Charge Translocation Research Articles

Structural basis for severe pain caused by mutations in the voltage sensors of sodium channel NaV1.7.

Voltage-gated sodium channels in peripheral nerves conduct nociceptive signals from nerve endings to the spinal cord. Mutations in voltage-gated sodium channel NaV1.7 are responsible for a number of severe inherited pain syndromes, including inherited erythromelalgia (IEM). Here, we describe the negative shifts in the voltage dependence of activation in the bacterial sodium channel NaVAb as a result of the incorporation of four different IEM mutations in the voltage sensor, which recapitulate the gain-of-function effects observed with these mutations in human NaV1.7. Crystal structures of NaVAb with these IEM mutations revealed that a mutation in the S1 segment of the voltage sensor facilitated the outward movement of S4 gating charges by widening the pathway for gating charge translocation. In contrast, mutations in the S4 segments modified hydrophobic interactions with surrounding amino acid side chains or membrane phospholipids that would enhance the outward movement of the gating charges. These results provide key structural insights into the mechanisms by which these IEM mutations in the voltage sensors can facilitate outward movements of the gating charges in the S4 segment and cause hyperexcitability and severe pain in IEM. Our work gives new insights into IEM pathogenesis at the near-atomic level and provides a molecular model for mutation-specific therapy of this debilitating disease.

Functional characterization of SGLT1 using SSM-based electrophysiology: Kinetics of sugar binding and translocation.

Beside the ongoing efforts to determine structural information, detailed functional studies on transporters are essential to entirely understand the underlying transport mechanisms. We recently found that solid supported membrane-based electrophysiology (SSME) enables the measurement of both sugar binding and transport in the Na+/sugar cotransporter SGLT1 (Bazzone et al, 2022a). Here, we continued with a detailed kinetic characterization of SGLT1 using SSME, determining KM and KD app for different sugars, kobs values for sugar-induced conformational transitions and the effects of Na+, Li+, H+ and Cl- on sugar binding and transport. We found that the sugar-induced pre-steady-state (PSS) charge translocation varies with the bound ion (Na+, Li+, H+ or Cl-), but not with the sugar species, indicating that the conformational state upon sugar binding depends on the ion. Rate constants for the sugar-induced conformational transitions upon binding to the Na+-bound carrier range from 208s-1 for D-glucose to 95s-1 for 3-OMG. In the absence of Na+, rate constants are decreased, but all sugars bind to the empty carrier. From the steady-state transport current, we found a sequence for sugar specificity (Vmax/KM): D-glucose > MDG > D-galactose > 3-OMG > D-xylose. While KM differs 160-fold across tested substrates and plays a major role in substrate specificity, Vmax only varies by a factor of 1.9. Interestingly, D-glucose has the lowest Vmax across all tested substrates, indicating a rate limiting step in the sugar translocation pathway following the fast sugar-induced electrogenic conformational transition. SGLT1 specificity for D-glucose is achieved by optimizing two ratios: the sugar affinity of the empty carrier for D-glucose is similarly low as for all tested sugars (KD,K app = 210mM). Affinity for D-glucose increases 14-fold (KD,Na app = 15mM) in the presence of sodium as a result of cooperativity. Apparent affinity for D-glucose during transport increases 8-fold (KM = 1.9mM) compared to KD,Na app due to optimized kinetics. In contrast, KM and KD app values for 3-OMG and D-xylose are of similar magnitude. Based on our findings we propose an 11-state kinetic model, introducing a random binding order and intermediate states corresponding to the electrogenic transitions detected via SSME upon substrate binding.

Electrostatic Filtering of Polypeptides Through Membrane Protein Pores.

Naturally-occurring membrane proteins have been engineered as nanopore sensors for the single-molecule detection of various biochemical molecules. Here, we present a natural bacterial porin, CymA containing a dynamic component and densely packed charged residues in the pore, shaping a unique structural conformation and charge feature. Using single-channel recordings, we investigated the translocation of charged polypeptides through native CymA and truncated CymA lacking the dynamic element. Cationic polypeptides bind to the pore with high affinity, specifically at low salt conditions indicating an electrostatic charge and voltage-dependent translocation. Anionic peptides did not bind to the pore, confirming the selective binding of polypeptides with the pore due to their specific charge distribution. Further, the distinct peptide translocation kinetics between native and truncated indicated the role of the dynamic segment in molecular transport. We suggest that these natural membrane pores that permit the selective translocation of cationic polypeptides are advantageous for nanopore proteomics applications.

Molecular basis of multistep voltage activation in plant two-pore channel 1

Voltage-gated ion channels confer excitability to biological membranes, initiating and propagating electrical signals across large distances on short timescales. Membrane excitation requires channels that respond to changes in electric field and couple the transmembrane voltage to gating of a central pore. To address the mechanism of this process in a voltage-gated ion channel, we determined structures of the plant two-pore channel 1 at different stages along its activation coordinate. These high-resolution structures of activation intermediates, when compared with the resting-state structure, portray a mechanism in which the voltage-sensing domain undergoes dilation and in-membrane plane rotation about the gating charge-bearing helix, followed by charge translocation across the charge transfer seal. These structures, in concert with patch-clamp electrophysiology, show that residues in the pore mouth sense inhibitory Ca2+ and are allosterically coupled to the voltage sensor. These conformational changes provide insight into the mechanism of voltage-sensor domain activation in which activation occurs vectorially over a series of elementary steps.

Modularity of membrane-bound charge-translocating protein complexes.

Energy transduction is the conversion of one form of energy into another; this makes life possible as we know it. Organisms have developed different systems for acquiring energy and storing it in useable forms: the so-called energy currencies. A universal energy currency is the transmembrane difference of electrochemical potential (Δμ~). This results from the translocation of charges across a membrane, powered by exergonic reactions. Different reactions may be coupled to charge-translocation and, in the majority of cases, these reactions are catalyzed by modular enzymes that always include a transmembrane subunit. The modular arrangement of these enzymes allows for different catalytic and charge-translocating modules to be combined. Thus, a transmembrane charge-translocating module can be associated with different catalytic subunits to form an energy-transducing complex. Likewise, the same catalytic subunit may be combined with a different membrane charge-translocating module. In this work, we analyze the modular arrangement of energy-transducing membrane complexes and discuss their different combinations, focusing on the charge-translocating module.

Energy landscapes and dynamics of ion translocation through membrane transporters: a meeting ground for physics, chemistry, and biology.

The dynamics of ion translocation through membrane transporters is visualized from a comprehensive point of view by a Gibbs energy landscape approach. The ΔG calculations have been performed with the Kirkwood-Tanford-Warshel (KTW) electrostatic theory that properly takes into account the self-energies of the ions. The Gibbs energy landscapes for translocation of a single charge and an ion pair are calculated, compared, and contrasted as a function of the order parameter, and the characteristics of the frustrated system with bistability for the ion pair are described and quantified in considerable detail. These calculations have been compared with experimental data on the ΔG of ion pairs in proteins. It is shown that, under suitable conditions, the adverse Gibbs energy barrier can be almost completely compensated by the sum of the electrostatic energy of the charge-charge interactions and the solvation energy of the ion pair. The maxima in ΔGKTW with interionic distance in the bound H+ -A- charge pair on the enzyme is interpreted in thermodynamic and molecular mechanistic terms, and biological implications for molecular mechanisms of ATP synthesis are discussed. The timescale at which the order parameter moves between two stable states has been estimated by solving the dynamical equations of motion, and a wealth of novel insights into energy transduction during ATP synthesis by the membrane-bound FOF1-ATP synthase transporter is offered. In summary, a unifying analytical framework that integrates physics, chemistry, and biology has been developed for ion translocation by membrane transporters for the first time by means of a Gibbs energy landscape approach.

Constraint-based modeling of yeast mitochondria reveals the dynamics of protein import and iron-sulfur cluster biogenesis

SummaryMitochondria are a hallmark of eukaryal cells and play an important role in cellular metabolism. There is a vast amount of knowledge available on mitochondrial metabolism and essential mitochondrial functions, such as protein import and iron-sulfur cluster biosynthesis, including multiple studies on the mitochondrial proteome. Therefore, there is a need for in silico approaches to facilitate the analysis of these data. Here, we present a detailed model of mitochondrial metabolism Saccharomyces cerevisiae, including protein import, iron-sulfur cluster biosynthesis, and a description of the coupling between charge translocation processes and ATP synthesis. Model analysis implied a dual dependence of absolute levels of proteins in protein import, iron-sulfur cluster biogenesis and cluster abundance on growth rate and respiratory activity. The model is instrumental in studying dynamics and perturbations in these processes and given the high conservation of mitochondrial metabolism in humans, it can provide insight into their role in human disease.

Analysis of Charged Peptide Loop-Flipping across a Lipid Bilayer Using the String Method with Swarms of Trajectories.

The hydrophobic core of the lipid bilayer is conventionally considered a barrier to the translocation of charged species such that the translocation of even single ions occurs on long time scales. In contrast, experiments have revealed that some materials, including peptides, proteins, and nanoparticles, can translocate multiple charged moieties across the bilayer on experimentally relevant time scales. Understanding the molecular mechanisms underlying this behavior is challenging because resolving corresponding free-energy landscapes with molecular simulation techniques is computationally expensive. To address this challenge, we use atomistic molecular dynamics simulations with the swarms-of-trajectories (SOT) string method to analyze charge translocation pathways across single-component lipid bilayers as a function of multiple collective variables. We first demonstrate that the SOT string method can reproduce the free-energy barrier for the translocation of a charged lysine amino acid analogue in good agreement with the literature. We then obtain minimum free-energy pathways for the translocation, or flipping, of charged peptide loops across the lipid bilayer by utilizing trajectories from prior temperature-accelerated molecular dynamics (TAMD) simulations as initial configurations. The corresponding potential of mean force calculations reveal that the protonation of a central membrane-exposed aspartate residue substantially reduces the free-energy barrier for flipping charged loops by modulating the water content of the bilayer. These results provide new insight into the thermodynamics underlying loop-flipping processes and highlight how the combination of TAMD and the SOT string method can be used to understand complex charge translocation mechanisms.

Mechanistic Dissection of Cu(I)‐translocating P‐type ATPase Pumps by I n‐vitro Real‐Time Transport in Artificial Lipid Bilayer Vesicles

Copper is an essential element in biological systems where it plays key roles as a co-factor in diverse cuproenzymes and in signaling processes. Cu(I) transporting P-type ATPases (CopA) are primary active transmembrane transporter proteins that utilize the energy of ATP hydrolysis to catalyze the extrusion of Cu(I) ions across cell membranes. Their activity plays a gatekeeper role in controlling intracellular Cu(I) levels and homeostasis, a key process for survival in all kingdoms of life. In humans, mutations in these Cu(I) pumps lead to the Menkes syndrome and the Wilson's disease. In pathogenic bacteria CopA proteins can act as virulence factors to overcome the Cu(I) stress generated by the host defense system, thereby representing a potential therapeutic target against pathogens causing various fatal diseases. Even though the structure and function of these transporters has been established, the overall mechanism for substrate translocation, their transport kinetic parameters and electrogenicity remain elusive. In this work we developed a novel coupled experimental platform, centered on multiple fluorescence reporter probes that permits to study substrate, the transport mechanism and translocation kinetics in real-time in Cu(I)-pumps reconstituted in a native-like environment. We reconstituted the model Cu(I)-pump from E. coli (EcCopA) and its inactive mutants in artificial small unilamellar vesicles (proteoliposomes). We quantitatively characterized the chemistry underlying substrate transport, putative counterions and charge translocation by encapsulating in the liposome lumen fluorescence detector probes (CTAP-3, Pyranine and Oxonol VI) responsive to diverse stimuli (Cu(I) substrate, pH and transmembrane potential). The study established that Cu(I) pumps are primary-active uniporters and electrogenic pumps that build a transmembrane potential by translocating one positive charge (Cu(I)) per ATP hydrolysis cycle without coupling it to counterion transport. This analysis further reveals mechanistic differences in substrate translocation by CopA proteins in comparison to other characterized P-type ATPase classes. Moreover, the developed approach can be extended as a general biophysical tool to investigate other P-type ATPase pumps in which the mechanism of substrate translocation has not yet been established.

Effect of Peptide Sequence Mutations on the Translocation of Charged Peptide Loops across Lipid Bilayers with Molecular Dynamics Simulations

The hydrophobic core of the cell membrane is considered largely impermeable to charged molecules because of the large free energy barrier and corresponding long timescale (∼hours) for charge translocation. Contradicting this view, a variety of water-soluble peptides are known to translocate charged groups across the cell membrane on a surprisingly rapid ∼seconds timescale, for at least some peptides. In this work, we study the interconversion of single α-helix peptides with charged flanking loops between a surface-adsorbed and membrane-embedded state, which requires the translocation (or “flipping”) of charged loops across the bilayer.

Mechanisms of Energy Transduction by Charge Translocating Membrane Proteins.

Life relies on the constant exchange of different forms of energy, i.e., on energy transduction. Therefore, organisms have evolved in a way to be able to harvest the energy made available by external sources (such as light or chemical compounds) and convert these into biological useable energy forms, such as the transmembrane difference of electrochemical potential (Δμ̃). Membrane proteins contribute to the establishment of Δμ̃ by coupling exergonic catalytic reactions to the translocation of charges (electrons/ions) across the membrane. Irrespectively of the energy source and consequent type of reaction, all charge-translocating proteins follow two molecular coupling mechanisms: direct- or indirect-coupling, depending on whether the translocated charge is involved in the driving reaction. In this review, we explore these two coupling mechanisms by thoroughly examining the different types of charge-translocating membrane proteins. For each protein, we analyze the respective reaction thermodynamics, electron transfer/catalytic processes, charge-translocating pathways, and ion/substrate stoichiometries.

Photovoltage generation by photosystem II core complexes immobilized onto a Millipore filter on an indium tin oxide electrode.

The light-induced functioning of photosynthetic pigment-protein complex of photosystem II (PSII) is linked to the vectorial translocation of charges across the membrane, which results in the formation of voltage. Direct measurement of the light-induced voltage (∆V) generated by spinach oxygen-evolving PSII core complexes adsorbed onto a Millipore membrane filter (MF) on an indium tin oxide (ITO) electrode under continuous illumination has been performed. PSII was shown to participate in electron transfer from water to the ITO electrode, resulting in ∆V generation. No photovoltage was detected in PSII deprived of the water-oxidizing complex. The maximal and stable photoelectric signal was observed in the presence of disaccharide trehalose and 2,6-dichloro-1,4-benzoquinone, acting as a redox mediator between the primary quinone acceptor QA of PSII and electrode surface. Long time preservation of the steady-state photoactivity at room temperature in a simple in design ITO|PSII-MF|ITO system may be related to the retention of water molecules attached to the PSII surface in the presence of trehalose.

Characterizing the Translocation of Charged Peptide Loops across Lipid Bilayers with Molecular Dynamics Simulations

The hydrophobic core of the cell membrane is considered largely impermeable to charged molecules because of the large free energy barrier and corresponding long timescale (∼hours) for charge translocation. Contradicting this view, a variety of water-soluble peptides are known to translocate charged groups across the cell membrane on a surprisingly rapid timescale, on the order of few seconds at least for some peptides. In this work, we study the interconversion of a peptide with charged flanking loops between a surface-adsorbed and membrane-embedded state, which requires the translocation (or “flipping”) of charged loops across the bilayer. We utilize all-atom Temperature Accelerated Molecular Dynamics (TAMD) simulations to predict the likelihood of loop flipping without predefining a specific translocation pathway. We show that membrane-exposed charged residues accelerate the flipping of charged peptide loops by stabilizing intramembrane water defects. We further demonstrate that this computational approach can identify multiple flipping pathways without specifying them a priori. The position of charged residues in the transmembrane helix also affects the flipping pathways and the apparent flipping free energy barrier. These detailed molecular-level insights of peptide translocation pathways may be valuable for designing small cationic peptides for applications in gene and drug delivery. Moreover, the methodology and findings discussed here can generalize to more complex behaviors, such as the large-scale conformational rearrangements of integral membrane proteins.

Dual-Self-Restricted GFP Chromophore Analogues with Significantly Enhanced Emission.

The tremendous gap of fluorescence emission of synthetic green fluorescent protein (GFP) chromophore to the protein itself makes it impossible to use for applications in molecular and cellular imaging. Here, we developed an efficient methodology to enhance the photoluminescence response of synthetic GFP chromophore analogues by constructing dual-self-restricted chromophores. Single self-restricted chromophores were first generated with 2,5-dimethoxy substitution on the aromatic ring, which were further modified with phenyl or 2,5-dimethoxy phenyl to form dual-self-restricted chromophores. These two chromophores showed an obvious solvatofluorochromic color palette across blue to yellow with a maximum emission Stokes shift of 95 nm and dramatically enhanced fluorescence emission in various aprotic solvents, especially in hexane, where the QY reached around 0.6. Importantly, in acetonitrile and dimethyl sulfoxide, the fluorescence QYs of both chromophores were over 0.22, which were the highest reported so far in high polar organic solvents. Meanwhile, the fluorescence lifetimes also improved obviously with the maximum of around 4.5 ns. Theoretical calculations revealed a more favorable Mülliken atomic charge translocation over the double-bond bridge and illustrated much higher energy barriers for the Z/E photoisomerization together with larger bond orders compared with the GFP core chromophore, p-HBDI. Our work significantly improved the fluorescence emission of synthetic GFP chromophore analogues in polar solvents while reserved the multicolor emitting function, providing a solid molecular motif for engineering high-performance fluorescent probes.

Quantitative Analysis of Factors Affecting the Event Rate in Glass Nanopore Sensors.

While the solid-state nanopore sensors have shown exceptional promise with their single-molecule sensitivity and label-free operations, one of the most significant challenges in the nanopore sensor is the limited analyte translocation event rate that leads to prolonged sensor response time. This issue is more pronounced when the analyte concentration is below the nanomolar (nM) range, owing to the diffusion-limited mass transport. In this work, we systematically studied the experimental factors beyond the intrinsic analyte concentration and electrophoretic mobility that affect the event rate in glass nanopore sensors. We developed a quantitative model to capture the impact of nanopore surface charge density, ionic strength, nanopore geometry, and translocation direction on the event rate. The synergistic effects of these factors on the event rates were investigated with the aim to find the optimized experimental conditions for operating the glass nanopore sensor from the response time standpoint. The findings in the study would provide useful and practical insight to enhance the device response time and achieve a lower detection limit for various glass nanopore-sensing experiments.

Characterizing the Molecular Mechanisms for Flipping Charged Peptide Flanking Loops across a Lipid Bilayer.

The cell membrane largely prevents the passive diffusion of charged molecules due to the large free energy barrier associated with translocating charged groups across the hydrophobic lipid bilayer core. Despite this barrier, some peptides can interconvert between transmembrane and surface-adsorbed states by "flipping" charged flanking loops across the bilayer on a surprisingly rapid second-minute time scale. The transmembrane helices of some multispanning membrane proteins undergo similar reorientation processes, suggesting that loop-flipping may be a mechanism for regulating membrane protein topology; however, the molecular mechanisms underlying this behavior remain unknown. In this work, we study the loop-flipping behavior exhibited by a peptide with a hydrophobic transmembrane helix, charged flanking loops, and a central, membrane-exposed aspartate residue of varying protonation state. We utilize all-atom temperature accelerated molecular dynamics simulations to predict the likelihood of loop-flipping without predefining specific loop-flipping pathways. We demonstrate that this approach can identify multiple possible flipping pathways, with the prevalence of each pathway depending on the protonation state of the central residue. In particular, we find that a charged central residue facilitates loop-flipping by stabilizing membrane water defects, enabling the "self-catalysis" of charge translocation. These findings provide detailed molecular-level insights into charged loop-flipping pathways that may generalize to other charge translocation processes, such as lipid flip-flop or the large-scale conformational rearrangements of multispanning membrane proteins.

Conformational memory in the association of the transmembrane protein phospholamban with the sarcoplasmic reticulum calcium pump SERCA

The sarcoplasmic reticulum Ca2+-ATPase SERCA promotes muscle relaxation by pumping calcium ions from the cytoplasm into the sarcoplasmic reticulum. SERCA activity is regulated by a variety of small transmembrane peptides, most notably by phospholamban in cardiac muscle and sarcolipin in skeletal muscle. However, how phospholamban and sarcolipin regulate SERCA is not fully understood. In the present study, we evaluated the effects of phospholamban and sarcolipin on calcium translocation and ATP hydrolysis by SERCA under conditions that mimic environments in sarcoplasmic reticulum membranes. For pre-steady-state current measurements, proteoliposomes containing SERCA and phospholamban or sarcolipin were adsorbed to a solid-supported membrane and activated by substrate concentration jumps. We observed that phospholamban altered ATP-dependent calcium translocation by SERCA within the first transport cycle, whereas sarcolipin did not. Using pre-steady-state charge (calcium) translocation and steady-state ATPase activity under substrate conditions (various calcium and/or ATP concentrations) promoting particular conformational states of SERCA, we found that the effect of phospholamban on SERCA depends on substrate preincubation conditions. Our results also indicated that phospholamban can establish an inhibitory interaction with multiple SERCA conformational states with distinct effects on SERCA's kinetic properties. Moreover, we noted multiple modes of interaction between SERCA and phospholamban and observed that once a particular mode of association is engaged it persists throughout the SERCA transport cycle and multiple turnover events. These observations are consistent with conformational memory in the interaction between SERCA and phospholamban, thus providing insights into the physiological role of phospholamban and its regulatory effect on SERCA transport activity.

Lysine 300 is essential for stability but not for electrogenic transport of the Escherichia coli NhaA Na+/H+ antiporter

Na+/H+ antiporters are located in the cytoplasmic and intracellular membranes and play crucial roles in regulating intracellular pH, Na+, and volume. The NhaA antiporter of Escherichia coli is the best studied member of the Na+/H+ exchanger family and a model system for all related Na+/H+ exchangers, including eukaryotic representatives. Several amino acid residues are important for the transport activity of NhaA, including Lys-300, a residue that has recently been proposed to carry one of the two H+ ions that NhaA exchanges for one Na+ ion during one transport cycle. Here, we sought to characterize the effects of mutating Lys-300 of NhaA to amino acid residues containing side chains of different polarity and length (i.e. Ala, Arg, Cys, His, Glu, and Leu) on transporter stability and function. Salt resistance assays, acridine-orange fluorescence dequenching, solid supported membrane-based electrophysiology, and differential scanning fluorometry were used to characterize Na+ and H+ transport, charge translocation, and thermal stability of the different variants. These studies revealed that NhaA could still perform electrogenic Na+/H+ exchange even in the absence of a protonatable residue at the Lys-300 position. However, all mutants displayed lower thermal stability and reduced ion transport activity compared with the wild-type enzyme, indicating the critical importance of Lys-300 for optimal NhaA structural stability and function. On the basis of these experimental data, we propose a tentative mechanism integrating the functional and structural role of Lys-300.

Charge translocation by mitochondrial NADH:ubiquinone oxidoreductase (complex I) from Yarrowia lipolytica measured on solid-supported membranes

The charge translocation by purified reconstituted mitochondrial complex I from the obligate aerobic yeast Yarrowia lipolytica was investigated after adsorption of proteoliposomes to solid-supported membranes. In presence of n-decylubiquinone (DBQ), pulses of NADH provided by rapid solution exchange induced charge transfer reflecting steady-state pumping activity of the reconstituted enzyme. The signal amplitude increased with time, indicating ‘deactive→active’ transition of the Yarrowia complex I. Furthermore, an increase of the membrane-conductivity after addition of 5-(N-ethyl-N-isopropyl)amiloride (EIPA) was detected which questiones the use of EIPA as an inhibitor of the Na+/H+-antiporter-like subunits of complex I. This investigation shows that electrical measurements on solid-supported membranes are a suitable method to analyze transport events and ‘active/deactive’ transition of mitochondrial complex I.

Space and Time Evolution of the Electrostatic Potential During the Activation of a Visual Pigment.

Animal and microbial retinal proteins employ the Schiff base of retinal as their chromophore. Here, the possible consequences of the charge translocation associated with the light-induced dynamics of the chromophore of a visual opsin are investigated along a representative semiclassical trajectory. We show that the evolution of the electrostatic potential projected by the chromophore onto the surrounding protein displays intense but topographically localized sudden variations in proximity of the decay region. pKa calculations carried out on selected snapshots used as probes, indicate that the only residue which may be sensitive to the electrostatic potential shift is Glu181. Accordingly, our results suggest that the frail Tyr191/268-Glu181-Wat2-Ser186 hydrogen bond network may be perturbed by the transient variations of the electrostatic potential.