The “www” of Xenopus laevis Oocytes: The Why, When, What of Xenopus laevis Oocytes in Membrane Transporters Research

Bhatt, Manan; Iacovo, Angela Di; Romanazzi, Tiziana; Roseti, Cristina; Cinquetti, Raffaella; Bossi, Elena

doi:10.3390/membranes12100927

Cited by 14 publications

(16 citation statements)

References 148 publications

(171 reference statements)

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“…Fully grown Xenopus laevis oocytes have a highly efficient biosynthetic apparatus that is able to perform all the post-translational modifications needed for correct protein targeting and function. We use the micro-injection technique to gain the heterologous expression of the protein of interest in Xenopus laevis oocytes [25]. BGT-1 is reported to transport betaine and GABA in a concentration-dependent manner, with higher affinity for GABA than betaine [26,27].…”

Section: Resultsmentioning

confidence: 99%

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

Bhatt,

Lazzarin,

Alberto-Silva

et al. 2024

Preprint

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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 <K0.5), it blocks the GABA reuptake. This GAT1 modulation occurs through the temporal inhibition of the transporter, i.e., the prolonged occupancy by betaine impedes the rapid transition of the transporter to the inward conformation. The temporal inhibition results in a crucial regulatory mechanism contributing to the maintenance of GABA homeostasis, preserving neurons from excitotoxicity.

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Section: Resultsmentioning

confidence: 99%

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

Bhatt,

Lazzarin,

Alberto-Silva

et al. 2024

Preprint

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“…Subsequent electrophysiological studies were performed using the two-electrode voltage clamp technique (Oocyte Clamp OC-725; Warner Instruments, Hamden, CT, USA) (Vacca et al . 2022; Bhatt et al . 2022).…”

Section: Methodsmentioning

confidence: 99%

Bioisosteric analogs of MDMA with improved pharmacological profile

Alberto-Silva,

Hemmer,

Bock

et al. 2024

Preprint

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Abstract3,4-Methylenedioxymethamphetamine (MDMA, ‘ecstasy’) is re-emerging in clinical settings as a candidate for the treatment of specific psychiatric 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 vitromethods included radiotracer assays, transporter electrophysiology, bioluminescence resonance energy transfer and fluorescence-based assays, pooled human liver microsome/S9 fraction incubation with isozyme mapping, and liquid chromatography coupled to high-resolution mass spectrometry.In silicomethods included molecular docking. Compared with MDMA, all three MDMA bioisosteres (ODMA, TDMA, and SeDMA) showed similar pharmacological activity at human serotonin and dopamine transporters (hSERT and hDAT, respectively) but decreased activity at 5-HT2A/2B/2Creceptors. Regarding their hepatic metabolism, they differed from MDMA, withN-demethylation being the only metabolic route shared, and without forming phase II metabolites. Additional screening for their interaction with human organic cation transporters (hOCTs) and plasma membrane transporter (hPMAT) revealed a weaker interaction of the MDMA analogs with hOCT1, hOCT2, and hPMAT. Our findings suggest that these new MDMA analogs might constitute appealing therapeutic alternatives to MDMA, sparing the primary pharmacological activity at hSERT and hDAT, but displaying a reduced activity at 5-HT2A/2B/2Creceptors and reduced hepatic metabolism. Whether these MDMA bioisosteres may pose lower risk alternatives to the clinically re-emerging MDMA warrants further studies.

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“…The electrophysiological measurements, apart from the steady-state currents, also let to record an exponentially decaying transient current, known as the pre-steady state current. They represent the intramembrane charge movement seen as a transient current in response to voltage or Na + concentration jumps and can be recorded by voltage clamp in X. laevis oocytes ( Bhatt et al, 2022 ) and in HEK cells ( Bicho and Grewer, 2005 ). These currents are associated with the initially synchronized out-of-equilibrium events such as binding and release of ions, conformational rearrangements, and charge movements across the membrane electric fields ( Wadiche et al, 1995 ; Peres et al, 2004 ).…”

Section: Role Of Ions and Substrates In Transport Functionmentioning

confidence: 99%

A comparative review on the well-studied GAT1 and the understudied BGT-1 in the brain

et al. 2023

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γ-aminobutyric acid (GABA) is the primary inhibitory neurotransmitter in the central nervous system (CNS). Its homeostasis is maintained by neuronal and glial GABA transporters (GATs). The four GATs identified in humans are GAT1 (SLC6A1), GAT2 (SLC6A13), GAT3 (SLC6A11), and betaine/GABA transporter-1 BGT-1 (SLC6A12) which are all members of the solute carrier 6 (SLC6) family of sodium-dependent transporters. While GAT1 has been investigated extensively, the other GABA transporters are less studied and their role in CNS is not clearly defined. Altered GABAergic neurotransmission is involved in different diseases, but the importance of the different transporters remained understudied and limits drug targeting. In this review, the well-studied GABA transporter GAT1 is compared with the less-studied BGT-1 with the aim to leverage the knowledge on GAT1 to shed new light on the open questions concerning BGT-1. The most recent knowledge on transporter structure, functions, expression, and localization is discussed along with their specific role as drug targets for neurological and neurodegenerative disorders. We review and discuss data on the binding sites for Na+, Cl−, substrates, and inhibitors by building on the recent cryo-EM structure of GAT1 to highlight specific molecular determinants of transporter functions. The role of the two proteins in GABA homeostasis is investigated by looking at the transport coupling mechanism, as well as structural and kinetic transport models. Furthermore, we review information on selective inhibitors together with the pharmacophore hypothesis of transporter substrates.

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The “www” of Xenopus laevis Oocytes: The Why, When, What of Xenopus laevis Oocytes in Membrane Transporters Research

Cited by 14 publications

References 148 publications

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

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

Bioisosteric analogs of MDMA with improved pharmacological profile

A comparative review on the well-studied GAT1 and the understudied BGT-1 in the brain

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