Perturbation Analysis of the Voltage-sensitive Conformational Changes of the Na+/Glucose Cotransporter

Abstract

Conformational changes of the human Na+/glucose cotransporter (hSGLT1) were studied using voltage-jump methods. The cotransporter was expressed in Xenopus laevis oocytes, and SGLT1 charge movements were measured in the micro- to millisecond time scale using the cut-open oocyte preparation and in the millisecond to second time scale using the two-electrode voltage clamp method. Simultaneous charge and fluorescence changes were studied using tetramethylrhodamine-6-maleimide-labeled hSGLT1 Q457C. In 100 mM external [Na+], depolarizing voltage steps evoked a charge movement that rose initially to a peak (with time constant τ = 0.17 ms) before decaying to steady state with two time constants (τ = 2–30 and 25–150 ms). The time to peak (0.9 ms) decreased with [Na+], and was not observed in 0 mM [Na+]. In absence of Na+, charge movement decayed monotonically to steady state with three time constants (0.2, 2, and 150 ms). Charge movement was accompanied by fluorescence changes with similar time courses, indicating that global conformational changes monitored by charge movement are reflected by local environmental changes at or near Q457C. Our results indicate that the major voltage-dependent step of the Na+/glucose transport cycle is the return of the empty carrier from inward to outward facing conformations. Finally, we observed subtle differences between time constants for charge movement and for optical changes, suggesting that optical recordings can be used to monitor local conformational changes that underlie the global conformational changes of cotransporters.

Functional identification and characterization of sodium binding sites in Na symporters

“…When membrane voltage was returned to V h , both the bilayer and the hSGLT1 transient currents were in the opposite direction. hSGLT1 pre-steady-state currents were blocked by phlorizin (a SGLT1-specific inhibitor) and glucose (10,22,24). In 10 mM Na + , the currents were smaller and the relaxations were faster.…”

“…*The Na + affinities of the two sites for the WT group are similar, because the three-state model fitted the data, and K d = √K dA K dB . † Loo et al (24). of the interaction between Y290 and glucose (21).…”

“…At each membrane voltage, SGLT1 charge movement (Q) was obtained from the integral of the pre-steady-state currents. The charge vs. voltage (Q-V) relations were fit to a single Boltzmann function (10,24,47),…”

Transmembrane IV of the high-affinity sodium-glucose cotransporter participates in sugar binding

“…SGLT1 pre-steady-state currents yields three time constants ( fast , medium , and slow ) (9,12), and the oocyte capacitive membrane current, which is not relevant to transporter activity, is Ͻ1 ms (2, 9). Loo et al (12) estimated WT hSGLT1 expression by adding the charge transferred associated with the medium and slow decays (Q med and Q slow ). We used the same approach to calculate the charge transferred in nC from the medium and slow decays for K157C and rWT (K157C 3.8 Ϯ 0.7 nC, n ϭ 8 vs. WT 12.8 Ϯ 0.4 nC, n ϭ 3).…”

Forces and Dynamics of Glucose and Inhibitor Binding to Sodium Glucose Co-transporter SGLT1 Studied by Single Molecule Force Spectroscopy

“…Phlorizin acts as a competitive inhibitor of SGLT1 with an apparent K i of 1 M (22). The phlorizin carrier complex represents a dead end conformation of the transporter in which it is locked into a condensed, rigid conformation unable to mediate translocation (23,24). Phlorizin consists of a pyranose ring (sugar residue) and two aromatic rings joined by an alkyl spacer (the aglucon moiety, phloretin) (22).…”