Functional characterization of SGLT1 using SSM-based electrophysiology: Kinetics of sugar binding and translocation

Abstract

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.

Transport of metformin metabolites by guanidinium exporters of the small multidrug resistance family

“…Assuming that currents reflect steady-state transport and not pre-steady-state binding events (as confirmed for Gdx-Clo/Gdm + in Fig. 2 F ), the current amplitudes reflect the initial rate of transport ( Bazzone et al, 2017 , 2022 , 2023 ), and their concentration dependence follows Michaelis–Menten kinetics ( Fig. 3 and Table 1 ).…”

A Comparative Study on the Lysosomal Cation Channel TMEM175 Using Automated Whole-Cell Patch-Clamp, Lysosomal Patch-Clamp, and Solid Supported Membrane-Based Electrophysiology: Functional Characterization and High-Throughput Screening Assay Development

“…The decay time τ 2 is (10.7 ± 1.1) ms and significantly faster compared to the typical decay times recorded for transporters that are usually in the range of 30–100 ms, likely due to the high translocation rate of the channel. The current rise and decay are monophasic, indicating the absence of any pre-steady-state currents [ 40 ]. However, the fast current decay may be limited by the time resolution of the solution exchange, making it difficult to distinguish ion translocation through the channel and potential pre-steady-state events.…”

“…Related to data interpretation, it is important to note that pre-steady-state currents have different origins in APC and SSME recordings. In SSME assays, pre-steady-state currents resulting from ion/substrate binding to the target protein may affect the peak currents, leading to shifted EC 50 values, as shown recently for SGLT1 [ 40 ]. Here, peak currents reveal kinetic information about ion binding, enabling the conclusion of K D values.…”

Structural and mechanistic insights into human choline and ethanolamine transport

“…However, only for FLVCR2, a second current phase was observed, revealing a much slower decay Mme constant. This current phase is typical for transport events that slowly charge the membrane capacitor in SSME experiments 23,24 . Despite the structural similariMes described above, FLVCRs seem to have different kineMc fingerprints that might either originate from subtle structural differences idenMfied at the termini of the two transporters, or specific requirements to their surrounding environment.…”

“…For both dose response curves and pH measurements, the error bars represent the s.d. As the peak current signal can be a combined effect of electrogenic PSS and transport events 24 , we use the EC50 parameter to describe the concentration that generates the half maximum response. Analysis of the data was performed in OriginPro (OriginLab Corporation).…”