scholarly journals The transport mechanism of the human sodium/myo-inositol transporter 2 (SMIT2/SGLT6), a member of the LeuT structural family

2014 ◽  
Vol 307 (5) ◽  
pp. C431-C441 ◽  
Author(s):  
Louis J. Sasseville ◽  
Jean-Philippe Longpré ◽  
Bernadette Wallendorff ◽  
Jean-Yves Lapointe
Keyword(s):  
Steady State ◽  
Conformational Changes ◽  
Voltage Pulse ◽  
Transport Mechanism ◽  
Simulated Annealing Algorithm ◽  
Transient Current ◽  
Xenopus Laevis Oocytes ◽  
Structural Family ◽  
Novel Approach ◽  
Steady State Current

The sodium/ myo-inositol transporter 2 (SMIT2) is a member of the SLC5A gene family, which is believed to share the five-transmembrane segment inverted repeat of the LeuT structural family. The two-electrode voltage-clamp (TEVC) technique was used to measure the steady-state and the pre-steady-state currents mediated by human SMIT2 after expression in Xenopus laevis oocytes. Phlorizin is first shown to be a poor inhibitor of pre-steady-state currents for depolarizing voltage pulse. From an up to threefold difference between the apparent ON and OFF transferred charges during a voltage pulse, we also show that a fraction of the transient current recorded for very negative potentials is not a true pre-steady-state current coming from the cotransporter conformational changes. We suggest that this transient current comes from a time-dependent leak current that can reach large amplitudes when external Na+ concentration is reduced. A kinetic model was generated through a simulated annealing algorithm. This algorithm was used to identify the optimal connectivity among 19 different kinetic models and obtain the numerical values of the associated parameters. The proposed 5-state model includes cooperative binding of Na+ ions, strong apparent asymmetry of the energy barriers, a rate-limiting step that is likely associated with the translocation of the empty transporter, and a turnover rate of 21 s−1. The proposed model is a proof of concept for a novel approach to kinetic modeling of electrogenic transporters and allows insight into the transport mechanism of members of the LeuT structural family at the millisecond timescale.

scholarly journals Transient Current Density in a Pair of Long Parallel Conductors

2021 ◽  
Vol 11 (15) ◽  
pp. 6920
Author(s):  
Oldřich Coufal
Keyword(s):  
Steady State ◽  
Cross Section ◽  
Current Density ◽  
Circular Cross Section ◽  
Transient Current ◽  
Constant Current ◽  
Voltage Source ◽  
Sinusoidal Voltage ◽  
Steady State Current ◽  
Maximum Current

Two infinitely long parallel conductors of arbitrary cross section connected to a voltage source form a loop. If the source voltage depends on time, then due to induction there is no constant current density in the loop conductors. It is only recently that a method has been published for accurately calculating current density in a group of long parallel conductors. The method has thus far been applied to the calculation of steady-state current density in a loop connected to a sinusoidal voltage source. In the present article, the method is used for an accurate calculation of transient current using transient current density. The transient current is analysed when connecting and short-circuiting the sources of sinusoidal, constant and sawtooth voltages. For circular cross section conductors, the dependences of maximum current density, maximum current and the time of achieving steady state on the source frequency, the distance of the conductors and their resistivity when connecting the source of sinusoidal voltage are examined.

scholarly journals Current- and Voltage-Clamped Studies on Myxicola Giant Axons

1969 ◽  
Vol 54 (6) ◽  
pp. 730-740 ◽  
Author(s):  
L. Binstock ◽  
L. Goldman
Keyword(s):  
Steady State ◽  
Membrane Potential ◽  
Action Potential ◽  
Voltage Clamp ◽  
Transient Current ◽  
Resting Membrane Potential ◽  
Action Potential Amplitude ◽  
Giant Axons ◽  
Potential Amplitude ◽  
Steady State Current

A new dissection procedure for preparing Myxicola giant axons for observation under voltage clamp is described. Preparation time is generally 40–45 min. 65–70% of the preparations attempted may be brought through the entire procedure, including insertion of the long internal electrode, and support an initial action potential amplitude of 100 mv or greater. Mean values for axon diameter, resting membrane potential, action potential amplitude, maximum peak inward transient current, and resting membrane resistance are 560 µ, —66.5 mv, 112 mv, 0.87 ma/cm2 and 1.22 KΩ cm 2 respectively. Cut branches do not seem to be a problem in this preparation. Behavior under voltage clamp is reasonably stable over several hours. Reductions in maximum inward transient current of 10% and in steady-state current of 5–10% are expected in the absence of any particular treatment. Tetrodotoxin blocks the action potential and both the inward and outward transient current, but has no effect on either the resting membrane potential or the steady-state current. This selective action of tetrodotoxin on the transient current is taken as an indication that this current component is probably carried by Na.

Substrate interactions in the human type IIa sodium-phosphate cotransporter (NaPi-IIa)

2005 ◽  
Vol 288 (5) ◽  
pp. F969-F981 ◽  
Author(s):  
Leila V. Virkki ◽  
Ian C. Forster ◽  
Jürg Biber ◽  
Heini Murer
Keyword(s):  
Steady State ◽  
Sodium Phosphate ◽  
Complete Removal ◽  
Xenopus Laevis Oocytes ◽  
Charge Movement ◽  
Steady State Current ◽  
Transport Cycle ◽  
Voltage Dependent ◽  
Kinetics Of ◽  
State Charge

We have characterized the kinetics of substrate transport in the renal type IIa human sodium-phosphate cotransporter (NaPi-IIa). The transporter was expressed in Xenopus laevis oocytes, and steady-state and pre-steady-state currents and substrate uptakes were characterized by voltage-clamp and isotope flux. First, by measuring simultaneous uptake of a substrate (32Pi, 22Na) and charge in voltage-clamped oocytes, we established that the human NaPi-IIa isoform operates with a Na:Pi:charge stoichiometry of 3:1:1 and that the preferred transported Pi species is HPO42−. We then probed the complex interrelationship of substrates, pH, and voltage in the NaPi-IIa transport cycle by analyzing both steady-state and pre-steady-state currents. Steady-state current measurements show that the apparent HPO42− affinity is voltage dependent and that this voltage dependency is abrogated by lowering the pH or the Na+ concentration. In contrast, the voltage dependency of the apparent Na+ affinity increased when pH was lowered. Pre-steady-state current analysis shows that Na+ ions bind first and influence the preferred orientation of the transporter in the absence of Pi. Pre-steady-state charge movement was partially suppressed by complete removal of Na+ from the bath, by reducing extracellular pH (both in the presence and absence of Na+), or by adding Pi (in the presence of 100 mM Na). None of these conditions suppressed charge movement completely. The results allowed us to modify previous models for the transport cycle of NaPi-II transporters by including voltage dependency of HPO42− binding and proton modulation of the first Na+ binding step.

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

2012 ◽  
Vol 140 (4) ◽  
pp. 361-374 ◽  
Author(s):  
Jean-Philippe Longpré ◽  
Louis J. Sasseville ◽  
Jean-Yves Lapointe
Keyword(s):  
Simulated Annealing ◽  
Steady State ◽  
Conformational Changes ◽  
Time Course ◽  
Structural Information ◽  
State Model ◽  
Time Range ◽  
Large Set ◽  
Structural Family ◽  
Fold Family

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 (C1 to C7) where the binding of the two Na+ ions (C4→C5) 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 (C1) to the outward-facing Na-bound configuration (C5), 1.3 negative elementary charges are moved outward. Although extracellular glucose binding (C5→C6) is electroneutral, the next step (C6→C7) 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.

scholarly journals Interaction of DDT with the Components of Lobster Nerve Membrane Conductance

1968 ◽  
Vol 51 (2) ◽  
pp. 177-198 ◽  
Author(s):  
Toshio Narahashi ◽  
Hans G. Haas
Keyword(s):  
Steady State ◽  
Action Potentials ◽  
Membrane Conductance ◽  
Transient Current ◽  
Nerve Membrane ◽  
Exponential Functions ◽  
Time To Peak ◽  
Cardiac Action ◽  
Steady State Current ◽  
Single Exponential Function

The falling phase of action potential of lobster giant axons is markedly prolonged by treatment with DDT, and a plateau phase appears as in cardiac action potentials. Repetitive afterdischarge is very often superimposed on the plateau. Voltage-clamp experiments with the axons treated with DDT and with DDT plus tetrodotoxin or saxitoxin have revealed the following: DDT markedly slows the turning-off process of peak transient current and suppresses the steady-state current. The falling phase of the peak transient current in the DDT-poisoned axon is no longer expressed by a single exponential function as in normal axons, but by two or more exponential functions with much longer time constants. The maximum peak transient conductance is not significantly affected by DDT. DDT did not induce a shift of the curve relating the peak transient conductance to membrane potential along the potential axis. The time to peak transient current and the time for the steady-state current to reach its half-maximum are prolonged by DDT to a small extent. The finding that, under the influence of DDT, the steady-state current starts flowing while the peak transient current is partially maintained supports the hypothesis of two operationally separate ion channels in the nerve membrane.

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

2004 ◽  
Vol 125 (1) ◽  
pp. 13-36 ◽  
Author(s):  
Donald D.F. Loo ◽  
Bruce A. Hirayama ◽  
Albert Cha ◽  
Francisco Bezanilla ◽  
Ernest M. Wright
Keyword(s):  
Steady State ◽  
Conformational Changes ◽  
Time Scale ◽  
Environmental Changes ◽  
Xenopus Laevis Oocytes ◽  
Charge Movement ◽  
Similar Time ◽  
Time Constants ◽  
Time To Peak ◽  
A Charge

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.

scholarly journals Gat1 (Gaba:Na+:Cl−) Cotransport Function

1999 ◽  
Vol 114 (3) ◽  
pp. 445-458 ◽  
Author(s):  
Chin-Chih Lu ◽  
Donald W. Hilgemann
Keyword(s):  
Steady State ◽  
Conformational Changes ◽  
Outward Current ◽  
Forward Rate ◽  
Total Charge ◽  
Charge Movement ◽  
Turnover Rates ◽  
High Concentration ◽  
Steady State Current ◽  
Charge Movements

To explain cotransport function, the “alternating access” model requires that conformational changes of the empty transporter allow substrates to bind alternatively on opposite membrane sides. To test this principle for the GAT1 (GABA:Na+:Cl−) cotransporter, we have analyzed how its charge-moving partial reactions depend on substrates on both membrane sides in giant Xenopus oocyte membrane patches. (a) “Slow” charge movements, which require extracellular Na+ and probably reflect occlusion of Na+ by GAT1, were defined in three ways with similar results: by application of the high-affinity GAT1 blocker (NO-711), by application of a high concentration (120 mM) of cytoplasmic Cl−, and by removal of extracellular Na+ via pipette perfusion. (b) Three results indicate that cytoplasmic Cl− and extracellular Na+ bind to the transporter in a mutually exclusive fashion: first, cytoplasmic Cl− (5–140 mM) shifts the voltage dependence of the slow charge movement to more negative potentials, specifically by slowing its “forward” rate (i.e., extracellular Na+ occlusion); second, rapid application of cytoplasmic Cl− induces an outward current transient that requires extracellular Na+, consistent with extracellular Na+ being forced out of its binding site; third, fast charge-moving reactions, which can be monitored as a capacitance, are “immobilized” both by cytoplasmic Cl− binding and by extracellular Na+ occlusion (i.e., by the slow charge movement). (c) In the absence of extracellular Na+, three fast (submillisecond) charge movements have been identified, but no slow components. The addition of cytoplasmic Cl− suppresses two components (τ < 1 ms and 13 μs) and enables a faster component (τ < 1 μs). (d) We failed to identify charge movements of fully loaded GAT1 transporters (i.e., with all substrates on both sides). (e) Under zero-trans conditions, inward (forward) GAT1 current shows pronounced pre–steady state transients, while outward (reverse) GAT1 current does not. (f) Turnover rates for reverse GAT1 transport (33°C), calculated from the ratio of steady state current magnitude to total charge movement magnitude, can exceed 60 s−1 at positive potentials.

Molecular Basis of Glucose Transport Mechanism in Plants

2019 ◽  
Author(s):  
Balaji Selvam ◽  
Ya-Chi Yu ◽  
Liqing Chen ◽  
Diwakar Shukla
Keyword(s):  
Molecular Dynamics ◽  
Free Energy ◽  
Molecular Dynamics Simulations ◽  
Conformational Changes ◽  
Transport Mechanism ◽  
Conformational Dynamics ◽  
Site Directed Mutagenesis ◽  
Free Energy Barrier ◽  
Transport Cycle ◽  
Dynamics Simulations

<p>The SWEET family belongs to a class of transporters in plants that undergoes large conformational changes to facilitate transport of sugar molecules across the cell membrane. However, the structures of their functionally relevant conformational states in the transport cycle have not been reported. In this study, we have characterized the conformational dynamics and complete transport cycle of glucose in OsSWEET2b transporter using extensive molecular dynamics simulations. Using Markov state models, we estimated the free energy barrier associated with different states as well as 1 for the glucose the transport mechanism. SWEETs undergoes structural transition to outward-facing (OF), Occluded (OC) and inward-facing (IF) and strongly support alternate access transport mechanism. The glucose diffuses freely from outside to inside the cell without causing major conformational changes which means that the conformations of glucose unbound and bound snapshots are exactly same for OF, OC and IF states. We identified a network of hydrophobic core residues at the center of the transporter that restricts the glucose entry to the cytoplasmic side and act as an intracellular hydrophobic gate. The mechanistic predictions from molecular dynamics simulations are validated using site-directed mutagenesis experiments. Our simulation also revealed hourglass like intermediate states making the pore radius narrower at the center. This work provides new fundamental insights into how substrate-transporter interactions actively change the free energy landscape of the transport cycle to facilitate enhanced transport activity.</p>

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