A Solid Supported Membrane-Based Technology for Electrophysical Screening of B0AT1-Modulating Compounds

Gerbeth-Kreul, Carolin; Pommereau, Antje; Ruf, Sven; Kane, John L.; Kuntzweiler, Theresa A.; Heßler, Gerhard; Engel, C.K.; Shum, Patrick; Wei, Lin‐Li; Czech, Jörg; Licher, Thomas

doi:10.1177/24725552211011180

Cited by 8 publications

(17 citation statements)

References 41 publications

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“…Therefore, the impact of PSS currents on the total charge is negligible. The current integral represents an alternative read-out in SSME due to different advantages over the peak currents ( Pintschovius et al, 1999 ; Bazzone et al, 2017b ; Gerbeth-Kreul et al, 2021 ; Thomas et al, 2021 ). When plotting the concentration dependence of the translocated charge, we obtain a K M of 1.62 ± 0.13 mM for SGLT1 mediated D-glucose transport ( Figure 1E ).…”

Section: Resultsmentioning

confidence: 99%

Functional characterization of SGLT1 using SSM-based electrophysiology: Kinetics of sugar binding and translocation

et al. 2023

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Beside the ongoing efforts to determine structural information, detailed functional studies on transporters are essential to entirely understand the underlying transport mechanisms. We recently found that solid supported membrane-based electrophysiology (SSME) enables the measurement of both sugar binding and transport in the Na+/sugar cotransporter SGLT1 (Bazzone et al, 2022a). Here, we continued with a detailed kinetic characterization of SGLT1 using SSME, determining KM and KDapp for different sugars, kobs values for sugar-induced conformational transitions and the effects of Na+, Li+, H+ and Cl− on sugar binding and transport. We found that the sugar-induced pre-steady-state (PSS) charge translocation varies with the bound ion (Na+, Li+, H+ or Cl−), but not with the sugar species, indicating that the conformational state upon sugar binding depends on the ion. Rate constants for the sugar-induced conformational transitions upon binding to the Na+-bound carrier range from 208 s−1 for D-glucose to 95 s−1 for 3-OMG. In the absence of Na+, rate constants are decreased, but all sugars bind to the empty carrier. From the steady-state transport current, we found a sequence for sugar specificity (Vmax/KM): D-glucose > MDG > D-galactose > 3-OMG > D-xylose. While KM differs 160-fold across tested substrates and plays a major role in substrate specificity, Vmax only varies by a factor of 1.9. Interestingly, D-glucose has the lowest Vmax across all tested substrates, indicating a rate limiting step in the sugar translocation pathway following the fast sugar-induced electrogenic conformational transition. SGLT1 specificity for D-glucose is achieved by optimizing two ratios: the sugar affinity of the empty carrier for D-glucose is similarly low as for all tested sugars (KD,Kapp = 210 mM). Affinity for D-glucose increases 14-fold (KD,Naapp = 15 mM) in the presence of sodium as a result of cooperativity. Apparent affinity for D-glucose during transport increases 8-fold (KM = 1.9 mM) compared to KD,Naapp due to optimized kinetics. In contrast, KM and KDapp values for 3-OMG and D-xylose are of similar magnitude. Based on our findings we propose an 11-state kinetic model, introducing a random binding order and intermediate states corresponding to the electrogenic transitions detected via SSME upon substrate binding.

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Section: Resultsmentioning

confidence: 99%

Functional characterization of SGLT1 using SSM-based electrophysiology: Kinetics of sugar binding and translocation

et al. 2023

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“…Active compounds from primary screens of large compound libraries were validated and false-positive hits were identified. These results clearly demonstrate the suitability of the SSM-based technique as a direct electrophysiological method for rapid and automated identification of small molecules that can inhibit B0AT1 activity (Gerbeth-Kreul et al, 2021).…”

Section: Anticipated Resultsmentioning

confidence: 99%

“…(2021) evaluated the electrogenic amino acid transporter B0AT1 (SLC6A19). Electrophysiological characterization indicated that leucine‐induced currents are B0AT1 dependent and can be inhibited using an established in‐house tool compound (Gerbeth‐Kreul et al., 2021). Active compounds from primary screens of large compound libraries were validated and false‐positive hits were identified.…”

Section: Commentarymentioning

confidence: 99%

“…1 and 2) and allows the determination of parameters such as EC 50 or IC 50 values (Figs. 1 and 2; Dvorak et al., 2021; Gerbeth‐Kreul et al., 2021; Nanion Technologies, 2021). Measurements can be done with purified membranes or vesicles from cells or with proteoliposomes, enabling label‐free electrical measurements (Nanion Technologies, 2021).…”

Section: Commentarymentioning

confidence: 99%

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Solid‐Supported Membrane (SSM)‐Based Electrophysiology Assays Using Surface Electrogenic Event Reader Technology (SURFE²R) in Early Drug Discovery

2023

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This article presents detailed descriptions of procedures and troubleshooting tips for solid-supported membrane (SSM)-based electrophysiology assays (SURFE 2 R) to measure electrogenic solute carrier transporter proteins (SLCs) and assess the effects of compounds that modulate their activity. SURFE 2 R allows the use of the standard 96-well format, making it an ideal platform for tertiary assays in a drug-discovery campaign. The assays are performed with cellline-derived membrane fractions or proteoliposomes containing the transporter of interest. Three main protocols are described for the isolation of membrane fractions from cell culture and the generation of proteoliposomes containing the transporter of interest. Additionally, detailed protocols for SURFE 2 R single concentration and dose-response experiments are included to measure the potencies of test compounds in stimulating or inhibiting transporter function (EC 50 or IC 50 values, respectively) and kinetic functional assays to calculate apparent affinity (k M ) and maximal velocity (V max ) of substrate uptake.

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“…The method is suitable for the detection of any kind of reaction associated with a charge displacement or with the change in water accessibility close to a charge (electrogenic transporters listed in Supplementary Table S1). The SLC families which were measured using this method are for example SLC1, SLC6, SLC8, SLC15 or SLCO (Geibel et al, 2006;Bazzone et al, 2017;Gerbeth-Kreul et al, 2021;Nanion Technologies Munich, 2021). In addition, since SSM-based electrophysiology has been used to assess the function of mitochondrial proteins, this technique may be applied to intracellular SLCs (Watzke et al, 2010).…”

Section: Suitable Solute Carrier Familiesmentioning

confidence: 99%

An Overview of Cell-Based Assay Platforms for the Solute Carrier Family of Transporters

et al. 2021

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The solute carrier (SLC) superfamily represents the biggest family of transporters with important roles in health and disease. Despite being attractive and druggable targets, the majority of SLCs remains understudied. One major hurdle in research on SLCs is the lack of tools, such as cell-based assays to investigate their biological role and for drug discovery. Another challenge is the disperse and anecdotal information on assay strategies that are suitable for SLCs. This review provides a comprehensive overview of state-of-the-art cellular assay technologies for SLC research and discusses relevant SLC characteristics enabling the choice of an optimal assay technology. The Innovative Medicines Initiative consortium RESOLUTE intends to accelerate research on SLCs by providing the scientific community with high-quality reagents, assay technologies and data sets, and to ultimately unlock SLCs for drug discovery.

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A Solid Supported Membrane-Based Technology for Electrophysical Screening of B0AT1-Modulating Compounds

Cited by 8 publications

References 41 publications

Functional characterization of SGLT1 using SSM-based electrophysiology: Kinetics of sugar binding and translocation

Functional characterization of SGLT1 using SSM-based electrophysiology: Kinetics of sugar binding and translocation

Solid‐Supported Membrane (SSM)‐Based Electrophysiology Assays Using Surface Electrogenic Event Reader Technology (SURFE²R) in Early Drug Discovery

An Overview of Cell-Based Assay Platforms for the Solute Carrier Family of Transporters

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