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

2006 ◽  
Vol 127 (5) ◽  
pp. 539-555 ◽  
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.

scholarly journals Structure–Function Relations of the First and Fourth Extracellular Linkers of the Type IIa Na+/Pi Cotransporter

2004 ◽  
Vol 124 (5) ◽  
pp. 489-503 ◽  
Author(s):  
Colin Ehnes ◽  
Ian C. Forster ◽  
Andrea Bacconi ◽  
Katja Kohler ◽  
Jürg Biber ◽  
...  
Keyword(s):  
Steady State ◽  
Conformational Changes ◽  
Underlying Mechanism ◽  
Transport Kinetics ◽  
State Behavior ◽  
Voltage Dependent ◽  
Cysteine Scanning Mutagenesis ◽  
Charge Movements ◽  
Kinetics Of ◽  
State Kinetic

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.

scholarly journals Proton wires mediate the optical signal for ArcLight-type Genetically Encoded Voltage Indicators

2020 ◽  
Author(s):  
B.E Kang ◽  
L. M. Leong ◽  
Y. Kim ◽  
K. Miyazaki ◽  
W. N. Ross ◽  
...  
Keyword(s):  
Voltage Clamp ◽  
Conformational Changes ◽  
Electrostatic Interactions ◽  
Negative Charge ◽  
Random Mutagenesis ◽  
Cell Voltage ◽  
Optical Signal ◽  
Excitation Wavelength ◽  
Optical Signals ◽  
Voltage Dependent

AbstractThe genetically encoded voltage indicators, ArcLight and its derivatives, mediate voltage dependent optical signals by intermolecular, electrostatic interactions between neighboring fluorescent proteins (FPs) via proton wires. A random mutagenesis event placed a negative charge on the exterior of the FP resulting in a greater than 10-fold improvement of the voltage-dependent optical signal. Repositioning this negative charge on the exterior of the FP reversed the polarity of voltage-dependent optical signals suggesting the presence of ‘hot spots’ capable of interacting with the negative charge on a neighboring FP thereby changing the fluorescent output. To explore the potential effect on the chromophore state, voltage-clamp fluorometry was performed with alternating excitation at 390 nm followed by excitation at 470 nm resulting in several mutants exhibiting voltage-dependent, ratiometric optical signals of opposing polarities. However, the kinetics, voltage ranges, and optimal FP fusion sites were different depending on the wavelength of excitation. These results suggest that the FP has external, electrostatic pathways capable of quenching fluorescence that are wavelength specific. ArcLight-derived GEVIs may therefore offer a novel way to map how conditions external to the β-can structure can affect the fluorescence of the chromophore and transiently manipulate those pathways via conformational changes mediated by whole cell voltage clamp.Statement of SignificanceArcLight-type GEVIs utilize proton pathways that send charge information outside of the FP to the internal chromophore enabling voltage induced conformational changes to affect fluorescence. These pathways are excitation wavelength specific suggesting that different external positions affect the protonated and deprotonated states of the chromophore.

scholarly journals E3 Ubiquitin Ligase SPL2 Is a Lanthanide-Binding Protein

2021 ◽  
Vol 22 (11) ◽  
pp. 5712
Author(s):  
Michał Tracz ◽  
Ireneusz Górniak ◽  
Andrzej Szczepaniak ◽  
Wojciech Białek
Keyword(s):  
Ubiquitin Ligase ◽  
Conformational Changes ◽  
Protein Interactions ◽  
E3 Ubiquitin Ligase ◽  
Lanthanide Ions ◽  
E3 Ligases ◽  
Fluorescence Measurements ◽  
Partial Unfolding

The SPL2 protein is an E3 ubiquitin ligase of unknown function. It is one of only three types of E3 ligases found in the outer membrane of plant chloroplasts. In this study, we show that the cytosolic fragment of SPL2 binds lanthanide ions, as evidenced by fluorescence measurements and circular dichroism spectroscopy. We also report that SPL2 undergoes conformational changes upon binding of both Ca2+ and La3+, as evidenced by its partial unfolding. However, these structural rearrangements do not interfere with SPL2 enzymatic activity, as the protein retains its ability to auto-ubiquitinate in vitro. The possible applications of lanthanide-based probes to identify protein interactions in vivo are also discussed. Taken together, the results of this study reveal that the SPL2 protein contains a lanthanide-binding site, showing for the first time that at least some E3 ubiquitin ligases are also capable of binding lanthanide ions.

Insights into the roles of charged residues in substrate binding and mode of action of mannuronan C-5 epimerase AlgE4

Glycobiology ◽  
2021 ◽  
Author(s):  
Margrethe Gaardløs ◽  
Sergey A Samsonov ◽  
Marit Sletmoen ◽  
Maya Hjørnevik ◽  
Gerd Inger Sætrom ◽  
...  
Keyword(s):  
Optical Tweezers ◽  
Conformational Changes ◽  
Molecular Mechanisms ◽  
Hydrophobic Interactions ◽  
Substrate Binding ◽  
Interdisciplinary Approach ◽  
Product Formation ◽  
Titration Calorimetry ◽  
Binding Groove ◽  
Dynamics Simulations

Abstract Mannuronan C-5 epimerases catalyse the epimerization of monomer residues in the polysaccharide alginate, changing the physical properties of the biopolymer. The enzymes are utilized to tailor alginate to numerous biological functions by alginate-producing organisms. The underlying molecular mechanisms that control the processive movement of the epimerase along the substrate chain is still elusive. To study this, we have used an interdisciplinary approach combining molecular dynamics simulations with experimental methods from mutant studies of AlgE4, where initial epimerase activity and product formation were addressed with NMR spectroscopy, and characteristics of enzyme-substrate interactions were obtained with isothermal titration calorimetry and optical tweezers. Positive charges lining the substrate-binding groove of AlgE4 appear to control the initial binding of poly-mannuronate, and binding also seems to be mediated by both electrostatic and hydrophobic interactions. After the catalytic reaction, negatively charged enzyme residues might facilitate dissociation of alginate from the positive residues, working like electrostatic switches, allowing the substrate to translocate in the binding groove. Molecular simulations show translocation increments of two monosaccharide units before the next productive binding event resulting in MG-block formation, with the epimerase moving with its N-terminus towards the reducing end of the alginate chain. Our results indicate that the charge pair R343-D345 might be directly involved in conformational changes of a loop that can be important for binding and dissociation. The computational and experimental approaches used in this study complement each other, allowing for a better understanding of individual residues’ roles in binding and movement along the alginate chains.

scholarly journals Activation-inactivation of potassium channels and development of the potassium-channel spike in internally perfused squid giant axons.

1981 ◽  
Vol 78 (1) ◽  
pp. 43-61 ◽  
Author(s):  
I Inoue
Keyword(s):  
Potassium Channels ◽  
Potassium Channel ◽  
Voltage Clamp ◽  
Time Constant ◽  
Equilibrium Potential ◽  
Giant Axons ◽  
Dependent Inactivation ◽  
Voltage Dependent ◽  
Time Courses ◽  
Calcium Permeability

A spike that is the result of calcium permeability through potassium channels was separated from the action potential is squid giant axons internally perfused with a 30 mM NaF solution and bathed in a 100 mM CaCl2 solution by blocking sodium channels with tetrodotoxin. Currents through potassium channels were studied under voltage clamp. The records showed a clear voltage-dependent inactivation of the currents. The inactivation was composed of at least two components; one relatively fast, having a time constant of 20--30 ms, and the other very slow, having a time constant of 5--10 s. Voltage clamp was carried out with a variety of salt compositions in both the internal and external solutions. A similar voltage-dependent inactivation, also composed of the two components, was recognized in all the current through potassium channels. Although the direction and intensity of current strongly depended on the salt composition of the solutions, the time-courses of these currents at corresponding voltages were very similar. These results strongly suggest that the inactivation of the currents in attributable to an essential, dynamic property of potassium channels themselves. Thus, the generation of a potassium-channel spike can be understood as an event that occurs when the equilibrium potential across the potassium channel becomes positive.

scholarly journals Structure–Function Relations of the First and Fourth Predicted Extracellular Linkers of the Type IIa Na+/Pi Cotransporter

2004 ◽  
Vol 124 (5) ◽  
pp. 475-488 ◽  
Author(s):  
Colin Ehnes ◽  
Ian C. Forster ◽  
Katja Kohler ◽  
Andrea Bacconi ◽  
Gerti Stange ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Reaction Rates ◽  
Transport Pathway ◽  
Extracellular Medium ◽  
Voltage Range ◽  
Substituted Cysteine Accessibility ◽  
Voltage Dependent ◽  
Opposite Behavior ◽  
Rate Limiting ◽  
Kinetics Of

The putative first intracellular and third extracellular linkers are known to play important roles in defining the transport properties of the type IIa Na+-coupled phosphate cotransporter (Kohler, K., I.C. Forster, G. Stange, J. Biber, and H. Murer. 2002b. J. Gen. Physiol. 120:693–705). To investigate whether other stretches that link predicted transmembrane domains are also involved, the substituted cysteine accessibility method (SCAM) was applied to sites in the predicted first and fourth extracellular linkers (ECL-1 and ECL-4). Mutants based on the wild-type (WT) backbone, with substituted novel cysteines, were expressed in Xenopus oocytes, and their function was assayed by isotope uptake and electrophysiology. Functionally important sites were identified in both linkers by exposing cells to membrane permeant and impermeant methanethiosulfonate (MTS) reagents. The cysteine modification reaction rates for sites in ECL-1 were faster than those in ECL-4, which suggested that the latter were less accessible from the extracellular medium. Generally, a finite cotransport activity remained at the end of the modification reaction. The change in activity was due to altered voltage-dependent kinetics of the Pi-dependent current. For example, cys substitution at Gly-134 in ECL-1 resulted in rate-limiting, voltage-independent cotransport activity for V ≤ −80 mV, whereas the WT exhibited a linear voltage dependency. After cys modification, this mutant displayed a supralinear voltage dependency in the same voltage range. The opposite behavior was documented for cys substitution at Met-533 in ECL-4. Modification of cysteines at two other sites in ECL-1 (Ile-136 and Phe-137) also resulted in supralinear voltage dependencies for hyperpolarizing potentials. Taken together, these findings suggest that ECL-1 and ECL-4 may not directly form part of the transport pathway, but specific sites in these linkers can interact directly or indirectly with parts of NaPi-IIa that undergo voltage-dependent conformational changes and thereby influence the voltage dependency of cotransport.

Presynaptic inhibition produced by an identified presynaptic inhibitory neuron. I. Physiological mechanisms

1986 ◽  
Vol 55 (1) ◽  
pp. 113-130 ◽  
Author(s):  
R. Kretz ◽  
E. Shapiro ◽  
E. R. Kandel
Keyword(s):  
Voltage Clamp ◽  
Presynaptic Inhibition ◽  
Inhibitory Neuron ◽  
Outward Current ◽  
Membrane Potentials ◽  
Slow Inward Current ◽  
Synaptic Potential ◽  
Synaptic Current ◽  
Voltage Dependent ◽  
Stimulation Of

We have examined the synaptic conductance mechanisms underlying presynaptic inhibition in Aplysia californica in a circuit in which all the neural elements are identified cells (Fig. 1). L10 makes connections to identified follower cells (RB and left upper quadrant cells, L2-L6). These connections are presynaptically inhibited by stimulating cells of the L32 cluster (4). L32 cells produce a slow inhibitory synaptic potential on L10. This inhibitory synaptic potential is associated with an apparent increased membrane conductance in L10. Both the inhibitory postsynaptic potential (IPSP) and the conductance increase are voltage dependent; the IPSP could not be reversed by hyperpolarizing the membrane potentials to - 120 mV. The hyperpolarization of L10 induced by L32 reduces the transmitter output of L10 and thereby contributes to presynaptic inhibition. However, this hyperpolarization accounts for about 30% of the effect because presynaptic inhibition can still be observed even when the hyperpolarization of L10 by L32 is prevented by voltage clamping. When L10 is voltage clamped, stimulation of L32 produces a slow outward synaptic current associated with an apparent increased conductance. Both the synaptic current and conductance change measured under clamp are voltage dependent, and the outward current could not be reversed. This synaptic current is not mediated by an increase in C1- conductance. It is sensitive to external K+ concentration, especially at hyperpolarized membrane potentials. With L10 under voltage clamp, stimulation of L32 also reduces a slow inward current in L10. This current has time and voltage characteristics similar to those of the Ca2+ current. Presynaptic inhibition is still produced by L32 when L10 is voltage clamped, and transmitter release is elicited by depolarizing voltage-clamp pulses. This component of presynaptic inhibition, which accounts for approximately 70% of the inhibition, appears to be due to a decrease in the Ca2+ current in the presynaptic neuron.

scholarly journals The redox environment triggers conformational changes and aggregation of hIAPP in Type II Diabetes

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Diana C. Rodriguez Camargo ◽  
Konstantinos Tripsianes ◽  
Katalin Buday ◽  
Andras Franko ◽  
Christoph Göbl ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Type Ii Diabetes ◽  
Type Ii ◽  
Redox Environment

scholarly journals Free energy landscape for the entire transport cycle of triose-phosphate/phosphate translocator

2017 ◽  
Author(s):  
Mizuki Takemoto ◽  
Yongchan Lee ◽  
Ryuichiro Ishitani ◽  
Osamu Nureki
Keyword(s):  
Free Energy ◽  
Conformational Changes ◽  
Conformational Transition ◽  
Substrate Binding ◽  
Energy Landscape ◽  
Umbrella Sampling ◽  
Free Energy Landscape ◽  
Coupling Mechanism ◽  
Triose Phosphate ◽  
Phosphate Translocator

AbstractSecondary active transporters translocate their substrates using the electrochemical potentials of other chemicals, undergoing large-scale conformational changes. Despite extensive structural studies, the atomic details of the transport mechanism still remain elusive. Here we performed a series of all-atom molecular dynamics simulations of the triose-phosphate/phosphate translocator (TPT), which exports organic phosphates in the chloroplast stroma in strict counter exchange with inorganic phosphate (Pi). Biased sampling methods, including string method and umbrella sampling, successfully reproduced the conformational changes between the inward– and outward-facing states, along with the substrate binding. The free energy landscape of this entire TPT transition pathway demonstrated the alternating access and substrate translocation mechanisms, which revealed Pi is relayed by positively charged residues along the transition pathway. Furthermore, the conserved Glu207 functions as a “molecular switch”, linking the local substrate binding and the global conformational transition. Our results provide atomic-detailed insights into the energy coupling mechanism of antiporter.

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