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

Abstract

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.

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

“…LUVs are small nano‐compartments enabling the quantification of solute permeabilities across its membrane utilizing encapsulated environmental sensitive dyes. The adaptation of our recently published mathematical model [ 61 ] to the herein presented passive permeability sensors enabled us to predict solute concentrations over time in dependence of the experimental conditions. Comparing the sensitivity of such a passive permeability sensor in the neutral regime, at a pH of 7.0, to the acidic region, at a pH of 4.5, revealed an increased sensitivity to ions in the acidic region ( Figure ).…”

“…With water permeability P f , molar volume of water v w , (Table 1) and the osmotic gradient δ Osm [ 61 ] $$$$\begin{eqnarray} {\delta }_{{\rm{Osm}}} &=& \left( {\mathop \sum \limits_{{\rm{i}} = 0}^{\rm{k}} \left[ {X\left( t \right)} \right]_{\rm{i}}^{{\rm{inside}}} + \mathop \sum \limits_{{\rm{i}} = 0}^{\rm{n}} \left[ {Y\left( {t = 0} \right)} \right]_{\rm{i}}^{{\rm{inside}}} \cdot \frac{{{V}_0}}{{V\left( t \right)}}} \right)\nonumber\\ &&-\ \left( {\mathop \sum \limits_{{\rm{i}}\ = \ 0}^{\rm{m}} \left[ {Z\left( t \right)} \right]_{\rm{i}}^{{\rm{outside}}}} \right) \end{eqnarray}$$$$where k permeable [ X ( t )] inside and n impermeable [ Y ( t = 0)] inside substrate concentrations were inside the vesicle. [ Z ( t )] outside was the sum of all m substrate concentrations outside the vesicle.…”

“…With water permeability P f , molar volume of water v w , (Table 1) and the osmotic gradient 𝛿 Osm [61]…”

“…LUVs are small nano-compartments enabling the quantification of solute permeabilities across its membrane utilizing encapsulated environmental sensitive dyes. The adaptation of our recently published mathematical model [61] to the herein presented passive permeability sensors enabled us to predict solute concentrations over time in dependence of the experimental conditions.…”

Sugar binding of sodium–glucose cotransporters analyzed by voltage-clamp fluorometry

“…To date, substrate transport by SGLT in vivo has been analyzed using knockout mouse experiments ( 14 ), RNA interference techniques ( 15 ), and human pathogenic mutation analyses ( 5 ). In vitro identification of transported substrates has been performed by analyzing the uptake of radioisotope-labeled (RI) substrates ( 16 , 17 ), performing electrophysiological analyses using cotransported sodium as a charge carrier ( 18 , 19 , 20 ), and conducting liposome reconstitution system assays ( 21 ). Advances in structural biology have provided clues to explain the substrate specificity of glucose transporters.…”