FIGURE 9 - available via license: Creative Commons Attribution 4.0 International
Content may be subject to copyright.
| Vesicles containing SGLT1. Model of a unilamellar vesicle with SGLT1 incorporated in two different orientations. The inward-and outward facing configurations oriented towards the intra and extra luminal side are denoted as IN and OUT, respectively. In each transport cycle SGLT1 transports 2 Na + ions and one glucose molecule. The model takes into account passive permeation of osmotically active solutes (OS) (i.e., sucrose, glucose, and NMDG 0 ) and charged particles (CP) (i.e., Na + , K + , NMDG + , Cl − , and protons) as well as passive flux of water.
Source publication
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...
Context in source publication
Context 1
... transition rates used in the model were adapted from Table S3 in Adelman et al. (2016) and they are listed in Table 3. When we incorporated the kinetic model of SGLT1 into the vesicle we also accounted for: 1) transporter orientation (IN and OUT) after insertion into the vesicular membrane (see Figure 9) and 2) for passive water flux through the transporter. We implemented the two orientations of the transporter by switching in the equations, which constitute the kinetic model the concentration terms (i.e., [Na] OUT and [glucose] OUT became [Na] IN and [glucose] IN and vice versa) (Parent et al., 1992). ...
Citations
... Once the relation of the intensity and pH change is established, governing equations for solute transport or simplified versions can be used to fit the P s values similar to the procedure followed for calculating the permeability of water. The details of the procedure, including a complete mathematical description of the model system [211], and various important considerations concerning proton, weak acid/base, and ion permeabilities are available in SI Section 3. It is important to note however that the number of studies that used a comprehensive stopped flow-based analysis for quantifying solute permeabilities is low and the accuracy and sensitivity of some of these approaches are still under question. ...
... This approach was used to estimate passive Cl − permeability through peptide-appended hybrid [4] arene channels [13] and carbon nanotubes [212]. However, the accuracy of this approach under a wide range of experimental conditions needs to be established in comparison with a detailed modeling approach [211] described above for pH-sensitive dyes. ...
Understanding the determinants of permeability and selectivity in biological channels serves two general purposes. It provides fundamental biophysical insights and allows the evaluation of new membrane proteins and artificial channel designs for the development of biomimetic separation membranes. This understanding relies on accurate ways to quantify unitary (single channel) solute and solvent permeabilities. However, the current research in biomimetic and bioinspired membranes and in protein biophysics tends to focus on relative permeabilities. Further, many methods and approximations currently used result in erroneous permeability values which hinder and complicate the comparison between channels. Combined, these gaps in the available measurements obscure the biophysical insights and impede the development of novel high-performance channels. In this review, we summarize in vitro model membrane systems useful to functionally characterize artificial as well as biological channels. We also critically discuss biophysical techniques capable of extracting permeability values and membrane channel densities, which are fundamental parameters required to determine accurate unitary permeability values. Pertinent examples are provided, along with advantages, disadvantages, and potential improvements needed for specific techniques to acquire accurate unitary permeability values for a diverse set of small molecules, including water, ions, weak acids and bases, neutral molecules, and protons.
Sugar absorption is crucial for life and relies on glucose transporters, including sodium–glucose cotransporters (SGLTs). Although the structure of SGLTs has been resolved, the substrate selectivity of SGLTs across diverse isoforms has not been determined owing to the complex substrate-recognition processes and limited analysis methods. Therefore, this study used voltage-clamp fluorometry (VCF) to explore the substrate-binding affinities of human SGLT1 in Xenopus oocytes. VCF analysis revealed high-affinity binding of D-glucose and D-galactose, which are known transported substrates. D-fructose, which is not a transported substrate, also bound to SGLT1, suggesting potential recognition despite the lack of transport activity. VCF analysis using the T287N mutant of the substrate-binding pocket, which has reduced D-glucose transport capacity, showed that its D-galactose-binding affinity exceeded its D-glucose-binding affinity. This suggests that the change in the VCF signal was due to substrate binding to the binding pocket. Both D-fructose and L-sorbose showed similar binding affinities, indicating that SGLT1 preferentially binds to pyranose-form sugars, including D-fructopyranose. Electrophysiological analysis confirmed that D-fructose binding did not affect the SGLT1 transport function. The significance of the VCF assay lies in its ability to measure sugar–protein interactions in living cells, thereby bridging the gap between structural analyses and functional characterizations of sugar transporters. Our findings also provide insights into SGLT substrate selectivity and the potential for developing medicines with reduced side effects by targeting non-glucose sugars with low bioreactivity.
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 NH 3 /NH 4 ⁺ 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 NH 3 , NH 4 ⁺ , 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.
