SSM-based electrophysiology, a label-free real-time method reveals sugar binding &amp; transport events in SGLT1

Bazzone, Andre; Körner, Alexander; Meincke, Melanie; Bhatt, Manan; Dondapati, Srujan Kumar; Barthmes, Maria; Kubick, Stefan; Fertig, Niels

doi:10.1016/j.bios.2021.113763

Cited by 15 publications

(29 citation statements)

References 33 publications

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“…It has to be noted that similar PSS currents have been detected in the glucose transporter GlcP from S. epidermidis ( 37 ) and the melibiose/Na + transporter MelB ( 48 , 49 ). Recently, we found similar PSS currents in the human d -glucose/Na + transporter SGLT1 ( 50 ), which belongs to the solute sodium symporter family. We propose that the underlying electrogenic process of sugar binding may be conserved across all MFS sugar transporters and potentially even beyond.…”

Section: Discussionmentioning

confidence: 85%

Investigation of sugar binding kinetics of the E. coli sugar/H+ symporter XylE using solid-supported membrane-based electrophysiology

Bazzone

Tesmer

Kurt

et al. 2022

Journal of Biological Chemistry

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Bacterial transporters are difficult to study using conventional electrophysiology because of their low transport rates and the small size of bacterial cells. Here, we applied solid-supported membrane–based electrophysiology to derive kinetic parameters of sugar translocation by the Escherichia coli xylose permease (XylE), including functionally relevant mutants. Many aspects of the fucose permease (FucP) and lactose permease (LacY) have also been investigated, which allow for more comprehensive conclusions regarding the mechanism of sugar translocation by transporters of the major facilitator superfamily. In all three of these symporters, we observed sugar binding and transport in real time to determine K M , V max , K D , and k obs values for different sugar substrates. K D and k obs values were attainable because of a conserved sugar-induced electrogenic conformational transition within these transporters. We also analyzed interactions between the residues in the available X-ray sugar/H + symporter structures obtained with different bound sugars. We found that different sugars induce different conformational states, possibly correlating with different charge displacements in the electrophysiological assay upon sugar binding. Finally, we found that mutations in XylE altered the kinetics of glucose binding and transport, as Q175 and L297 are necessary for uncoupling H + and d -glucose translocation. Based on the rates for the electrogenic conformational transition upon sugar binding (>300 s −1 ) and for sugar translocation (2 s −1 − 30 s −1 for different substrates), we propose a multiple-step mechanism and postulate an energy profile for sugar translocation. We also suggest a mechanism by which d -glucose can act as an inhibitor for XylE.

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Section: Discussionmentioning

confidence: 85%

Investigation of sugar binding kinetics of the E. coli sugar/H+ symporter XylE using solid-supported membrane-based electrophysiology

Bazzone

Tesmer

Kurt

et al. 2022

Journal of Biological Chemistry

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“…For all experiments, purified membrane vesicles from CHO cells overexpressing SGLT1 were used. Membrane purification was performed via ultracentrifugation with a sucrose gradient as described previously ( Bazzone et al, 2022a ). Vesicles of total protein concentrations between 2 and 5 mg/mL were stored in buffer containing 30 mM HEPES, 130 mM NMDG-Cl, 10 mM KCl, 5 mM MgCl 2 at −80°C.…”

Section: Methodsmentioning

confidence: 99%

“…Despite these prominent details about the transport mechanism, sugar binding to SGLT1 has not yet been measured directly. We recently developed a solid supported membrane-based electrophysiology (SSME) assay for SGLT1, enabling the analysis of both, binding and transport properties at the same time ( Bazzone et al, 2022a ). In SSME, transporter activity is triggered by substrate concentration jumps at 0 mV with the possibility to apply co-substrate gradients as an additional driving force.…”

Section: Introductionmentioning

confidence: 99%

“…In SSME all recorded currents are transient and decay to zero, usually within a second, due to the capacitive read-out ( Bazzone et al, 2017a ; Bazzone and Barthmes, 2020 ). Sugar concentration jumps on SGLT1 generate biphasic transient currents comprised of a fast decaying PSS component and a slowly decaying transport component ( Bazzone et al, 2022a ). While the fast PSS component reflects a sugar binding induced electrogenic transition and can be also observed in the absence of Na + , the slowly decaying transport component is only observed when sugar binding is followed by Na + translocation across the membrane.…”

Section: Introductionmentioning

confidence: 99%

“… 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 K M and K D app for different sugars, k obs values for sugar-induced conformational transitions and the effects of Na + , Li + , H + and Cl − on sugar binding and transport.…”

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confidence: 99%

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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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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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SSM-based electrophysiology, a label-free real-time method reveals sugar binding & transport events in SGLT1

Cited by 15 publications

References 33 publications

Investigation of sugar binding kinetics of the E. coli sugar/H+ symporter XylE using solid-supported membrane-based electrophysiology

Investigation of sugar binding kinetics of the E. coli sugar/H+ symporter XylE using solid-supported membrane-based electrophysiology

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

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