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"Gradisch, Ralph"
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Structural basis of organic cation transporter-3 inhibition
Organic cation transporters (OCTs) facilitate the translocation of catecholamines, drugs and xenobiotics across the plasma membrane in various tissues throughout the human body. OCT3 plays a key role in low-affinity, high-capacity uptake of monoamines in most tissues including heart, brain and liver. Its deregulation plays a role in diseases. Despite its importance, the structural basis of OCT3 function and its inhibition has remained enigmatic. Here we describe the cryo-EM structure of human OCT3 at 3.2 Å resolution. Structures of OCT3 bound to two inhibitors, corticosterone and decynium-22, define the ligand binding pocket and reveal common features of major facilitator transporter inhibitors. In addition, we relate the functional characteristics of an extensive collection of previously uncharacterized human genetic variants to structural features, thereby providing a basis for understanding the impact of OCT3 polymorphisms.
The current work reports the structure of the human organic cation transporter 3 (OCT3 / SLC22A3) and provides the structural basis of its inhibition by two specific inhibitors, decynium-22 and corticosterone.
Journal Article
Ligand coupling mechanism of the human serotonin transporter differentiates substrates from inhibitors
The presynaptic serotonin transporter (SERT) clears extracellular serotonin following vesicular release to ensure temporal and spatial regulation of serotonergic signalling and neurotransmitter homeostasis. Prescription drugs used to treat neurobehavioral disorders, including depression, anxiety, and obsessive-compulsive disorder, trap SERT by blocking the transport cycle. In contrast, illicit drugs of abuse like amphetamines reverse SERT directionality, causing serotonin efflux. Both processes result in increased extracellular serotonin levels. By combining molecular dynamics simulations with biochemical experiments and using a homologous series of serotonin analogues, we uncovered the coupling mechanism between the substrate and the transporter, which triggers the uptake of serotonin. Free energy analysis showed that only scaffold-bound substrates could initiate SERT occlusion through attractive long-range electrostatic interactions acting on the bundle domain. The associated spatial requirements define substrate and inhibitor properties, enabling additional possibilities for rational drug design approaches.
The serotonin transporter, targeted by several medications, terminates neurotransmission by clearing serotonin from the synaptic cleft. Combining biochemical results with in silico data, the authors show the key interactions that initiate substrate transport.
Journal Article
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 K
0.5
. 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.
Journal Article
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.
Journal Article
De novo variants in GABRA4 are associated with a neurological phenotype including developmental delay, behavioral abnormalities and epilepsy
Nine out of 19 genes encoding GABA A receptor subunits have been linked to monogenic syndromes characterized by seizures and developmental disorders. Previously, we reported the de novo variant p.(Thr300Ile) in GABRA4 in a patient with epilepsy and neurodevelopmental abnormalities. However, no new cases have been reported since then. Through an international collaboration, we collected molecular and phenotype data of individuals carrying de novo variants in GABRA4 . Patients and their parents were investigated either by exome or genome sequencing, followed by targeted Sanger sequencing in some cases. All variants within the transmembrane domain, including the previously reported p.(Thr300Ile) variant, were characterized in silico and analyzed by molecular dynamics (MD) simulation studies. We identified three novel de novo missense variants in GABRA4 (NM_000809.4): c.797 C > T, p.(Pro266Leu), c.899 C > A, p.(Thr300Asn), and c.634 G > A, p.(Val212Ile). The p.(Thr300Asn) variant impacts the same codon as the previously reported variant p.(Thr300Ile) and likely arose post-zygotically as evidenced by sequencing oral mucosal cells. Overlapping phenotypes among affected individuals included developmental delay (4/4), epileptiform EEG abnormalities (3/4), attention deficits (3/4), seizures (2/4), autistic features (2/4) and structural brain abnormalities (2/4). MD simulations of the three variants within the transmembrane domain of the receptor indicate that sub-microsecond scale dynamics differ between wild-type and mutated subunits. Taken together, our findings further corroborate an association between GABRA4 and a neurological phenotype including variable neurodevelopmental, behavioral and epileptic abnormalities.
Journal Article
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. Using electrophysiology, mass spectroscopy, radiolabelled cellular assay, and molecular dynamics simulation we demonstrate that betaine at mM concentration acts as a slow substrate of GAT1 (slc6a1), the predominant GABA transporter in the central nervous system. Intriguingly, when betaine is present at low concentration (0.01-3 mM) with GABA (at concentration
Paper
Interaction of GAT1 with sodium ions: from efficient recruitment to stabilisation of substrate and conformation
The human GABA transporter (GAT1) is a membrane transporter that mediates the reuptake of the neurotransmitter GABA from the synaptic cleft into neurons and glial cells. Dysregulation of the transport cycle has been associated with epilepsy and neuropsychiatric disorders, highlighting the crucial role of the transporter in maintaining homeostasis of brain GABA levels. GAT1 is a secondary active transporter that couples the movement of substrate to the simultaneous transport of sodium and chloride ions along their electrochemical gradients. Using MD simulations, we identified a novel sodium recruiting site at the entrance to the outer vestibule, which attracts positively charged ions and increases the local sodium concentration, thereby indirectly increasing sodium affinity. Mutations of negatively charged residues at the recruiting site slowed the binding kinetics, while experimental data revealed a change in sodium dependency of GABA uptake and a reduction of sodium affinity. Simulation showed that sodium displays a higher affinity for the sodium binding site NA2, which plays a role in stabilisation of the outward-open conformation. We directly show that the presence of a sodium ion bound to NA2 increases the stability of the closed inner gate and restrains motions of TM5. We find that sodium is only weakly bound to NA1 in the absence of GABA, while the presence of the substrate strengthens the interaction due to the completed ion coordinating shell, explaining cooperativity between GABA and sodium.Competing Interest StatementThe authors have declared no competing interest.Footnotes* We thank our reviewer for their thorough evaluation and their constructive comments, which have helped to improve the manuscript. We have enhanced the description of the sodium recruiting site, created new figures, which clearly show structural details and sodium density. We conducted the suggested experiments, created the single GAT1 mutants D281A and E283A at the sodium recruiting site and measured surface expression, GABA uptake and sodium dependence. The data shows that E283A is wild-type like, while D281A is intermediate between wild-type and the D281A-E283A double mutant for all measured properties. A sequence alignment of all human SLC6 transporters corroborates these experimental data. The conservation of E283 is low, while D281 is an aspartate in all GABA transporters and a glutamate in all monoamine transporters. We now also give standard deviations and/or p-values for all experimental data. The SI now shows traces of distances between the sodium ion and the recruiting site for every trajectory in which sodium binds to GAT1. We have improved the visualisation of most structural figures to enhance clarity. For the analysis of the principal components (PC), we created new panels in figure 6 that visualise the motions described by the first two PCs. We have added a paragraph describing potential limitations of the AlphaFold model. The observed long-range effect of sodium binding to GAT1 and destabilisation of the inner gate has, based on our data, a causal effect. Motions of TM5a were among the 2 largest motions identified by PCA, which suggest to reflect relevant motions. To directly quantify the structural dynamics of the inner gate, we measured informative distances at the inner gate of GAT1, as shown in Figure 5i,j,k and separated data according to the presence of sodium in NA2. For the following reasons we exclude that the results are a consequence of structural inconsistencies introduced by AlphaFold and therefore not reflecting functionally relevant effect: If depending on the model instead of sodium binding, the effect should not be correlated with the presence of sodium in the NA2 binding site. We have observed the same property in SERT, for which we used experimental structures as starting positions (doi: 10.1038/s41467-023-44637-6), suggesting that this could be a generally mechanism. All available structures from the entire SLC6 family are consistent with structural effects of TM5a in response to bundle domain motions and therefore to binding of sodium to NA2 as stabilizing the outward-open state.* https://doi.org/10.5281/zenodo.10686813
Paper
Structural basis of organic cation transporter-3 inhibition
Organic cation transporters (OCTs) facilitate the translocation of catecholamines and xenobiotics across the plasma membrane in various tissues throughout the human body. OCT3 plays a key role in low-affinity, high-capacity uptake of monoamines in most tissues including heart, brain and liver. Its deregulation plays a role in diseases. Despite its importance, the structural basis of OCT3 function and its inhibition has remained enigmatic. Here we describe the cryo-EM structure of human OCT3 at 3.2 A resolution. Structures of OCT3 bound to two inhibitors, corticosterone and decynium-22, define the ligand binding pocket and reveal common features of major facilitator transporter inhibitors. In addition, we relate the functional characteristics of an extensive collection of previously uncharacterized human genetic variants to structural features, thereby providing a basis for understanding the impact of OCT3 polymorphisms. Competing Interest Statement The authors have declared no competing interest.
Paper