Transmembrane Charge Transfer Research Articles

Biomedical Implants with Charge-Transfer Monitoring and Regulating Abilities.

Transmembrane charge (ion/electron) transfer is essential for maintaining cellular homeostasis and is involved in many biological processes, from protein synthesis to embryonic development in organisms. Designing implant devices that can detect or regulate cellular transmembrane charge transfer is expected to sense and modulate the behaviors of host cells and tissues. Thus, charge transfer can be regarded as a bridge connecting living systems and human‐made implantable devices. This review describes the mode and mechanism of charge transfer between organisms and nonliving materials, and summarizes the strategies to endow implants with charge‐transfer regulating or monitoring abilities. Furthermore, three major charge‐transfer controlling systems, including wired, self‐activated, and stimuli‐responsive biomedical implants, as well as the design principles and pivotal materials are systematically elaborated. The clinical challenges and the prospects for future development of these implant devices are also discussed.

Control of transmembrane charge transfer in cytochrome c oxidase by the membrane potential

The respiratory chain in mitochondria is composed of membrane-bound proteins that couple electron transfer to proton translocation across the inner membrane. These charge-transfer reactions are regulated by the proton electrochemical gradient that is generated and maintained by the transmembrane charge transfer. Here, we investigate this feedback mechanism in cytochrome c oxidase in intact inner mitochondrial membranes upon generation of an electrochemical potential by hydrolysis of ATP. The data indicate that a reaction step that involves proton uptake to the catalytic site and presumably proton translocation is impaired by the potential, but electron transfer is not affected. These results define the order of electron and proton-transfer reactions and suggest that the proton pump is regulated by the transmembrane electrochemical gradient through control of internal proton transfer rather than by control of electron transfer.

Control of transmembrane charge transfer in cytochrome c oxidase by the membrane potential

The respiratory chain in mitochondria is composed of membrane-bound proteins that couple electron transfer to proton translocation across the inner membrane. These charge-transfer reactions are regulated by the proton electrochemical gradient that is generated and maintained by the transmembrane charge transfer. Here, we investigate this feedback mechanism in cytochrome c oxidase in intact inner mitochondrial membranes upon generation of an electrochemical potential by hydrolysis of ATP. The data indicate that a reaction step that involves proton uptake to the catalytic site and presumably proton translocation is impaired by the potential, but electron transfer is not affected. These results define the order of electron and proton-transfer reactions and suggest that the proton pump is regulated by the transmembrane electrochemical gradient through control of internal proton transfer rather than by control of electron transfer.

The soluble loop BC region guides, but not dictates, the assembly of the transmembrane cytochrome b6.

Studying folding and assembly of naturally occurring α-helical transmembrane proteins can inspire the design of membrane proteins with defined functions. Thus far, most studies have focused on the role of membrane-integrated protein regions. However, to fully understand folding pathways and stabilization of α–helical membrane proteins, it is vital to also include the role of soluble loops. We have analyzed the impact of interhelical loops on folding, assembly and stability of the heme-containing four-helix bundle transmembrane protein cytochrome b6 that is involved in charge transfer across biomembranes. Cytochrome b6 consists of two transmembrane helical hairpins that sandwich two heme molecules. Our analyses strongly suggest that the loop connecting the helical hairpins is not crucial for positioning the two protein “halves” for proper folding and assembly of the holo-protein. Furthermore, proteolytic removal of any of the remaining two loops, which connect the two transmembrane helices of a hairpin structure, appears to also not crucially effect folding and assembly. Overall, the transmembrane four-helix bundle appears to be mainly stabilized via interhelical interactions in the transmembrane regions, while the soluble loop regions guide assembly and stabilize the holo-protein. The results of this study might steer future strategies aiming at designing heme-binding four-helix bundle structures, involved in transmembrane charge transfer reactions.

Gating Charge Calculations: Probing Voltage-Sensing Proteins through Computational Electrophysiology

Sensitivity to voltage stimuli is a defining property of electrical excitability and is found in ion channels with dedicated voltage sensors, but also in other channels, transporters and even metabotropic receptors. Voltage dependence is conferred by an effective transmembrane charge transfer that can be attributed to global or localized conformational changes, ion or ligand binding, protonation or changes in water accessibility that reshape the electric field.The Computational Electrophysiology protocol, CompEl, (Kutzner et al., Biophys J. 2011), introduced into GROMACS (Abraham et al., 2015, SoftwareX), allows for simulations of realistic transmembrane potentials by all-atom MD. CompEl generates well-defined ion driving forces by maintaining slight ion number differences between two aqueous compartments in a two-bilayer simulation system and has proven successful in the investigation of ion conduction and selectivity of various ion channels (Kopfer et al., Science 2014; Machtens et al., Cell 2015).Gating charge calculations provide insights into molecular bases of voltage sensing and the comparison with experiments furthermore allows refining models of protein structures. We here present an efficient protocol for gating charge calculations using CompEl. Using an antiparallel, symmetric system and considering the bilayer as a capacitor, we simulate the process of membrane charging by ions to investigate the resulting V-Δq relationship. Thereby we directly determine the gating charge and the change in capacitance associated with any conformational change, taking all possible causes of voltage sensitivity explicitly into account. The decomposition of the gating charges into additive contributions from, e.g., protein, water or solutes, permits direct mechanistic insights into the utilized mechanisms of voltage sensing. The contribution of individual side chains to the total gating charge can be easily determined. We illustrate the method on the K+ channel Kv1.2 and the voltage sensor of the voltage-sensitive phosphate Ci-VSP.

Halothane Inhibition of Recombinant Cardiac L-type Ca2+ Channels Expressed in HEK-293 Cells

Volatile anesthetics depress cardiac contractility, which involves inhibition of cardiac L-type calcium channels. To explore the role of voltage-dependent inactivation, the authors analyzed halothane effects on recombinant cardiac L-type calcium channels (alpha1Cbeta2a and alpha1Cbeta2aalpha2/delta1), which differ by the alpha2/delta1 subunit and consequently voltage-dependent inactivation. HEK-293 cells were transiently cotransfected with complementary DNAs encoding alpha1C tagged with green fluorescent protein and beta2a, with and without alpha2/delta1. Halothane effects on macroscopic barium currents were recorded using patch clamp methodology from cells expressing alpha1Cbeta2a and alpha1Cbeta2aalpha2/delta1 as identified by fluorescence microscopy. Halothane inhibited peak current (I(peak)) and enhanced apparent inactivation (reported by end pulse current amplitude of 300-ms depolarizations [I300]) in a concentration-dependent manner in both channel types. alpha2/delta1 coexpression shifted relations leftward as reported by the 50% inhibitory concentration of I(peak) and I300/I(peak)for alpha1Cbeta2a (1.8 and 14.5 mm, respectively) and alpha1Cbeta2aalpha2/delta1 (0.74 and 1.36 mm, respectively). Halothane reduced transmembrane charge transfer primarily through I(peak) depression and not by enhancement of macroscopic inactivation for both channels. The results indicate that phenotypic features arising from alpha2/delta1 coexpression play a key role in halothane inhibition of cardiac L-type calcium channels. These features included marked effects on I(peak) inhibition, which is the principal determinant of charge transfer reductions. I(peak) depression arises primarily from transitions to nonactivatable states at resting membrane potentials. The findings point to the importance of halothane interactions with states present at resting membrane potential and discount the role of inactivation apparent in current time courses in determining transmembrane charge transfer.

Halothane Inhibition of Recombinant Cardiac L-type Ca2+ Channels Expressed in HEK-293 Cells

Background. Volatile anesthetics depress cardiac contractility, which involves inhibition of cardiac L-type calcium channels. To explore the role of voltage-dependent inactivation, the authors analyzed halothane effects on recombinant cardiac L-type calcium channels (alpha1Cbeta2a and alpha1Cbeta2aalpha2/delta1), which differ by the alpha2/delta1 subunit and consequently voltage-dependent inactivation.Methods. HEK-293 cells were transiently cotransfected with complementary DNAs encoding alpha1C tagged with green fluorescent protein and beta2a, with and without alpha2/delta1. Halothane effects on macroscopic barium currents were recorded using patch clamp methodology from cells expressing alpha1Cbeta2a and alpha1Cbeta2aalpha2/delta1 as identified by fluorescence microscopy.Results. Halothane inhibited peak current (I(peak)) and enhanced apparent inactivation (reported by end pulse current amplitude of 300-ms depolarizations [I300]) in a concentration-dependent manner in both channel types. alpha2/delta1 coexpression shifted relations leftward as reported by the 50% inhibitory concentration of I(peak) and I300/I(peak)for alpha1Cbeta2a (1.8 and 14.5 mm, respectively) and alpha1Cbeta2aalpha2/delta1 (0.74 and 1.36 mm, respectively). Halothane reduced transmembrane charge transfer primarily through I(peak) depression and not by enhancement of macroscopic inactivation for both channels.Conclusions. The results indicate that phenotypic features arising from alpha2/delta1 coexpression play a key role in halothane inhibition of cardiac L-type calcium channels. These features included marked effects on I(peak) inhibition, which is the principal determinant of charge transfer reductions. I(peak) depression arises primarily from transitions to nonactivatable states at resting membrane potentials. The findings point to the importance of halothane interactions with states present at resting membrane potential and discount the role of inactivation apparent in current time courses in determining transmembrane charge transfer.

Photoelectric studies of the transmembrane charge transfer reactions in photosystem I pigment-protein complexes

The results of studies of charge transfer in cyanobacterial photosystem I (PS I) using the photoelectric method are reviewed. The electrogenicity in the PS I complex and its interaction with natural donors (plastocyanin, cytochrome c6), natural acceptors (ferredoxin, flavodoxin), or artificial acceptors and donors (methyl viologen and other redox dyes) were studied. The operating dielectric constant values in the vicinity of the charge transfer carriers in situ were calculated. The profile of distribution of the dielectric constant along the PS I pigment–protein complex (from plastocyanin or cytochrome c6 through the chlorophyll dimer P700 to the acceptor complex) was estimated, and possible mechanisms of correlation between the local dielectric constant and electron transfer rate constant were discussed.

Scanning electrochemical microscopy of living cells. 3. Rhodobacter sphaeroides.

The scanning electrochemical microscope (SECM) was used to probe the redox activity of individual purple bacteria (Rhodobacter sphaeroides). The approaches developed in our previous studies of mammalian cells were expanded to measure the rates and investigate the pathway of transmembrane charge transfer in bacteria. The two groups of redox mediators (i.e., hydrophilic and hydrophobic redox species) were used to shuttle the electrons between the SECM tip electrode in solution and the redox centers inside the cell. The analysis of the dependencies of the measured rate constant on formal potential and concentration of mediator species in solution yielded information about the permeability of the outer cell membrane to different ionic species and intracellular redox properties. The maps of redox reactivity of the cell surface were obtained with a micrometer or submicrometer spatial resolution.

Coupling of proton flow to ATP synthesis in Rhodobacter capsulatus: F 0F 1-ATP synthase is absent from about half of chromatophores

F 0F 1-ATP synthase (H +-ATP synthase, F 0F 1) utilizes the transmembrane protonmotive force to catalyze the formation of ATP from ADP and inorganic phosphate (P i). Structurally the enzyme consists of a membrane-embedded proton-translocating F 0 portion and a protruding hydrophilic F 1 part that catalyzes the synthesis of ATP. In photosynthetic purple bacteria a single turnover of the photosynthetic reaction centers (driven by a short saturating flash of light) generates protonmotive force that is sufficiently large to drive ATP synthesis. Using isolated chromatophore vesicles of Rhodobacter capsulatus, we monitored the flash induced ATP synthesis (by chemoluminescence of luciferin/luciferase) in parallel to the transmembrane charge transfer through F 0F 1 (by following the decay of electrochromic bandshifts of intrinsic carotenoids). With the help of specific inhibitors of F 1 (efrapeptin) and of F 0 (venturicidin), we decomposed the kinetics of the total proton flow through F 0F 1 into (i) those coupled to the ATP synthesis and (ii) the de-coupled proton escape through F 0. Taking the coupled proton flow, we calculated the H +/ATP ratio; it was found to be 3.3±0.6 at a large driving force (after one saturating flash of light) but to increase up to 5.1±0.9 at a smaller driving force (after a half-saturating flash). From the results obtained, we conclude that our routine chromatophore preparations contained three subsets of chromatophore vesicles. Chromatophores with coupled F 0F 1 dominated in fresh material. Freezing/thawing or pre-illumination in the absence of ADP and P i led to an increase in the fraction of chromatophores with at least one de-coupled F 0(F 1). The disclosed fraction of chromatophores that lacked proton-conducting F 0(F 1) (approx. 40% of the total amount) remained constant upon these treatments.

Noninvasive Probing of Redox Reactions in Single Living Cells by Scanning Electrochemical Microscopy (SECM)

We reported recently the first application of the scanning electrochemical microscope (SECM) to probe the redox activity of individual living cells. The possibilities of measuring the rate and investigating the pathway of transmembrane charge transfer (CT) were demonstrated. Significant differences were detected in the redox responses given by normal human breast epithelial cells (MCF-10A) and metastatic breast cancer cells (MDA-MB-231). Here, we demonstrate that the intracellular redox potentials and concentrations of redox components in the cell can be evaluated noninvasively by the SECM. Such measurements provide mechanistic explanation of the observed differences in cell redox activities.In SECM, a microscopic electrochemical sensor, usually called the tip, is scanned over the surface of a sample, and topographic images and maps of chemical reactivity across the surface are obtained. The schematic diagram of such measurements is shown in Figure 1. The oxidized (or reduced) form of redox species initially present in solution reacts at the surface of the ultramicroelectrode tip.

Scanning electrochemical microscopy of living cells: different redox activities of nonmetastatic and metastatic human breast cells.

Electrochemical methods have been widely used to monitor physiologically important molecules in biological systems. This report describes the first application of the scanning electrochemical microscope (SECM) to probe the redox activity of individual living cells. The possibilities of measuring the rate and investigating the pathway of transmembrane charge transfer are demonstrated. By this approach, significant differences are detected in the redox responses given by nonmotile, nontransformed human breast epithelial cells, breast cells with a high level of motility (engendered by overexpression of protein kinase Calpha), and highly metastatic breast cancer cells. SECM analysis of the three cell lines reveals reproducible differences with respect to the kinetics of charge transfer by several redox mediators.

Mechanism and kinetic analysis of the photo-induced electron transfer mediated by a stacked metallotriporphyrin in planar lipid bilayers

The photo-induced transmembrane charge transfer across planar lipid bilayers sensitized by a stacked ZnCuZn triporphyrin and enclosed between two aqueous phases is shown to occur via monomers spanning the membrane. The first step of the proposed mechanism involves the oxidation of the excited state of the porphyrin ring which faces the aqueous oxidizing phase. The positive charge then shifts from the resulting radical cation toward the porphyrin ring facing the reducing phase. This mechanism for the intramolecular electron transfer is preferred to the one based on the formation of a charge-separated state such as P +-P −-P. The reaction scheme selected is supported by fluorescence and UV-visible absorption data. The kinetic equations derived fit the experimental time course of the photocurrents and photopotentials. A numerical simulation provides a lower estimation of the rate constant of the photo-induced intramolecular electron-transfer step.

Mechanism and kinetic analysis of the photo-induced electron transfer mediated by a stacked metallotriporphyrin in planar lipid bilayers

The photo-induced transmembrane charge transfer across planar lipid bilayers sensitized by a stacked ZnCuZn triporphyrin and enclosed between two aqueous phases is shown to occur via monomers spanning the membrane. The first step of the proposed mechanism involves the oxidation of the excited state of the porphyrin ring which faces the aqueous oxidizing phase. The positive charge then shifts from the resulting radical cation toward the porphyrin ring facing the reducing phase. This mechanism for the intramolecular electron transfer is preferred to the one based on the formation of a charge-separated state such as P+—P−—P. The reaction scheme selected is supported by fluorescence and UV—visible absorption data. The kinetic equations derived fit the experimental time course of the photocurrents and photopotentials. A numerical simulation provides a lower estimation of the rate constant of the photo-induced intramolecular electron-transfer step.

Electrical conductivity, transfer of hydrogen ions in lipid bilayer membranes and uncoupling effect induced by pentachlorobenzenethiol (pentachlorothiophenol).

Pentachlorobenzenethiol (PCBT) has been considered an anomalous uncoupler. It was reported as active in mitochondria, but not effective in inducing electrical conductivity in lipid bilayer membranes. We have overcome the experimental difficulties associated with accurate determination of the induced conductivity. The main contributing factors to the difficulties, we discovered, are the photolability and the low solubility of the compound in aqueous medium. We have conclusively demonstrated that PCBT does induce conductivity in lipid bilayers and compared this conductance with its uncoupling activity reported by other investigators in the literature. We present the results of steady-state current-voltage measurements: conductance dependence on applied voltage for various values of pH, buffer strength and PCBT concentration, as well as the dependence of the conductance on pH, buffer strength and PCBT concentration in the limit of zero applied voltage. We have also compared the above results with those obtained previously with pentachlorophenol. Our experimental results on PCBT-induced membrane conductance suggest that PCBT belongs to class II uncouplers and that "disulfide dimer" of PCBT is membrane inactive. Thus the replacement of oxygen in molecular structure of pentachlorophenol (R-OH) by sulfur (R-SH) does not change the protonophoretic activity of the compound. The conductivity of a membrane is due to PCBT-induced hydrogen ion transfer and it was found to be limited by the kinetics of reactions coupled to transmembrane charge transfer.(ABSTRACT TRUNCATED AT 250 WORDS)

On the mechanism of photosynthetic electron transfer in Rhodopseudomonas capsulata and Rhodopseudomonas sphaeroides

(1) Current models for the mechanism of cyclic electron transport in Rhodopseudomonas sphaeroides and Rhodopseudomonas capsulata have been investigated by observing the kinetics of electron transport in the presence of inhibitors, or in photosynthetically incompetent mutant strains. (2) In addition to its well-characterized effect on the Rieske-type iron sulfur center, 5-( n-undecyl)-6-hydroxy-4,7-dioxobenzothiazole (UHDBT) inhibits both cytochrome b 50 and cytochrome b −90 reduction induced by flash excitation in Rps. sphaeroides and Rps. capsulata. The concentration dependency of the inhibition in the presence of antimycin (approx. 2.7 mol UHDBT/mol reaction center for 50% inhibition of extent) is very similar to that of its inhibition of the antimycin-insensitive phase of ferricytochrome c re-reduction. UHDBT did not inhibit electron transfer between the reduced primary acceptor ubiquinone (Q ⨪ I) and the secondary acceptor ubiquinone (Q II) of the reaction center acceptor complex. A mutant of Rps. capsulata, strain R126, lacked both the UHDBT and antimycin-sensitive phases of cytochrome c re-reduction, and ferricytochrome b 50 reduction on flash excitation. (3) In the presence of antimycin, the initial rate of cytochrome b 50 reduction increased about 10-fold as the E h(7.0) was lowered below 180 mV. A plot of the rate at the fastest point in each trace against redox potential resembles the Nernst plot for a two-electron carrier with E m(7.0) ≈ 125 ± 15 mV. Following flash excitation there was a lag of 100–500 μs before cytochrome b 50 reduction began. However, there was a considerably longer lag before significant reduction of cytochrome c by the antimycin-sensitive pathway occurred. (4) The herbicide ametryne inhibited electron transfer between Q ⨪ I and Q II. It was an effective inhibitor of cytochrome b 50 photoreduction at E h(7.0) 390 mV, but not at E h(7.0) 100 mV. At the latter E h, low concentrations of ametryne inhibited turnover after one flash in only half of the photochemical reaction centers. By analogy with the response to o-phenanthroline, it is suggested that ametryne is ineffective at inhibiting electron transfer from Q ⨪ I to the secondary acceptor ubiquinone when the latter is reduced to the semiquinone form before excitation. (5) At E h(7.0) > 200 mV, antimycin had a marked effect on the cytochrome b 50 reduction-oxidation kinetics but not on the cytochrome c and reaction center changes or the slow phase III of the electrochromic carotenoid change on a 10-ms time scale. This observation appears to rule out a mechanism in which cytochrome b 50 oxidation is obligatorily and kinetically linked to the antimycin-sensitive phase of cytochrome c reduction in a reaction involving transmembrane charge transfer at high E h values. However, at lower redox potentials, cytochrome b 50 oxidation is more rapid, and may be linked to the antimycin-sensitive reduction of cytochrome c. (6) It is concluded that neither a simple linear scheme nor a simple Q-cycle model can account adequately for all the observations. Future models will have to take account of a possible heterogeneity of redox chains resulting from the two-electron gate at the level of the secondary quinone, and of the involvement of cytochrome b −90 in the rapid reactions of the cyclic electron transfer chain