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

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

doi:10.1016/j.bios.2021.113763

Abstract

Here, we present a solid-supported membrane (SSM)-based electrophysiological approach to study sugar binding and Na+/glucose cotransport by SGLT1 in membrane vesicles. SSM-based electrophysiology delivers a cumulative real-time current readout from numerous SGLT1 proteins simultaneously using a gold-coated sensor chip.In contrast to conventional techniques, which mainly operate with voltage steps, currents are triggered by sugar or sodium addition. Sugar concentration jumps in the presence of sodium lead to transport currents between 5 and 10 nA. Remarkably, in the absence of sodium (i.e. no transport), we observed fast pre-steady-state (PSS) currents with time constants between 3 and 10 ms. These PSS currents mainly originate from sugar binding. Sodium binding does not induce PSS currents. Due to high time resolution, PSS currents were distinguished from transport and eventually correlated with conformational transitions within the sugar translocation pathway.In addition, we analyzed the impact of driving forces on transport and binding currents, showing that membrane voltage and sodium concentration gradients lead to an increased transport rate without affecting sugar binding kinetics. We also compared Na+/sugar efflux with physiologically relevant influx and found similar transport rates, but lower affinity in efflux mode.SSM-based electrophysiology is a powerful technique, which overcomes bottlenecks for transport measurements observed in other techniques such as the requirement of labels or the lack of real-time data. Rapid solution exchange enables the observation of substrate-induced electrogenic events like conformational transitions, opening novel perspectives for in-depth functional studies of SGLT1 and other transporters.

Highlights

Diabetes is an expanding 21st century affliction of public health worldwide
To have a reliable and robust sample available on demand, SGLT1expressing cells were used for preparation of membrane vesicles
Conventional approaches to study SGLT1 such as Two-Electrode voltage-clamp (TEVC) apply voltage steps and produce electrogenic signals, which have been assigned to the empty carrier translocation and Na+ binding/dissociation (Fig. 1A, steps 5–8) (Loo et al 2005, 2006)

Summary

Introduction

Diabetes is an expanding 21st century affliction of public health worldwide. Pharmacological targets aimed at treating this disease are of increasing interest among the scientific community. A commonly used kinetic scheme for SGLT1-mediated transport is the 6-state alternating access model postulated by Parent et al (Parent et al, 1992b) and further developed into an 8-state model by Loo et al, (2006) Both models assume that the accessibility of the substrate binding sites changes from inward facing to outward facing by a major conforma tional transition within the transporter, known as alternating access (Jardetzky, 1966). Sugar binding (CoutNa2S) generates an occluded trans porter intermediate (CoccNa2S), followed by the conformational transi tion to the inward facing, fully loaded carrier (CinNa2S). Assuming saturating substrate concentrations and a negative membrane potential, the whole cycle completes in about 36 ms, resulting in a turnover rate of 28 sugar molecules per second (Loo et al, 2005)

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Discussion

Conclusion