Longpré JP et al. (2012), Simulated annealing reveals the kinetic activit...

XB-ART-46545

J Gen Physiol 2012 Oct 01;1404:361-74. doi: 10.1085/jgp.201210822.

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Simulated annealing reveals the kinetic activity of SGLT1, a member of the LeuT structural family.

Longpré JP , Sasseville LJ , Lapointe JY .

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The Na(+)/glucose cotransporter (SGLT1) is the archetype of membrane proteins that use the electrochemical Na(+) gradient to drive uphill transport of a substrate. The crystal structure recently obtained for vSGLT strongly suggests that SGLT1 adopts the inverted repeat fold of the LeuT structural family for which several crystal structures are now available. What is largely missing is an accurate view of the rates at which SGLT1 transits between its different conformational states. In the present study, we used simulated annealing to analyze a large set of steady-state and pre-steady-state currents measured for human SGLT1 at different membrane potentials, and in the presence of different Na(+) and α-methyl-d-glucose (αMG) concentrations. The simplest kinetic model that could accurately reproduce the time course of the measured currents (down to the 2 ms time range) is a seven-state model (C(1) to C(7)) where the binding of the two Na(+) ions (C(4)→C(5)) is highly cooperative. In the forward direction (Na(+)/glucose influx), the model is characterized by two slow, electroneutral conformational changes (59 and 100 s(-1)) which represent reorientation of the free and of the fully loaded carrier between inside-facing and outside-facing conformations. From the inward-facing (C(1)) to the outward-facing Na-bound configuration (C(5)), 1.3 negative elementary charges are moved outward. Although extracellular glucose binding (C(5)→C(6)) is electroneutral, the next step (C(6)→C(7)) carries 0.7 positive charges inside the cell. Alignment of the seven-state model with a generalized model suggested by the structural data of the LeuT fold family suggests that electrogenic steps are associated with the movement of the so-called thin gates on each side of the substrate binding site. To our knowledge, this is the first model that can quantitatively describe the behavior of SGLT1 down to the 2 ms time domain. The model is highly symmetrical and in good agreement with the structural information obtained from the LeuT structural family.

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Species referenced: Xenopus
Genes referenced: slc5a1.2 tbx2

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	Figure 1. Cotransport mechanism of SGLT1 suggested by the structural data of the LeuT fold family. In state S1, the cotransporter is in its inward open conformation. The transition from S1 to S2 closes the intracellular thin gate (orange stick), thus occluding the binding sites. A major conformational change (S2→S3) then reorients SGLT1 to its outward facing state. The subsequent opening of the extracellular thin gate (S3→S4) makes it possible for SGLT1 to bind Na+ ions (green circles) and glucose (yellow hexagon; S4→S5). The extracellular thin gate is then free to close (S5→S6), which provokes a structural rearrangement of the fully loaded transporter leading to the intracellular facing occluded state S7. This is followed by the opening of the intracellular thin gate (S7→S8), allowing intracellular release of the substrates (S8→S1).
	Figure 2. The Metropolis algorithm and the principal parameters characterizing a SA run. (A) A random modification of one of the parameters is made and the resulting fitting error is compared with the error obtained with the original parameter set. If the new error is smaller than the previous one, the new parameter value is accepted. In contrast, if the new error is larger, it is not necessarily rejected. Instead, a temperature-dependent transition probability is calculated, and if its value is greater than a random number between 0 and 1, the change is accepted. Then, the loop starts again and a new parameter to be modified is randomly chosen. (B) Dynamic adjustment of the maximal amplitude of random changes applied to the kinetic parameters of the model. The amplitude decreases when the system is cooled down to keep an acceptance ratio of ∼50%. (C) Evolution of the fitting error throughout a simulation.
	Figure 3. Complete seven-state model used to simulate SGLT1 transient and steady-state currents. Intracellular binding of Na+ and αMG have been lumped into a single transition (k17). The scheme includes a five-state model (gray box) which is the simplest structure leading to an accurate fit of the transient currents in the absence of αMG. The different states suggested by the LeuT structural family are labeled S1 to S8 and are presented above and below the kinetic model.
	Figure 4. Simulation of the pre–steady-state ON and OFF currents (solid lines) using the five-state model. The simulation is generated in the presence of (A) 90 mM, (B) 50 mM, and (C) 10 mM external Na+ concentrations ([Na+]o). Symbols represent the mean experimental data ± SEM (inset) The Q(Vm) curves corresponding to the integrals of the simulated and experimental OFF transient currents are compared at 90, 50, and 10 mM [Na+]o.
	Figure 5. The simulated steady-state and pre–steady-state ON and OFF currents in the presence of 90 mM [Na+]o and αMG. Simulated currents obtained with (A) 5 mM or (B) 0.1 mM [αMG]o are compared with experimental data. (Inset) Q(Vm) curves obtained from integration of the OFF transient currents in the presence of 90 mM [Na+]o and 5 mM or 0.1 mM [αMG]o.
	Figure 6. Comparison of αMG and Na+ affinity constants predicted by the seven-state model with experimental data from a published study. (A) KmαMG, in the presence of 100 mM [Na+]o, and (B) KmNa, in the presence of 25 mM [αMG]o, are calculated as a function of membrane potential using the seven-state model and the parameters given in Table 1 (filled squares without error bars). These values are compared with the data published by Wright et al. (2011) for a typical hSGLT1-expressing oocyte (filled circle with error bars representing the fitting uncertainty).
	Figure 7. The conformational time course of SGLT1 in different conditions. The time courses were simulated for 10 s (events occurring during the first 0.5 s are depicted) in the presence of 90 mM [Na+]o and (A) 0.1 mM [αMG]o at −50 mV, (B) 5 mM [αMG]o at −50 mV, and (C) 5 mM [αMG]o at −150 mV. Short arrows above each panel indicate the completion of a cotransport cycle and conformational change from C7 to C1. The occupational probabilities of the seven states during the 10 s period are shown on the right side of each panel. In addition, conformations suggested by the structural data of the LeuT fold family have been assigned to states C1 through C7 to illustrate the behavior of SGLT1. The so-called thin gates are represented by orange sticks, and the green circles and yellow hexagons represent Na+ ions and glucose molecules.

References [+] :

Bissonnette, Functional expression of tagged human Na+-glucose cotransporter in Xenopus laevis oocytes. 1999, Pubmed, Xenbase