Correlating Charge Movements with Local Conformational Changes of a Na+-Coupled Cotransporter

Functionally important sites in the predicted first and fourth extracellular linkers of the type IIa Na+/Pi cotransporter (NaPi-IIa) were identified by cysteine scanning mutagenesis (Ehnes et al., 2004). Cysteine substitution or modification with impermeant and permeant methanethiosulfonate (MTS) reagents at certain sites resulted in changes to the steady-state voltage dependency of the cotransport mode (1 mM Pi, 100 mM Na+ at pH 7.4) of the mutants. At Gly-134 (ECL-1) and Met-533 (ECL-4), complementary behavior of the voltage dependency was documented with respect to the effect of cys-substitution and modification. G134C had a weak voltage dependency that became even stronger than that of the wild type (WT) after MTS incubation. M533C showed a WT-like voltage dependency that became markedly weaker after MTS incubation. To elucidate the underlying mechanism, the steady-state and presteady-state kinetics of these mutants were studied in detail. The apparent affinity constants for Pi and Na+ did not show large changes after MTS exposure. However, the dependency on external protons was changed in a complementary manner for each mutant. This suggested that cys substitution at Gly-134 or modification of Cys-533 had induced similar conformational changes to alter the proton modulation of transport kinetics. The changes in steady-state voltage dependency correlated with changes in the kinetics of presteady-state charge movements determined in the absence of Pi, which suggested that voltage-dependent transitions in the transport cycle were altered. The steady-state and presteady-state behavior was simulated using an eight-state kinetic model in which the transition rate constants of the empty carrier and translocation of the fully loaded carrier were found to be critical determinants of the transport kinetics. The simulations predict that cys substitution at Gly-134 or cys modification of Cys-533 alters the preferred orientation of the empty carrier from an inward to outward-facing conformation for hyperpolarizing voltages.

Download Full-text

Voltage Clamp Fluorometric Measurements on a Type II Na+-coupled Pi Cotransporter: Shedding Light on Substrate Binding Order

The Journal of General Physiology ◽

10.1085/jgp.200609496 ◽

2006 ◽

Vol 127 (5) ◽

pp. 539-555 ◽

Cited By ~ 39

Author(s):

Leila V. Virkki ◽

Heini Murer ◽

Ian C. Forster

Keyword(s):

Voltage Clamp ◽

Conformational Changes ◽

Substrate Binding ◽

Type Ii ◽

New Model ◽

Fluorescence Measurements ◽

Voltage Dependent ◽

Electrophysiological Studies ◽

Ion Substitution ◽

Charge Movements

Voltage clamp fluorometry (VCF) combines conventional two-electrode voltage clamp with fluorescence measurements to detect protein conformational changes, as sensed by a fluorophore covalently attached to the protein. We have applied VCF to a type IIb Na+-coupled phosphate cotransporter (NaPi-IIb), in which a novel cysteine was introduced in the putative third extracellular loop and expressed in Xenopus oocytes. Labeling this cysteine (S448C) with methanethiosulfonate (MTS) reagents blocked cotransport function, however previous electrophysiological studies (Lambert G., I.C. Forster, G. Stange, J. Biber, and H. Murer. 1999. J. Gen. Physiol. 114:637–651) suggest that substrate interactions with the protein can still occur, thus permitting study of a limited subset of states. After labeling S448C with the fluorophore tetramethylrhodamine MTS, we detected voltage- and substrate-dependent changes in fluorescence (ΔF), which suggested that this site lies in an environment that is affected by conformational change in the protein. ΔF was substrate dependent (no ΔF was detectable in 0 mM Na+) and showed little correlation with presteady-state charge movements, indicating that the two signals provide insight into different underlying physical processes. Interpretation of ion substitution experiments indicated that the substrate binding order differs from our previous model (Forster, I., N. Hernando, J. Biber, and H. Murer. 1998. J. Gen. Physiol. 112:1–18). In the new model, two (rather than one) Na+ ions precede Pi binding, and only the second Na+ binding transition is voltage dependent. Moreover, we show that Li+, which does not drive cotransport, interacts with the first Na+ binding transition. The results were incorporated in a new model of the transport cycle of type II Na+/Pi cotransporters, the validity of which is supported by simulations that successfully predict the voltage and substrate dependency of the experimentally determined fluorescence changes.

Download Full-text

Active site voltage clamp fluorometry of the sodium glucose cotransporter hSGLT1

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1713899114 ◽

2017 ◽

Vol 114 (46) ◽

pp. E9980-E9988 ◽

Cited By ~ 5

Author(s):

Edurne Gorraitz ◽

Bruce A. Hirayama ◽

Aviv Paz ◽

Ernest M. Wright ◽

Donald D. F. Loo

Keyword(s):

Binding Site ◽

Conformational Changes ◽

Side Chains ◽

Mutant Proteins ◽

Sugar Binding ◽

Internal Surfaces ◽

Sodium Glucose Cotransporter ◽

Time Courses ◽

Covalently Linked ◽

Charge Movements

In the human sodium glucose cotransporter (hSGLT1) cycle, the protein undergoes conformational changes where the sugar-binding site alternatively faces the external and internal surfaces. Functional site-directed fluorometry was used to probe the conformational changes at the sugar-binding site. Residues (Y290, T287, H83, and N78) were mutated to cysteines. The mutants were expressed in Xenopus laevis oocytes and tagged with environmentally sensitive fluorescent rhodamines [e.g., tetramethylrhodamine (TMR)-thiols]. The fluorescence intensity was recorded as the mutants were driven into different conformations using voltage jumps. Sugar binding and transport by the fluorophore-tagged mutants were blocked, but Na+ binding and the voltage-dependent conformational transitions were unaffected. Structural models indicated that external Na+ binding opened a large aqueous vestibule (600 Å3) leading to the sugar-binding site. The fluorescence of TMR covalently linked to Y290C, T287C, and H83C decreased as the mutant proteins were driven from the inward to the outward open Na+-bound conformation. The time courses of fluorescence changes (milliseconds) were close to the SGLT1 capacitive charge movements. The quench in rhodamine fluorescence indicated that the environment of the chromophores became more polar with opening of the external gates as the protein transitioned from the inward to outward facing state. Structural analyses showed an increase in polar side chains and a decrease in hydrophobic side chains lining the vestibule, and this was reflected in solvation of the chromophore. The results demonstrate the opening and closing of external gates in real time, with the accompanying changes of polarity of the sugar vestibule.

Download Full-text

Correlating Charge Movements with Local Conformational Changes of a Na-Coupled Cotransporter

Biophysical Journal ◽

10.1016/j.bpj.2013.11.1336 ◽

2014 ◽

Vol 106 (2) ◽

pp. 228a

Author(s):

Monica Patti ◽

Ian C. Forster

Keyword(s):

Conformational Changes ◽

Charge Movements

Download Full-text

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

The Journal of General Physiology ◽

10.1085/jgp.114.3.445 ◽

1999 ◽

Vol 114 (3) ◽

pp. 445-458 ◽

Cited By ~ 54

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.

Download Full-text

Influence of Calcium Ions on the Plant Cell Surface: Membrane Fusions and Conformational Changes

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100050780 ◽

1974 ◽

Vol 32 ◽

pp. 154-155

Author(s):

D. James Morré ◽

Charles E. Bracker ◽

William J. VanDerWoude

Keyword(s):

Cell Wall ◽

Cell Surface ◽

Conformational Changes ◽

Calcium Ions ◽

Surface Membrane ◽

Carboxyl Groups ◽

Cross Linking ◽

Ultrastructural Evidence ◽

Glycine Max L ◽

Wall Extensibility

Calcium ions in the concentration range 5-100 mM inhibit auxin-induced cell elongation and wall extensibility of plant stems. Inhibition of wall extensibility requires that the tissue be living; growth inhibition cannot be explained on the basis of cross-linking of carboxyl groups of cell wall uronides by calcium ions. In this study, ultrastructural evidence was sought for an interaction of calcium ions with some component other than the wall at the cell surface of soybean (Glycine max (L.) Merr.) hypocotyls.

Download Full-text

Role of spectrin in membrane fusion and in shape change of human erythrocyte ghost

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100082509 ◽

1978 ◽

Vol 36 (2) ◽

pp. 328-329

Author(s):

Hideo Hayashi ◽

Yoshikazu Hirai ◽

John T. Penniston

Keyword(s):

Conformational Changes ◽

Human Erythrocyte ◽

Shape Change ◽

Membrane Surface ◽

Slight Modification ◽

Active Role ◽

Main Role ◽

Bank Blood ◽

Human Erythrocyte Ghost

Spectrin is a membrane associated protein most of which properties have been tentatively elucidated. A main role of the protein has been assumed to give a supporting structure to inside of the membrane. As reported previously, however, the isolated spectrin molecule underwent self assemble to form such as fibrous, meshwork, dispersed or aggregated arrangements depending upon the buffer suspended and was suggested to play an active role in the membrane conformational changes. In this study, the role of spectrin and actin was examined in terms of the molecular arrangements on the erythrocyte membrane surface with correlation to the functional states of the ghosts.Human erythrocyte ghosts were prepared from either freshly drawn or stocked bank blood by the method of Dodge et al with a slight modification as described before. Anti-spectrin antibody was raised against rabbit by injection of purified spectrin and partially purified.

Download Full-text

Pinocytotic-Like Forms of Mitochondria From Rat Anterior Pituitary

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100100470 ◽

1968 ◽

Vol 26 ◽

pp. 146-147

Author(s):

Burton B. Silver

Keyword(s):

Electron Micrographs ◽

Conformational Changes ◽

Anterior Pituitary ◽

Dynamic State ◽

Surrounding Cell ◽

The Matrix ◽

The Moment ◽

Cell Medium ◽

Sectioned Tissue ◽

State Of Activity

Sectioned tissue rarely indicates evidence of what is probably a highly dynamic state of activity in mitochondria which have been reported to undergo a variety of movements such as streaming, divisions and coalescence. Recently, mitochondria from the rat anterior pituitary have been fixed in a variety of configurations which suggest that conformational changes were occurring at the moment of fixation. Pinocytotic-like vacuoles which may be taking in or expelling materials from the surrounding cell medium, appear to be forming in some of the mitochondria. In some cases, pores extend into the matrix of the mitochondria. In other forms, the remains of what seems to be pinched off vacuoles are evident in the mitochondrial interior. Dense materials, resembling secretory droplets, appear at the junction of the pores and the cytoplasm. The droplets are similar to the secretory materials commonly identified in electron micrographs of the anterior pituitary.

Download Full-text

Electron Microscopy and image reconstruction reveal the structural basis for spectrin's elastic properties

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100122952 ◽

1992 ◽

Vol 50 (1) ◽

pp. 510-511

Author(s):

Amy M. McGough ◽

Robert Josephs

Keyword(s):

Electron Microscopy ◽

Elastic Properties ◽

Conformational Changes ◽

Quaternary Structure ◽

Three Dimensional ◽

Membrane Skeleton ◽

Projection Algorithm ◽

Structural Basis ◽

Iterative Approximation ◽

Membrane Skeletons

The remarkable deformability of the erythrocyte derives in large part from the elastic properties of spectrin, the major component of the membrane skeleton. It is generally accepted that spectrin's elasticity arises from marked conformational changes which include variations in its overall length (1). In this work the structure of spectrin in partially expanded membrane skeletons was studied by electron microscopy to determine the molecular basis for spectrin's elastic properties. Spectrin molecules were analysed with respect to three features: length, conformation, and quaternary structure. The results of these studies lead to a model of how spectrin mediates the elastic deformation of the erythrocyte.Membrane skeletons were isolated from erythrocyte membrane ghosts, negatively stained, and examined by transmission electron microscopy (2). Particle lengths and end-to-end distances were measured from enlarged prints using the computer program MACMEASURE. Spectrin conformation (straightness) was assessed by calculating the particles’ correlation length by iterative approximation (3). Digitised spectrin images were correlation averaged or Fourier filtered to improve their signal-to-noise ratios. Three-dimensional reconstructions were performed using a suite of programs which were based on the filtered back-projection algorithm and executed on a cluster of Microvax 3200 workstations (4).

Download Full-text

Protein-induced conformational changes in 16S rRNA during assembly of the Escherichia Coli 30S ribosomal subunit: A STEM study

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100128833 ◽

1987 ◽

Vol 45 ◽

pp. 910-911

Author(s):

M. Boublik ◽

V. Mandiyan ◽

J.F. Hainfeld ◽

J.S. Wall

Keyword(s):

16S Rrna ◽

Conformational Changes ◽

Ribosomal Proteins ◽

Structural Changes ◽

Ribosomal Subunit ◽

Compact Structure ◽

E Coli ◽

Freeze Dried ◽

16S Rna ◽

30S Subunit

The aim of this study is to understand the mechanism of 16S rRNA folding into the compact structure of the small 30S subunit of E. coli ribosome. The assembly of the 30S E. coli ribosomal subunit is a sequence of specific interactions of 16S rRNA with 21 ribosomal proteins (S1-S21). Using dedicated high resolution STEM we have monitored structural changes induced in 16S rRNA by the proteins S4, S8, S15 and S20 which are involved in the initial steps of 30S subunit assembly. S4 is the first protein to bind directly and stoichiometrically to 16S rRNA. Direct binding also occurs individually between 16S RNA and S8 and S15. However, binding of S20 requires the presence of S4 and S8. The RNA-protein complexes are prepared by the standard reconstitution procedure, dialyzed against 60 mM KCl, 2 mM Mg(OAc)2, 10 mM-Hepes-KOH pH 7.5 (Buffer A), freeze-dried and observed unstained in dark field at -160°.

Download Full-text