRPC3 currents during repetitive activation by DAG. Importantly, initial DAG exposure generates a subsequently sensitized channel state that is associated with significantly faster activation kinetics. TRPC3 sensitization is specifically promoted by mutations within L2, with G652A exhibiting sensitization at very low levels of active DAG. We demonstrate the ability of TRPC3 to adopt a closed state conformation that features partial lipidation of L2 sites by DAG and enables fast activation of the channel by the phospholipase C-DAG pathway.

Occlusion of the human serotonin transporter is mediated by serotonin-induced conformational changes in the bundle domain.

The human serotonin transporter (hSERT) terminates neurotransmission by removing serotonin (5HT) from the synaptic cleft, an essential process for proper functioning of serotonergic neurons. Structures of the hSERT have revealed its molecular architecture in four conformations, including the outward-open and occluded states, and show the transporter's engagement with co-transported ions and the binding mode of inhibitors. In this study, we investigated the molecular mechanism by which the hSERT occludes and sequesters the substrate 5HT. This first step of substrate uptake into cells is a structural change consisting of the transition from the outward-open to the occluded state. Inhibitors such as the antidepressants citalopram, fluoxetine, and sertraline inhibit this step of the transport cycle. Using molecular dynamics simulations, we reached a fully occluded state, in which the transporter-bound 5HT becomes fully shielded from both sides of the membrane by two closed hydrophobic gates. Analysis of 5HT-triggered occlusion showed that bound 5HT serves as an essential trigger for transporter occlusion. Moreover, simulations revealed a complex sequence of steps and showed that movements of bundle domain helices are only partially correlated. 5HT-triggered occlusion is initially dominated by movements of transmembrane helix 1b, while in the final step, only transmembrane helix 6a moves and relaxes an intermediate change in its secondary structure.

YASARA Model-Interactive Molecular Modeling from Two Dimensions to Virtual Realities.

The industry's transition from three-dimensional (3D) glasses to virtual reality (VR) headsets has left modelers stranded without hardware supply, since walking around and waving arms in a virtual world is a great experience but also very tiring when doing time-intensive modeling work. We present a novel software implementation that uses a VR headset while sitting at a desk in front of the normal screen, which is beamed into the virtual reality together with keyboard, mouse, and chair using the headset's cameras and an extra tracker attached to the seat-back. Compared to 3D glasses, this yields a comparably relaxing but much more immersive workplace and provides additional possibilities such as taking molecules into one's hands, standing up, and walking or teleporting through the models. This VR functionality has been combined with a molecular graphics engine based on Vulkan, a next-generation cross-platform application programming interface (API) for GPUs and the successor of the widely used Open Graphics Library (OpenGL). It is built into the YASARA Model program, which includes many features like small and large molecule builders, electron densities, partial surfaces, contact analysis, coordinate manipulation, and animations. Interactive tutorials are provided to guide modelers into VR and familiarize them with the molecular modeling features. YASARA Model is available for Linux, Windows, Android, and MacOS (the latter without VR) with an introductory video at www.YASARA.org/vr.

Human ABCB1 with an ABCB11-like degenerate nucleotide binding site maintains transport activity by avoiding nucleotide occlusion.

Several ABC exporters carry a degenerate nucleotide binding site (NBS) that is unable to hydrolyze ATP at a rate sufficient for sustaining transport activity. A hallmark of a degenerate NBS is the lack of the catalytic glutamate in the Walker B motif in the nucleotide binding domain (NBD). The multidrug resistance transporter ABCB1 (P-glycoprotein) has two canonical NBSs, and mutation of the catalytic glutamate E556 in NBS1 renders ABCB1 transport-incompetent. In contrast, the closely related bile salt export pump ABCB11 (BSEP), which shares 49% sequence identity with ABCB1, naturally contains a methionine in place of the catalytic glutamate. The NBD-NBD interfaces of ABCB1 and ABCB11 differ only in four residues, all within NBS1. Mutation of the catalytic glutamate in ABCB1 results in the occlusion of ATP in NBS1, leading to the arrest of the transport cycle. Here we show that despite the catalytic glutamate mutation (E556M), ABCB1 regains its ATP-dependent transport activity, when three additional diverging residues are also replaced. Molecular dynamics simulations revealed that the rescue of ATPase activity is due to the modified geometry of NBS1, resulting in a weaker interaction with ATP, which allows the quadruple mutant to evade the conformationally locked pre-hydrolytic state to proceed to ATP-driven transport. In summary, we show that ABCB1 can be transformed into an active transporter with only one functional catalytic site by preventing the formation of the ATP-locked pre-hydrolytic state in the non-canonical site.

Thermal Unfolding of the Human Serotonin Transporter: Differential Effect by Stabilizing and Destabilizing Mutations and Cholesterol on Thermodynamic and Kinetic Stability.

Folding-deficient mutants of solute carrier 6 (SLC6) family members have been linked to human diseases. The serotonin transporter [(SERT)/SLC6A4] is an important drug target in the treatment of depression, anxiety, and obsessive-compulsive disorders and-with structural information in several conformational states-one of the best understood transporters. Here, we surmised that thermal unfolding offered a glimpse on the folding energy landscape of SLC6 transporters. We carried out molecular dynamic (MD) simulations to understand the mechanistic basis for enhanced and reduced stability, respectively, of the thermostabilized variant SERT-Y110A/I291A/T439S, which had previously been used for crystallization of human SERT in the outward-facing state, and of the folding-deficient SERT-P601A/G602A. We also examined the hydrophobic mismatch caused by the absence of cholesterol to explore the contribution of cholesterol to protein stability. When compared with wild type SERT, the thermodynamic and kinetic stability of SERT-Y110A/I291A/T439S was enhanced. In the other instances, changes in these two components were not correlated: the mutations in SERT-P601A/G602A led to a drop in thermodynamic but an increase in kinetic stability. The divergence was even more pronounced after cholesterol depletion, which reduced thermodynamic stability but increased the kinetic stability of wild type SERT to a level comparable to that of SERT-Y110A/I291A/T439S. We conclude that the low cholesterol content of the endoplasmic reticulum facilitates progression of the folding trajectory by reducing the energy difference between folding intermediates and the native state. SIGNIFICANCE STATEMENT: Point mutations in solute carrier 6 (SLC6) family members cause folding diseases. The serotonin transporter [(SERT)/SLC6A4] is a target for antidepressants and the best understood SLC6. This study produced molecular dynamics simulations and examined thermal unfolding of wild type and mutant SERT variants to understand their folding energy landscape. In the folding-deficient SERT-P012A/G602A, changes in kinetic and thermodynamic stability were not correlated. Similarly, cholesterol depletion lowered thermodynamic but enhanced kinetic stability. These observations allow for rationalizing the action of pharmacochaperones.

Structural, functional, and behavioral insights of dopamine dysfunction revealed by a deletion in SLC6A3.

The human dopamine (DA) transporter (hDAT) mediates clearance of DA. Genetic variants in hDAT have been associated with DA dysfunction, a complication associated with several brain disorders, including autism spectrum disorder (ASD). Here, we investigated the structural and behavioral bases of an ASD-associated in-frame deletion in hDAT at N336 (∆N336). We uncovered that the deletion promoted a previously unobserved conformation of the intracellular gate of the transporter, likely representing the rate-limiting step of the transport process. It is defined by a "half-open and inward-facing" state (HOIF) of the intracellular gate that is stabilized by a network of interactions conserved phylogenetically, as we demonstrated in hDAT by Rosetta molecular modeling and fine-grained simulations, as well as in its bacterial homolog leucine transporter by electron paramagnetic resonance analysis and X-ray crystallography. The stabilization of the HOIF state is associated both with DA dysfunctions demonstrated in isolated brains of Drosophila melanogaster expressing hDAT ∆N336 and with abnormal behaviors observed at high-time resolution. These flies display increased fear, impaired social interactions, and locomotion traits we associate with DA dysfunction and the HOIF state. Together, our results describe how a genetic variation causes DA dysfunction and abnormal behaviors by stabilizing a HOIF state of the transporter.

SLC6 transporter oligomerization.

Transporters of the solute carrier 6 (SLC6) family mediate the reuptake of neurotransmitters such as dopamine, norepinephrine, serotonin, GABA, and glycine. SLC6 family members are 12 transmembrane helix-spanning proteins that operate using the transmembrane sodium gradient for transport. These transporters assume various quaternary arrangements ranging from monomers to complex stoichiometries with multiple subunits. Dopamine and serotonin transporter oligomerization has been implicated in trafficking of newly formed proteins from the endoplasmic reticulum to the plasma membrane with a pre-fixed assembly. Once at the plasma membrane, oligomers are kept fixed in their quaternary assembly by interaction with phosphoinositides. While it remains unclear how oligomer formation precisely affects physiological transporter function, it has been shown that oligomerization supports the activity of release-type psychostimulants. Most recently, single molecule microscopy experiments unveiled that the stoichiometry differs between individual members of the SLC6 family. The present overview summarizes our understanding of the influence of plasma membrane constituents on transporter oligomerization, describes the known interfaces between protomers and discusses open questions.

Assembly of Biomolecular Gigastructures and Visualization with the Vulkan Graphics API.

Building and displaying all-atom models of biomolecular structures with millions or billions of atoms, like virus particles or cells, remain a challenge due to the sheer size of the data, the required levels of automated building, and the visualization limits of today's graphics hardware. Based on concepts introduced with the CellPack program, we report new algorithms to create such large-scale models using an intermediate coarse-grained "pet representation" of biomolecules with 1/10th the normal size. Pet atoms are placed such that they optimally trace the surface of the original molecule with just ∼1/50th the original atom number and are joined with covalent bonds. Molecular dynamics simulations of pet molecules allow for efficient packing optimization, as well as the generation of realistic DNA/RNA conformations. This pet world can be expanded back to the all-atom representation to be explored and visualized with full details. Essential for the efficient interactive visualization of gigastructures is the use of multiple levels of detail (LODs), where distant molecules are drawn with a heavily reduced polygon count. We present a grid-based algorithm to create such LODs for all common molecular graphics styles (including ball-and-sticks, ribbons, and cartoons) that do not require monochrome molecules to hide LOD transitions. As a practical application, we built all-atom models of SARS-CoV-2, HIV, and an entire presynaptic bouton with 1 µm diameter and 3.6 billion atoms, using modular building blocks to significantly reduce GPU memory requirements through instancing. We employ the Vulkan graphics API to maximize performance on consumer grade hardware and describe how to use the mmCIF format to efficiently store such giant models. An implementation is available as part of the YASARA molecular modeling and simulation program from www.YASARA.org. The free YASARA View program can be used to explore the presented models, which can be downloaded from www.YASARA.org/petworld, a Creative Commons platform for sharing giant biomolecular structures.

The Bile Salt Export Pump: Molecular Structure, Study Models and Small-Molecule Drugs for the Treatment of Inherited BSEP Deficiencies.

The bile salt export pump (BSEP/ABCB11) is responsible for the transport of bile salts from hepatocytes into bile canaliculi. Malfunction of this transporter results in progressive familial intrahepatic cholestasis type 2 (PFIC2), benign recurrent intrahepatic cholestasis type 2 (BRIC2) and intrahepatic cholestasis of pregnancy (ICP). Over the past few years, several small molecular weight compounds have been identified, which hold the potential to treat these genetic diseases (chaperones and potentiators). As the treatment response is mutation-specific, genetic analysis of the patients and their families is required. Furthermore, some of the mutations are refractory to therapy, with the only remaining treatment option being liver transplantation. In this review, we will focus on the molecular structure of ABCB11, reported mutations involved in cholestasis and current treatment options for inherited BSEP deficiencies.

Analysis of Sequence Divergence in Mammalian ABCGs Predicts a Structural Network of Residues That Underlies Functional Divergence.

The five members of the mammalian G subfamily of ATP-binding cassette transporters differ greatly in their substrate specificity. Four members of the subfamily are important in lipid transport and the wide substrate specificity of one of the members, ABCG2, is of significance due to its role in multidrug resistance. To explore the origin of substrate selectivity in members 1, 2, 4, 5 and 8 of this subfamily, we have analysed the differences in conservation between members in a multiple sequence alignment of ABCG sequences from mammals. Mapping sets of residues with similar patterns of conservation onto the resolved 3D structure of ABCG2 reveals possible explanations for differences in function, via a connected network of residues from the cytoplasmic to transmembrane domains. In ABCG2, this network of residues may confer extra conformational flexibility, enabling it to transport a wider array of substrates.

Functional Impact of the G279S Substitution in the Adenosine A1-Receptor (A1R-G279S7.44), a Mutation Associated with Parkinson's Disease.

In medium-size, spiny striatal neurons of the direct pathway, dopamine D1- and adenosine A1-receptors are coexpressed and are mutually antagonistic. Recently, a mutation in the gene encoding the A1-receptor (A1R), A1R-G279S7.44, was identified in an Iranian family: two affected offspring suffered from early-onset l-DOPA-responsive Parkinson's disease. The link between the mutation and the phenotype is unclear. Here, we explored the functional consequence of the G279S substitution on the activity of the A1-receptor after heterologous expression in HEK293 cells. The mutation did not affect surface expression and ligand binding but changed the susceptibility to heat denaturation: the thermodynamic stability of A1R-G279S7.44 was enhanced by about 2 and 8 K when compared with wild-type A1-receptor and A1R-Y288A7.53 (a folding-deficient variant used as a reference), respectively. In contrast, the kinetic stability was reduced, indicating a lower energy barrier for conformational transitions in A1R-G279S7.44 (73 ± 23 kJ/mol) than in wild-type A1R (135 ± 4 kJ/mol) or in A1R-Y288A7.53 (184 ± 24 kJ/mol). Consistent with this lower energy barrier, A1R-G279S7.44 was more effective in promoting guanine nucleotide exchange than wild-type A1R. We detected similar levels of complexes formed between D1-receptors and wild-type A1R or A1R-G279S7.44 by coimmunoprecipitation and bioluminescence resonance energy transfer. However, lower concentrations of agonist were required for half-maximum inhibition of dopamine-induced cAMP accumulation in cells coexpressing D1-receptor and A1R-G279S7.44 than in those coexpressing wild-type A1R. These observations predict enhanced inhibition of dopaminergic signaling by A1R-G279S7.44 in vivo, consistent with a pathogenic role in Parkinson's disease. SIGNIFICANCE STATEMENT: Parkinson's disease is caused by a loss of dopaminergic input from the substantia nigra to the caudate nucleus and the putamen. Activation of the adenosine A1-receptor antagonizes responses elicited by dopamine D1-receptor. We show that this activity is more pronounced in a mutant version of the A1-receptor (A1R-G279S7.44), which was identified in individuals suffering from early-onset Parkinson's disease.

Dopamine transporter forms stable dimers in the live cell plasma membrane in a phosphatidylinositol 4,5-bisphosphate-independent manner.

The human dopamine transporter (hDAT) regulates the level of the neurotransmitter dopamine (DA) in the synaptic cleft and recycles DA for storage in the presynaptic vesicular pool. Many neurotransmitter transporters exist as oligomers, but the physiological role of oligomerization remains unclear; for example, it has been speculated to be a prerequisite for amphetamine-induced release and protein trafficking. Previous studies point to an oligomeric quaternary structure of hDAT; however, the exact stoichiometry and the fraction of co-existing oligomeric states are not known. Here, we used single-molecule brightness analysis to quantify the degree of oligomerization of heterologously expressed hDAT fused to monomeric GFP (mGFP-hDAT) in Chinese hamster ovary (CHO) cells. We observed that monomers and dimers of mGFP-hDAT co-exist and that higher-order molecular complexes of mGFP-hDAT are absent at the plasma membrane. The mGFP-hDAT dimers were stable over several minutes, and the fraction of dimers was independent of the mGFP-hDAT surface density. Furthermore, neither oxidation nor depletion of cholesterol had any effect on the fraction of dimers. Unlike for the human serotonin transporter (hSERT), in which direct binding of phosphatidylinositol 4,5-bisphosphate (PIP2) stabilized the oligomers, the stability of mGFP-hDAT dimers was PIP2 independent.

An optically controlled probe identifies lipid-gating fenestrations within the TRPC3 channel.

Transient receptor potential canonical (TRPC) channels TRPC3, TRPC6 and TRPC7 are able to sense the lipid messenger diacylglycerol (DAG). The DAG-sensing and lipid-gating processes in these ion channels are still unknown. To gain insights into the lipid-sensing principle, we generated a DAG photoswitch, OptoDArG, that enabled efficient control of TRPC3 by light. A structure-guided mutagenesis screen of the TRPC3 pore domain unveiled a single glycine residue behind the selectivity filter (G652) that is exposed to lipid through a subunit-joining fenestration. Exchange of G652 with larger residues altered the ability of TRPC3 to discriminate between different DAG molecules. Light-controlled activation-deactivation cycling of TRPC3 channels by an OptoDArG-mediated optical 'lipid clamp' identified pore domain fenestrations as pivotal elements of the channel´s lipid-sensing machinery. We provide evidence for a novel concept of lipid sensing by TRPC channels based on a lateral fenestration in the pore domain that accommodates lipid mediators to control gating.

The Amino Terminus of LeuT Changes Conformation in an Environment Sensitive Manner.

Neurotransmitter:sodium symporters are highly expressed in the human brain and catalyze the uptake of substrate through the plasma membrane by using the electrochemical gradient of sodium as the energy source. The bacterial homolog LeuT, a small amino acid transporter isolated from the bacteria Aquifex aeolicus, is the founding member of the family and has been crystallized in three conformations. The N-terminus is structurally well defined and strongly interacts with the transporter core in the outward-facing conformations. However, it could not be resolved in the inward-facing conformation, which indicates enhanced mobility. Here we investigate conformations and dynamics of the N-terminus, by combining molecular dynamics simulations with experimental verification using distance measurements and accessibility studies. We found strongly increased dynamics of the N-terminus, but also that helix TM1A is subject to enhanced mobility. TM1A moves towards the transporter core in the membrane environment, reaching a conformation that is closer to the structure of LeuT with wild type sequence, indicating that the mutation introduced to create the inward-facing structure might have altered the position of helix TM1A. The mobile N-terminus avoids entering the open vestibule of the inward-facing state, as accessibility studies do not show any reduction of quenching by iodide of a fluorophore attached to the N-terminus.

Both reentrant loops of the sodium-coupled glutamate transporters contain molecular determinants of cation selectivity.

In the brain, glutamate transporters terminate excitatory neurotransmission by removing this neurotransmitter from the synapse via cotransport with three sodium ions into the surrounding cells. Structural studies have identified the binding sites of the three sodium ions in glutamate transporters. The residue side-chains directly interact with the sodium ions at the Na1 and Na3 sites and are fully conserved from archaeal to eukaryotic glutamate transporters. The Na2 site is formed by three main-chain oxygens on the extracellular reentrant hairpin loop HP2 and one on transmembrane helix 7. A glycine residue on HP2 is located closely to the three main-chain oxygens in all glutamate transporters, except for the astroglial transporter GLT-1, which has a serine residue at that position. Unlike for WT GLT-1, substitution of the serine residue to glycine enables sustained glutamate transport also when sodium is replaced by lithium. Here, using functional and simulation studies, we studied the role of this serine/glycine switch on cation selectivity of substrate transport. Our results indicate that the side-chain oxygen of the serine residues can form a hydrogen bond with a main-chain oxygen on transmembrane helix 7. This leads to an expansion of the Na2 site such that water can participate in sodium coordination at Na2. Furthermore, we found other molecular determinants of cation selectivity on the nearby HP1 loop. We conclude that subtle changes in the composition of the two reentrant hairpin loops determine the cation specificity of acidic amino acid transport by glutamate transporters.

Dopamine transporter oligomerization involves the scaffold domain, but spares the bundle domain.

The human dopamine transporter (hDAT) is located on presynaptic neurons, where it plays an essential role in limiting dopaminergic signaling by temporarily curtailing high neurotransmitter concentration through rapid re-uptake. Transport by hDAT is energized by transmembrane ionic gradients. Dysfunction of this transporter leads to disease states, such as Parkinson's disease, bipolar disorder or depression. It has been shown that hDAT and other members of the monoamine transporter family exist in oligomeric forms at the plasma membrane. Several residues are known to be involved in oligomerization, but interaction interfaces, oligomer orientation and the quarternary arrangement in the plasma membrane remain poorly understood. Here we examine oligomeric forms of hDAT using a direct approach, by following dimerization of two randomly-oriented hDAT transporters in 512 independent simulations, each being 2 µs in length. We employed the DAFT (docking assay for transmembrane components) approach, which is an unbiased molecular dynamics simulation method to identify oligomers, their conformations and populations. The overall ensemble of a total of >1 ms simulation time revealed a limited number of symmetric and asymmetric dimers. The identified dimer interfaces include all residues known to be involved in dimerization. Importantly, we find that the surface of the bundle domain is largely excluded from engaging in dimeric interfaces. Such an interaction would typically lead to inhibition by stabilization of one conformation, while substrate transport relies on a large scale rotation between the inward-facing and the outward-facing state.