Mechanism Of Ion Translocation Research Articles

Mechanism of Ion Translocation by Na+-Rhodopsin.

This review provides a brief description of the structure and transport function of the recently discovered family of retinal-containing Na+-translocating rhodopsins. The main emphasis is put on the kinetics of generation of electric potential difference in the membrane during a single transporter turnover. According to the proposed transport mechanism of Na+-rhodopsin, the driving force for the Na+ translocation from the cytoplasm is the local electric field created by the H+ movement from the Schiff base.

Deciphering ion transport and ATPase coupling in the intersubunit tunnel of KdpFABC

KdpFABC, a high-affinity K+ pump, combines the ion channel KdpA and the P-type ATPase KdpB to secure survival at K+ limitation. Here, we apply a combination of cryo-EM, biochemical assays, and MD simulations to illuminate the mechanisms underlying transport and the coupling to ATP hydrolysis. We show that ions are transported via an intersubunit tunnel through KdpA and KdpB. At the subunit interface, the tunnel is constricted by a phenylalanine, which, by polarized cation-π stacking, controls K+ entry into the canonical substrate binding site (CBS) of KdpB. Within the CBS, ATPase coupling is mediated by the charge distribution between an aspartate and a lysine. Interestingly, individual elements of the ion translocation mechanism of KdpFABC identified here are conserved among a wide variety of P-type ATPases from different families. This leads us to the hypothesis that KdpB might represent an early descendant of a common ancestor of cation pumps.

Metal Substrate Selectivity and Mechanism of Transport in a Transmembrane Zn‐pump Revealed by in‐vitro Real‐time Transport

P1B-type ATPases are primary active transmembrane metal pumps conserved in all kingdoms of life that play a pivotal role in controlling cellular metal ion levels and homeostasis. In bacteria, they are responsible for selectively exporting essential and toxic metal ions across the cell membrane to maintain concentrations below toxic levels. They are critical virulence factors in pathogenic bacteria and allow selected microorganisms to survive in extreme metal-rich environments. The P1B-2-type Zn(II) ATPase pump subfamily possesses a wide promiscuity towards metal substrate translocation by plastic selection of Zn(II)/Cd(II)/Hg(II)/Pb(II) at their high-affinity transmembrane metal binding site. Despite these substrates stimulate ATP-hydrolysis, direct translocation of all these metals and their mechanism of transport remain to be demonstrated. In this work, we established a platform for purification and in-vitro functional reconstitution in artificial phospholipid bilayer vesicles (proteoliposomes) of ZntA from Cupriavidus metallidurans to dissect the molecular mechanism of divalent metal ion translocation and substrate promiscuity towards first-, second- and the third-row transition metals. Metal-dependent ATPase activity assays of CmΔZntA166–794 in detergent micelles and proteoliposomes revealed stimulation of the ATPase activity by Pb(II), Zn(II), Cd(II) and Hg(II) substrates with the maximal velocities (Vmax) following the order Pb(II) > Zn(II) > Cd(II) > Hg(II), confirming the successful functional reconstitution in lipid vesicles. By selecting and encapsulating divalent metal sensitive fluorescent probes in the proteoliposome lumen, we performed real-time metal transport assays to reveal the ATP-dependent translocation of Pb(II), Zn(II), Cd(II) and Hg(II) across the lipid bilayer, establishing that the translocation rates correlate to the order observed following ATP hydrolysis. Furthermore, to dissect the substrate transport mechanism, we encapsulated the pH sensitive fluorescent probe pyranine to demonstrated that, differently than other P-type ATPases, translocation is not coupled to H+ counter-flux for all the M(II) substrates. By encapsulating the membrane potential sensitive fluorescent probe Oxonol-VI we also revealed that, with all substrates, Zn-pumps act as primary active electrogenic uniporters capable of generating a transmembrane potential. This work establishes a platform to dissect the mechanisms of translocation of various metal substrates in P-type ATPases which is applicable to study other uncharacterized transporter families and to reveal the bioinorganic chemistry of metal ion transport at molecular level.

Functional and Structural Roles of the Major Facilitator Superfamily Bacterial Multidrug Efflux Pumps.

Pathogenic microorganisms that are multidrug-resistant can pose severe clinical and public health concerns. In particular, bacterial multidrug efflux transporters of the major facilitator superfamily constitute a notable group of drug resistance mechanisms primarily because multidrug-resistant pathogens can become refractory to antimicrobial agents, thus resulting in potentially untreatable bacterial infections. The major facilitator superfamily is composed of thousands of solute transporters that are related in terms of their phylogenetic relationships, primary amino acid sequences, two- and three-dimensional structures, modes of energization (passive and secondary active), and in their mechanisms of solute and ion translocation across the membrane. The major facilitator superfamily is also composed of numerous families and sub-families of homologous transporters that are conserved across all living taxa, from bacteria to humans. Members of this superfamily share several classes of highly conserved amino acid sequence motifs that play essential mechanistic roles during transport. The structural and functional importance of multidrug efflux pumps that belong to the major facilitator family and that are harbored by Gram-negative and -positive bacterial pathogens are considered here.

Ion-Ion Repulsions and Charge-Shielding Effects Dominate the Permeation Mechanism through the OmpF Porin Channel.

OmpF is a wide channel bacterial porin frequently employed to study selective ionic translocation. The cationic preference of this porin is mainly determined by electrostatic forces between the translocated ion and the protein and the formation of ion pairs (e.g., K+···Cl-) being previously pointed as the main cause to favor the cationic transport through the constriction zone. Hybrid quantum mechanics/molecular mechanics-molecular dynamics simulations, which have provided polarization-containing potentials of mean force profiles for different permeation scenarios, reveal significant new insights related with the ion translocation mechanism. Results show that the permeation is dominated by electrostatic interactions, which in turn affect ion-protein interactions at the constriction zone. However, it is observed that ion flow is favored by ion-ion repulsions and, in a lesser extent, by charge-shielding effects, instead of the previously pointed ionic pair formation.

Molecular Characterization of the CTR Copper Transporter

Copper (Cu) is an essential trace element for cells but it is also highly toxic. Therefore, Cu uptake, cellular distribution, and efflux are tightly regulated. CTR (copper transporter) protein is one of the major players in cellular Cu uptake. It is a membrane protein selectively transporting Cu ions into the cytosol. CTR has no homology to other known transition metal ion transporters, and our understanding of its transport mechanism is still limited. A current CTR model at 7 Å resolution shows that it forms a homotrimer with a central pore. However, the resolution is not sufficient to reveal details on ion binding sites and the mechanism of ion translocation. Previous studies have also suggested that CTR favors Cu+ over Cu2+, however, Cu+ is sensitive to oxygen and its transport has not been studied in any detail. We developed assays to measure binding and transport of Cu+ and other transitional metal ions in CTR, and will report the results from these studies.

Ligand-induced conformational dynamics of the Escherichia coli Na+/H+ antiporter NhaA revealed by hydrogen/deuterium exchange mass spectrometry

Na+/H+ antiporters comprise a family of membrane proteins evolutionarily conserved in all kingdoms of life and play an essential role in cellular ion homeostasis. The NhaA crystal structure of Escherichia coli has become the paradigm for this class of secondary active transporters. However, structural data are only available at low pH, where NhaA is inactive. Here, we adapted hydrogen/deuterium-exchange mass spectrometry (HDX-MS) to analyze conformational changes in NhaA upon Li+ binding at physiological pH. Our analysis revealed a global conformational change in NhaA with two sets of movements around an immobile binding site. Based on these results, we propose a model for the ion translocation mechanism that explains previously controversial data for this antiporter. Furthermore, these findings contribute to our understanding of related human transporters that have been linked to various diseases.

High-resolution structure and substrate and ion translocation mechanism of a di-carboxylate transporter

High-resolution structure and substrate and ion translocation mechanism of a di-carboxylate transporter

The Cardiac Ryanodine Receptor (RyR2): Investigating Mechanisms of Gating at the Selectivity Filter

Ryanodine receptors (RyR) provide the pathway for release of intracellular calcium ions (Ca2+) that initiate muscle contraction. Mutations in cardiac RyRs, (RyR2) underlie arrhythmia and sudden cardiac death. RyRs are therefore an attractive therapeutic target, however more information regarding structure and function is required. The enormous size of RyRs precludes detailed structural analysis. Models, based on potassium channel templates, exist for the RyR pore-forming region (PFR) providing an invaluable framework in which predictions of specific processes of ion translocation and gating mechanisms can be tested.Potassium channels have a conserved gating mechanism involving a distinct hydrogen-bonding network of residues at the selectivity filter that is responsible for holding the filter in an inactive, non-conducting conformation. This study examines interactions of equivalent residues in RyR2 to ascertain whether a similar gating mechanism exists.Three alanine (D4829A, Y4813A, Y4839A) and one conserved tryptophan (Y4839W) RyR2 mutations were constructed to assess a proposed hydrogen-bonding network. Mutated channels formed functional homotetramers in vivo whereby they released Ca2+ upon caffeine addition. Differences in [3H]-ryanodine binding on isolated WT and mutant mix membrane populations revealed 1) altered ryanodine binding site and/or 2) altered calcium sensitivity for mutant channels. Preliminary single-channel experiments assessing ion handling and gating properties under steady state conditions suggest that the selectivity filer has a role in channel gating. Conductance and open probability (Po) for Y4839A was reduced by 15 % and 76 % respectively compared to WT RyR2. No single-channel experiments were performed for Y4813A due to inherent protein instability when purified. Four unique gating modes including subconductance states for D4829A were observed. Further experiments are required to assess the role of the selectivity filter in gating.Study supported by the British Heart Foundation.

Infrared Spectroscopic Evidence of a Redox-Dependent Conformational Change Involving Ion Binding Residue NqrB-D397 in the Na+-Pumping NADH:Quinone Oxidoreductase from Vibrio cholerae

The Na(+)-pumping NADH:quinone oxidoreductase (Na(+)-NQR) is a unique respiratory enzyme that conserves energy by translocating Na(+) through the plasma membrane. Found only in prokaryotes, the enzyme serves as the point of entry of electrons into the respiratory chain in many pathogens, including Vibrio cholerae and Yersinia pestis. In this study, a combined electrochemical and Fourier transform infrared (FTIR) spectroscopic approach revealed that Na(+)-NQR undergoes significant conformational changes upon oxidoreduction, depending on the monovalent cation present (Na(+), Li(+), K(+), or Rb(+)). In the presence of the inhibitor Rb(+), additional conformational changes are evident, indicating a changed accessibility of the sodium binding sites. In electrochemically induced FTIR difference spectra, the involvement of deprotonated acid residues in the binding of cations, together with the spectral features, that point toward a monodentate binding mode for these acid residues in the oxidized form of the enzyme and bidentate binding in the reduced form could be identified. The measurements confirmed that NqrB-D397 is one of the acid residues involved in Na(+) and Li(+) binding. In the NqrB-D397E mutant, the spectral features characteristic of COO(-) groups are shifted, and a weakening of the hydrogen binding of the ion binding cluster is revealed. Finally, H-D exchange kinetics of amide protons confirmed that Na(+)-NQR adopts different conformations, with different accessibilities to the aqueous environment, depending on the cation present, which contributes to the selectivity mechanism of ion translocation.

E. Coli F1Fo ATP Synthase Subunit C: Solution NMR Structure by CS-Rosetta

The ATP synthase is the main source of ATP in cells, whose function is to inter-convert chemical energy and transmembrane electrochemical potential difference. The enzyme contains two domains: a soluble F1 domain where ATP is synthesized/hydrolyzed and a transmembrane FO domain, which functions as an ion pump. Multiple (9-15) copies of subunit c form an oligomeric ring in the FO domain. The ion translocation activity takes place at the interface of c-ring and subunit a where an essential acidic residue in middle of the outer helix in subunit c undergoes a protonation/deprotonation process, which couples with the relative rotation of c-ring to subunit a. Although high-resolution X-ray structures of the c-rings from several organisms have been solved, the mechanism of ion translocation still needs to be clarified at atomic resolution.We aim to solve the structures of E. coli subunit c at both protonation states of the essential Asp61 and elucidate any conformational changes at the active site during the ion translocation process. Due to the difficulty of obtaining unambiguous long-range NOE constraints in membrane proteins, traditional solution NMR methods were unsuccessful in this case. We employed CS-Rosetta, which utilizes minimal NMR constraints, in our study.Our study of the subunit c in the protonated state shows that the choice of proper scoring weights is essential in the application of CS-Rosetta to membrane proteins. Using limited NMR restraints this method converges on a structure for E. coli subunit c that is similar to the X-ray structures of subunit c from other organisms. Further improving the quality of the structure may be achieved by optimizing the weights in the calculation. We are also using this approach on deprotonated subunit c and hoping to model the c-ring from our results.

The Molecular Determinants of the Zinc Transporter, hZIP4

Zinc is an essential micronutrient which is required for the function of hundreds of cellular enzymes. In addition, zinc is the second most abundant transition metal found in biological systems (iron is most abundant). However, the concentration of free zinc is nano to picomolar since most zinc is bound to proteins. This makes investigating the mechanism of zinc transport across the plasma membrane a challenge. Our interest has been to elucidate the mechanism of zinc transport mediated by one member of the ZIP family of proteins, hZIP4. hZIP4 is located at the primary location of zinc uptake in humans and has been directly implicated in multiple disease states including Acrodermatitis enteropathica and pancreatic cancer. However the mechanism of transport is not known. We have previously shown that Zn2+, Ni2+ and Cu2+ can be transported by hZIP4, following heterologous expression in X. laevis oocytes, where there are two binding affinities (in the nM and μM range of biometal). Currently, our research interests are to investigate the mechanism of ion translocation using a mixture of biochemical and biophysical techniques.

Structure of the rotor ring modified with N , N ′ -dicyclohexylcarbodiimide of the Na + -transporting vacuolar ATPase

The prokaryotic V-ATPase of Enterococcus hirae, closely related to the eukaryotic enzymes, provides a unique opportunity to study the ion-translocation mechanism because it transports Na(+), which can be detected by radioisotope (22Na(+)) experiments and X-ray crystallography. In this study, we demonstrated that the binding affinity of the rotor ring (K ring) for 22Na(+) decreased approximately 30-fold by reaction with N,N(')-dicyclohexylcarbodiimide (DCCD), and determined the crystal structures of Na(+)-bound and Na(+)-unbound K rings modified with DCCD at 2.4- and 3.1-Å resolutions, respectively. Overall these structures were similar, indicating that there is no global conformational change associated with release of Na(+) from the DCCD-K ring. A conserved glutamate residue (E139) within all 10 ion-binding pockets of the K ring was neutralized by modification with DCCD, and formed an "open" conformation by losing hydrogen bonds with the Y68 and T64 side chains, resulting in low affinity for Na(+). This open conformation is likely to be comparable to that of neutralized E139 forming a salt bridge with the conserved arginine of the stator during the ion-translocation process. Based on these findings, we proposed the ion-translocation model that the binding affinity for Na(+) decreases due to the neutralization of E139, thus releasing bound Na(+), and that the structures of Na(+)-bound and Na(+)-unbound DCCD-K rings are corresponding to intermediate states before and after release of Na(+) during rotational catalysis of V-ATPase, respectively.

Microscopic rotary mechanism of ion translocation in the Fo complex of ATP synthases

The microscopic mechanism of coupled c-ring rotation and ion translocation in F(1)F(o)-ATP synthases is unknown. Here we present conclusive evidence supporting the notion that the ability of c-rings to rotate within the F(o) complex derives from the interplay between the ion-binding sites and their nonhomogenous microenvironment. This evidence rests on three atomic structures of the c(15) rotor from crystals grown at low pH, soaked at high pH and, after N,N'-dicyclohexylcarbodiimide (DCCD) modification, resolved at 1.8, 3.0 and 2.2 Å, respectively. Alongside a quantitative DCCD-labeling assay and free-energy molecular dynamics calculations, these data demonstrate how the thermodynamic stability of the so-called proton-locked state is maximized by the lipid membrane. By contrast, a hydrophilic environment at the a-subunit-c-ring interface appears to unlock the binding-site conformation and promotes proton exchange with the surrounding solution. Rotation thus occurs as c-subunits stochastically alternate between these environments, directionally biased by the electrochemical transmembrane gradient.

Molecular Mechanism of Light-driven Ion Pumps

Crystallographic studies of archaeal light-driven ion pumps, including bacteriorhodopsin (BR) and halorhodopsin (HR), have shown that water molecules entrapped in protein cavities play an active role in the ion-translocation mechanism. On the basis of structural data of the reaction intermediates, we have proposed the hypothesis that BR functions as a proton/water antiporter, while HR functions as a proton/HCl antiporter.

Channelrhodopsin-2 Variants with Accelerated and Decelerated Channel Kinetics

The light-activated cation channel Channelrhodopsin-2 (ChR2) is a powerful tool for controlling neuronal activity. Its genetic information is carried into neurons which express the protein. Channel activation by blue light exposure causes membrane depolarizations that immediately trigger action potentials. We genetically modified ChR2 wildtype and created variants with decelerated and accelerated channel kinetics as well as changed ion selectivities. ChR2 mutations with new features broaden the toolbox for neuroscientists but the mechanisms of channel activation and ion translocation are still unclear. We use a combination of theoretical approaches like molecular and mathematical modeling as well as experimental techniques like UV/vis spectroscopy, flashlight photolysis and two electrode voltage clamp to reveal how mutations affect the channel properties.

Use Of Shaker B K+ Channel NH2-inactivation Peptides To Probe The Ryanodine Receptor Ca2+ Release Channel Pore

Inactivation of K+ channels occurs by the interaction of the pore with NH2-terminal sequences. Synthetic peptides corresponding to these sequences were applied to the purified mouse cardiac ryanodine receptor type 2 (RyR2) and single channel activity was recorded in planar lipid bilayers. The wild type (WT) Shaker B NH2-peptide MAAVAGLYGLGEDRQHRKKQ induced a block of the open RyR2 channel in a concentration and voltage-dependent manner, but also when the channel displayed a ryanodine-modified state [Mead et al., J. Membrane Biol., 1998]. In the latter condition, in a 200 mM KCl buffer when the holding potential was at +50 mV, the open probability decreased by a factor of 2 (p=0.001, n=10 channels for control and n=5 for WT peptide) mainly due to a 4-fold reduction (p<0.01, n=10 channels for control and n=5 for WT peptide) of the mean open time in the presence of 20 μM WT peptide. It is noteworthy that the peptide at 20 μM had no effect in a 600 mM KCl buffer, suggesting the importance of charged residues in the blocking effect on RyR2. Furthermore we designed a mutant peptide where the amino acid Alanine in positions 3 and 5 was replaced by Glutamine residues, giving a less hydrophobic feature to the peptide. The mutant peptide at 20 μM in a 200 mM KCl buffer was less effective than the WT peptide, while showing a different mechanism of action. These findings are consistent with the idea that RyR2 contains well organised charged and hydrophobic residues in its conduction pathway and that they may play a general role in the ion translocation mechanisms of the sarcoplasmic reticulum Ca2+ release channel.Acknowledgement: This work is funded by the British Heart Foundation.

An Ion Selectivity Filter in the Extracellular Domain of Cys-loop Receptors Reveals Determinants for Ion Conductance

Neurotransmitter binding to Cys-loop receptors promotes a prodigious transmembrane flux of several million ions/s, but to date, structural determinants of ion flux have been identified flanking the membrane-spanning region. Using x-ray crystallography, sequence analysis, and single-channel recording, we identified a novel determinant of ion conductance near the point of entry of permeant ions. Co-crystallization of acetylcholine-binding protein with sulfate anions revealed coordination of SO4(2-) with a ring of lysines at a position equivalent to 24 A above the lipid membrane in homologous Cys-loop receptors. Analysis of multiple sequence alignments revealed that residues equivalent to the ring of lysines are negatively charged in cation-selective receptors but are positively charged in anion-selective receptors. Charge reversal of side chains at homologous positions in the nicotinic receptor from the motor end plate decreases unitary conductance up to 80%. Selectivity filters stemming from transmembrane alpha-helices have similar pore diameters and compositions of amino acids. These findings establish that when the channel opens under a physiological electrochemical gradient, permeant ions are initially stabilized within the extracellular vestibule of Cys-loop receptors, and this stabilization is a major determinant of ion conductance.

Molecular physiology of renal ClC chloride channels/transporters

Recent findings relevant to the renal ClC chloride channels/transporters are reviewed with a focus on structure-function relationships, regulation of trafficking, role in blood pressure control, and pharmacology. The ClC proteins include plasma membrane Cl channels and vesicular Cl/H exchangers. Recent experiments have revealed further details regarding the structure and mechanism of the permeation path. X-ray crystallographic and electrophysiological studies have identified two glutamate residues required for gated Cl movement and proton permeation in bacterial and two mammalian (ClC-4, ClC-5) ClC transporters. In renal ClC channels (ClC-Ka, ClC-Kb), both glutamate residues are replaced by valine, leading to speculation about critical differences between transporter and channel members of the ClC family. New information about the physiological regulation of renal ClC proteins has implicated the Nedd4 ubiquitin ligases and serum and glucocorticoid-inducible kinases in controlling functional levels of ClC-5 and ClC-K/barttin in renal cells. ClC proteins are critical for many clinically relevant physiological events. New insights into fundamental structure-function relationships, mechanisms of ion translocation, cellular regulation, and roles in human disease have increased attention on ClC proteins as important potential therapeutic targets.

PH-induced structural change in a sodium/proton antiporter from Methanococcus jannaschii

Na+/H+ antiporters are pH-dependent membrane transport proteins that maintain the homeostasis of H+ and Na+ in living cells. MjNhaP1 from Methanococcus jannaschii, a hyperthermophilic archaeon that grows optimally at 85 degrees C, was cloned and expressed in Escherichia coli. Two-dimensional crystals were obtained from purified protein at pH 4. Electron cryomicroscopy yielded an 8 A projection map. Like the related E. coli antiporter NhaA, MjNhaP1 is a dimer, but otherwise the structures of the two antiporters differ significantly. The map of MjNhaP1 shows elongated densities in the centre of the dimer and a cluster of density peaks on either side of the dimer core, indicative of a bundle of 4-6 membrane-spanning helices. The effect of pH on the structure of MjNhaP1 was studied in situ. A major change in density distribution within the helix bundle, and an approximately 2 A shift in the position of the helix bundle relative to the dimer core occurred at pH 6 and above. The two conformations at low and high pH most likely represent the closed and open states of the antiporter.