Liposome-based measurement of light-driven chloride transport kinetics of halorhodopsin

Feroz, Hasin; Ferlez, Bryan; Oh, Hyeonji; Mohammadiarani, Hossein; Ren, Tingwei; Baker, Carol S.; Gajewski, Joseph J.; Lugar, Daniel J.; Gaudana, Sandeep B.; Butler, Peter J.; Hühn, Jonas; Lamping, Matthias; Parak, Wolfgang J.; Blatt, Michael R.; Kerfeld, Cheryl A.; Smirnoff, Nicholas; Vashisth, Harish; Golbeck, John H.; Kumar, Manish

doi:10.1016/j.bbamem.2021.183637

Cited by 4 publications

(3 citation statements)

References 84 publications

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“…The latter is performed via intra luminal trapped fluorophores as volume readout. The time course and extent of the change in the concentration of the individual ion species and the magnitude of the transmembrane potential are accessible via ion-( Feroz et al, 2021 ) or voltage- ( Cortes et al, 2018 ; Gest et al, 2021 ) sensitive fluorophores. These parameters can be measured for different intra and extraluminal ion concentrations in the presence and absence of SGLT1, with and without substrate.…”

Section: Discussionmentioning

confidence: 99%

Modeling of SGLT1 in Reconstituted Systems Reveals Apparent Ion-Dependencies of Glucose Uptake and Strengthens the Notion of Water-Permeable Apo States

et al. 2022

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The reconstitution of secondary active transporters into liposomes shed light on their molecular transport mechanism. The latter are either symporters, antiporters or exchangers, which use the energy contained in the electrochemical gradient of ions to fuel concentrative uptake of their cognate substrate. In liposomal preparations, these gradients can be set by the experimenter. However, due to passive diffusion of the ions and solutes through the membrane, the gradients are not stable and little is known on the time course by which they dissipate and how the presence of a transporter affects this process. Gradient dissipation can also generate a transmembrane potential (VM). Because it is the effective ion gradient, which together with VM fuels concentrative uptake, knowledge on how these parameters change within the time frame of the conducted experiment is key to understanding experimental outcomes. Here, we addressed this problem by resorting to a modelling approach. To this end, we mathematically modeled the liposome in the assumed presence and absence of the sodium glucose transporter 1 (SGLT1). We show that 1) the model can prevent us from reaching erroneous conclusions on the driving forces of substrate uptake and we 2) demonstrate utility of the model in the assignment of the states of SGLT1, which harbor a water channel.

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Section: Discussionmentioning

confidence: 99%

Modeling of SGLT1 in Reconstituted Systems Reveals Apparent Ion-Dependencies of Glucose Uptake and Strengthens the Notion of Water-Permeable Apo States

et al. 2022

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“…The latter being a prerequisite to characterize protein function in the acidic pH region or to increase the sensitivity for ions, e.g., NH 4 + . As ion permeation creates a membrane potential in LUVs, ion permeabilities can only be assessed by either measuring the initial slope of the measurement signal at t = 0 [ 33 ] or using membrane potential decouplers. However, efficient decoupling via the latter crucially depends on the lipid to decoupler ratio.…”

Section: Introductionmentioning

confidence: 99%

Quantification of Fundamental Weak Base and Ion Permeabilities in the Acidic pH Region Utilizing Conjugated Oregon Green in Liposome‐Based Assays

Barta

Seiser

Hörner

2023

Advanced Sensor Research

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Ammonia and ammonium play an essential role in the metabolism of almost all living organisms. Despite their importance for human health as well as plant growth, knowledge about passive NH3/NH4+ membrane permeabilities is scarce. Large unilamellar vesicles (LUVs), a popular model membrane system, have the potential to be a game changer. However, despite a variety of environmental sensitive dyes awaiting encapsulation into membrane vesicles, a pH sensor for the acidic pH region is currently missing. This gap is filled by introducing conjugated Oregon Green (OG) which can be encapsulated into lipid‐ or polymer‐based vesicles. This allows the quantification of proton or weak base permeabilities across the vesicular membrane and functionally characterizes pH sensitive membrane proteins at an acidic pH as low as pH 4.0. Furthermore, the expanded pH range enables simultaneous estimation of ion permeabilities without the use of membrane potential uncouplers due to an increased ion sensitivity. The utility of the sensor is demonstrated by quantifying passive NH3, NH4+, and Cl− permeabilities through vastly different lipid and polymer membranes. Moreover, its performance is benchmarked against carboxyfluorescein, an established pH‐sensor in the neutral pH‐range.

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“…The first discovered microbial rhodopsins (M-Rho) are typically seven-pass transmembrane helix region bound with retinal to absorb light energy for ion translocation or phototaxis response [ 4 , 5 ]. The photochemical properties of ion-translocating rhodopsins fall into several categories, such as the light-driven outward proton transport bacteriorhodopsin (BR) [ 4 , 6 ], the light-driven inward chloride transport halorhodopsin (HR) [ 7 , 8 ], outward sodium transport (KR2) [ 9 ], and the nonselective cation channel channelrhodopsin II (ChR2) [ 10 , 11 ].…”

Section: Introductionmentioning

confidence: 99%

Engineered Bacteriorhodopsin May Induce Lung Cancer Cell Cycle Arrest and Suppress Their Proliferation and Migration

Wong

Huang

et al. 2021

Molecules

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Highly expressible bacteriorhodopsin (HEBR) is a light-triggered protein (optogenetic protein) that has seven transmembrane regions with retinal bound as their chromophore to sense light. HEBR has controllable photochemical properties and regulates activity on proton pumping. In this study, we generated HEBR protein and incubated with lung cancer cell lines (A549 and H1299) to evaluate if there was a growth-inhibitory effect with or without light illumination. The data revealed that the HEBR protein suppressed cell proliferation and induced the G0/G1 cell cycle arrest without light illumination. Moreover, the migration abilities of A549 and H1299 cells were reduced by ~17% and ~31% after incubation with HEBR (40 μg/mL) for 4 h. The Snail-1 gene expression level of the A549 cells was significantly downregulated by ~50% after the treatment of HEBR. In addition, HEBR significantly inhibited the gene expression of Sox-2 and Oct-4 in H1299 cells. These results suggested that the HEBR protein may inhibit cell proliferation and cell cycle progression of lung cancer cells, reduce their migration activity, and suppress some stemness-related genes. These findings also suggested the potential of HEBR protein to regulate the growth and migration of tumor cells, which may offer the possibility for an anticancer drug.

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Liposome-based measurement of light-driven chloride transport kinetics of halorhodopsin

Cited by 4 publications

References 84 publications

Modeling of SGLT1 in Reconstituted Systems Reveals Apparent Ion-Dependencies of Glucose Uptake and Strengthens the Notion of Water-Permeable Apo States

Modeling of SGLT1 in Reconstituted Systems Reveals Apparent Ion-Dependencies of Glucose Uptake and Strengthens the Notion of Water-Permeable Apo States

Quantification of Fundamental Weak Base and Ion Permeabilities in the Acidic pH Region Utilizing Conjugated Oregon Green in Liposome‐Based Assays

Engineered Bacteriorhodopsin May Induce Lung Cancer Cell Cycle Arrest and Suppress Their Proliferation and Migration

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