Bioinspired Artificial Ion Pumps.

Ion pumps are important membrane-spanning transporters that pump ions against the electrochemical gradient across the cell membrane. In biological systems, ion pumping is essential to maintain intracellular osmotic pressure, to respond to external stimuli, and to regulate physiological activities by consuming adenosine triphosphate. In recent decades, artificial ion pumping systems with diverse geometric structures and functions have been developing rapidly with the progress of advanced materials and nanotechnology. In this Review, bioinspired artificial ion pumps, including four categories: asymmetric structure-driven ion pumps, pH gradient-driven ion pumps, light-driven ion pumps, and electron-driven ion pumps, are summarized. The working mechanisms, functions, and applications of those artificial ion pumping systems are discussed. Finally, a brief conclusion of underpinning challenges and outlook for future research are tentatively discussed.

Engineering Smart Nanofluidic Systems for Artificial Ion Channels and Ion Pumps: From Single-Pore to Multichannel Membranes.

Biological ion channels and ion pumps with intricate ion transport functions widely exist in living organisms and play irreplaceable roles in almost all physiological functions. Nanofluidics provides exciting opportunities to mimic these working processes, which not only helps understand ion transport in biological systems but also paves the way for the applications of artificial devices in many valuable areas. Recent progress in the engineering of smart nanofluidic systems for artificial ion channels and ion pumps is summarized. The artificial systems range from chemically and structurally diverse lipid-membrane-based nanopores to robust and scalable solid-state nanopores. A generic strategy of gate location design is proposed. The single-pore-based platform concept can be rationally extended into multichannel membrane systems and shows unprecedented potential in many application areas, such as single-molecule analysis, smart mass delivery, and energy conversion. Finally, some present underpinning issues that need to be addressed are discussed.

An Electrically Actuated, Carbon-Nanotube-Based Biomimetic Ion Pump.

Single-walled carbon nanotubes (SWCNTs) are well-established transporters of electronic current, electrolyte, and ions. In this work, we demonstrate an electrically actuated biomimetic ion pump by combining these electronic and nanofluidic transport capabilities within an individual SWCNT device. Ion pumping is driven by a solid-state electronic input, as Coulomb drag coupling transduces electrical energy from solid-state charge along the SWCNT shell to electrolyte inside the SWCNT core. Short-circuit ionic currents, measured without an electrolyte potential difference, exceed 1 nA and scale larger with increasing ion concentrations through 1 M, demonstrating applicability under physiological (∼140 mM) and saltwater (∼600 mM) conditions. The interlayer coupling allows ionic currents to be tuned with the source-drain potential difference and electronic currents to be tuned with the electrolyte potential difference. This combined electronic-nanofluidic SWCNT device presents intriguing applications as a biomimetic ion pump or component of an artificial membrane.

Structural dynamics of P-type ATPase ion pumps.

P-type ATPases transport ions across biological membranes against concentration gradients and are essential for all cells. They use the energy from ATP hydrolysis to propel large intramolecular movements, which drive vectorial transport of ions. Tight coordination of the motions of the pump is required to couple the two spatially distant processes of ion binding and ATP hydrolysis. Here, we review our current understanding of the structural dynamics of P-type ATPases, focusing primarily on Ca2+ pumps. We integrate different types of information that report on structural dynamics, primarily time-resolved fluorescence experiments including single-molecule Förster resonance energy transfer and molecular dynamics simulations, and interpret them in the framework provided by the numerous crystal structures of sarco/endoplasmic reticulum Ca2+-ATPase. We discuss the challenges in characterizing the dynamics of membrane pumps, and the likely impact of new technologies on the field.

The natural history of Get3-like chaperones.

Get3 in yeast or TRC40 in mammals is an ATPase that, in eukaryotes, is a central element of the GET or TRC pathway involved in the targeting of tail-anchored proteins. Get3 has also been shown to possess chaperone holdase activity. A bioinformatic assessment was performed across all domains of life on functionally important regions of Get3 including the TRC40-insert and the hydrophobic groove essential for tail-anchored protein binding. We find that such a hydrophobic groove is much more common in bacterial Get3 homologs than previously appreciated based on a directed comparison of bacterial ArsA and yeast Get3. Furthermore, our analysis shows that the region containing the TRC40-insert varies in length and methionine content to an unexpected extent within eukaryotes and also between different phylogenetic groups. In fact, since the TRC40-insert is present in all domains of life, we suggest that its presence does not automatically predict a tail-anchored protein targeting function. This opens up a new perspective on the function of organellar Get3 homologs in plants which feature the TRC40-insert but have not been demonstrated to function in tail-anchored protein targeting. Our analysis also highlights a large diversity of the ways Get3 homologs dimerize. Thus, based on the structural features of Get3 homologs, these proteins may have an unexplored functional diversity in all domains of life.

Artificial light-driven ion pump for photoelectric energy conversion.

Biological light-driven ion pumps move ions against a concentration gradient to create a membrane potential, thus converting sunlight energy directly into an osmotic potential. Here, we describe an artificial light-driven ion pump system in which a carbon nitride nanotube membrane can drive ions thermodynamically uphill against an up to 5000-fold concentration gradient by illumination. The separation of electrons and holes in the membrane under illumination results in a transmembrane potential which is thought to be the foundation for the pumping phenomenon. When used for harvesting solar energy, a sustained open circuit voltage of 550 mV and a current density of 2.4 µA/cm2 can reliably be generated, which can be further scaled up through series and parallel circuits of multiple membranes. The ion transport based photovoltaic system proposed here offers a roadmap for the development of devices by using simple, cheap, and stable polymeric carbon nitride.

Quantum Modeling: A Bridge between the Pumping and Signaling Functions of Na/K-ATPase.

Although the signaling function of Na/K-ATPase has been studied for decades, the chasm between the pumping function and the signaling function of Na/K-ATPase is still an open issue. This article explores the relationship between ion pumping and signaling with attention to the amplification of oxidants through this signaling function. We specifically consider the Na/K-ATPase with respect to its signaling function as a superposition of different states described for its pumping function. We then examine how alterations in the relative amounts of these states could alter signaling through the Src-EGFR-ROS pathway. Using assumptions based on some experimental observations published by our laboratories and others, we develop some predictions regarding cellular oxidant stress.

Primary Transfer Step in the Light-Driven Ion Pump Bacteriorhodopsin: An Irreversible U-Turn Revealed by Dynamic Nuclear Polarization-Enhanced Magic Angle Spinning NMR.

Despite much attention, the path of the highly consequential primary proton transfer in the light-driven ion pump bacteriorhodopsin (bR) remains mysterious. Here we use DNP-enhanced magic angle spinning (MAS) NMR to study critical elements of the active site just before the Schiff base (SB) deprotonates (in the L intermediate), immediately after the SB has deprotonated and Asp85 has become protonated (in the Mo intermediate), and just after the SB has reprotonated and Asp96 has deprotonated (in the N intermediate). An essential feature that made these experiments possible is the 75-fold signal enhancement through DNP. 15N(SB)-1H correlations reveal that the newly deprotonated SB is accepting a hydrogen bond from an alcohol and 13C-13C correlations show that Asp85 draws close to Thr89 before the primary proton transfer. Concurrently, 15N-13C correlations between the SB and Asp85 show that helices C and G draw closer together just prior to the proton transfer and relax thereafter. Together, these results indicate that Thr89 serves to relay the SB proton to Asp85 and that creating this pathway involves rapprochement between the C and G helices as well as chromophore torsion.

Bioinspired Heterogeneous Ion Pump Membranes: Unidirectional Selective Pumping and Controllable Gating Properties Stemming from Asymmetric Ionic Group Distribution.

The creation of an artificial solid-state ion pump that mimics the delicate ion transport behaviors of a biological protein-based ion pump is drawing more and more research attention due to its potential applications in energy conversion, biosensor, and desalination. However, the reported bioinspired double-gated ion pump systems are generally very primary and can only realize nonselective ion pumping functions with no directionality and uncontrollable ion gating functions, which are far from their biological counterparts. To make the bioinspired device "smart" in a real sense, the implementation of high-level selectivity and directionality in the ion pumping process, while achieving great controllability in the ion gating process, is a necessity. Here, we developed a bioinspired heterogeneous ion pump membrane by combining block copolymer membrane sacrificial coating and plasma grafting technique. The system has unidirectional selective ion pumping and controllable ion gating properties. The introduction of asymmetric ionic group distribution is the key reason for its novel transport behaviors. Such a heterogeneous ion pump could not only provide a basic platform that potentially sparks further efforts to simulate the smart ion transport processes in living bodies but also promote the application of artificial nanofluidic devices in energy conversion, water treatment, and biosensing.

Low-temperature FTIR spectroscopy provides evidence for protein-bound water molecules in eubacterial light-driven ion pumps.

Light-driven H+, Na+ and Cl- pumps have been found in eubacteria, which convert light energy into a transmembrane electrochemical potential. A recent mutation study revealed asymmetric functional conversion between the two pumps, where successful functional conversions are achieved exclusively when mutagenesis reverses the evolutionary amino acid sequence changes. Although this fact suggests that the essential structural mechanism of an ancestral function is retained even after gaining a new function, questions regarding the essential structural mechanism remain unanswered. Light-induced difference FTIR spectroscopy was used to monitor the presence of strongly hydrogen-bonded water molecules for all eubacterial H+, Na+ and Cl- pumps, including a functionally converted mutant. This fact suggests that the strongly hydrogen-bonded water molecules are maintained for these new functions during evolution, which could be the reason for successful functional conversion from Na+ to H+, and from Cl- to H+ pumps. This also explains the successful conversion of the Cl- to the H+ pump only for eubacteria, but not for archaea. It is concluded that water-containing hydrogen-bonding networks constitute one of the essential structural mechanisms in eubacterial light-driven ion pumps.

Nanoscale Ion Pump Derived from a Biological Water Channel.

Biological molecular machines perform the work of supporting life at the smallest of scales, including the work of shuttling ions across cell boundaries and against chemical gradients. Systems of artificial channels at the nanoscale can likewise control ionic concentration by way of ionic current rectification, species selectivity, and voltage gating mechanisms. Here, we theoretically show that a voltage-gated, ion species-selective, and rectifying ion channel can be built using the components of a biological water channel aquaporin. Through all-atom molecular dynamics simulations, we show that the ionic conductance of a truncated aquaporin channel nonlinearly increases with the bias magnitude, depends on the channel's orientation, and is highly cation specific but only for one polarity of the transmembrane bias. Further, we show that such an unusually complex response of the channel to transmembrane bias arises from mechanical motion of a positively charged gate that blocks cation transport. By combining two truncated aquaporins, we demonstrate a molecular system that pumps ions against their chemical gradients when subject to an alternating transmembrane bias. Our work sets the stage for future biomimicry efforts directed toward reproducing the function of biological ion pumps using synthetic components.

Crystal structure and functional characterization of a light-driven chloride pump having an NTQ motif.

A novel light-driven chloride-pumping rhodopsin (ClR) containing an 'NTQ motif' in its putative ion conduction pathway has been discovered and functionally characterized in a genomic analysis study of a marine bacterium. Here we report the crystal structure of ClR from the flavobacterium Nonlabens marinus S1-08(T) determined under two conditions at 2.0 and 1.56 Å resolutions. The structures reveal two chloride-binding sites, one around the protonated Schiff base and the other on a cytoplasmic loop. We identify a '3 omega motif' formed by three non-consecutive aromatic amino acids that is correlated with the B-C loop orientation. Detailed ClR structural analyses with functional studies in E. coli reveal the chloride ion transduction pathway. Our results help understand the molecular mechanism and physiological role of ClR and provide a structural basis for optogenetic applications.

Asymmetric Functional Conversion of Eubacterial Light-driven Ion Pumps.

In addition to the well-known light-driven outward proton pumps, novel ion-pumping rhodopsins functioning as outward Na(+) and inward Cl(-) pumps have been recently found in eubacteria. They convert light energy into transmembrane electrochemical potential difference, similar to the prototypical archaeal H(+) pump bacteriorhodopsin (BR) and Cl(-) pump halorhodopsin (HR). The H(+), Na(+), and Cl(-) pumps possess the conserved respective DTE, NDQ, and NTQ motifs in the helix C, which likely serve as their functional determinants. To verify this hypothesis, we attempted functional interconversion between selected pumps from each category by mutagenesis. Introduction of the proton-pumping motif resulted in successful Na(+) → H(+) functional conversion. Introduction of the respective characteristic motifs with several additional mutations leads to successful Na(+) → Cl(-) and Cl(-) → H(+) functional conversions, whereas remaining conversions (H(+) → Na(+), H(+) → Cl(-), Cl(-) → Na(+)) were unsuccessful when mutagenesis of 4-6 residues was used. Phylogenetic analysis suggests that a H(+) pump is the common ancestor of all of these rhodopsins, from which Cl(-) pumps emerged followed by Na(+) pumps. We propose that successful functional conversions of these ion pumps are achieved exclusively when mutagenesis reverses the evolutionary amino acid sequence changes. Dependence of the observed functional conversions on the direction of evolution strongly suggests that the essential structural mechanism of an ancestral function is retained even after the gain of a new function during natural evolution, which can be evoked by a few mutations. By contrast, the gain of a new function needs accumulation of multiple mutations, which may not be easily reproduced by limited mutagenesis in vitro.

Hybrid integrated biological-solid-state system powered with adenosine triphosphate.

There is enormous potential in combining the capabilities of the biological and the solid state to create hybrid engineered systems. While there have been recent efforts to harness power from naturally occurring potentials in living systems in plants and animals to power complementary metal-oxide-semiconductor integrated circuits, here we report the first successful effort to isolate the energetics of an electrogenic ion pump in an engineered in vitro environment to power such an artificial system. An integrated circuit is powered by adenosine triphosphate through the action of Na(+)/K(+) adenosine triphosphatases in an integrated in vitro lipid bilayer membrane. The ion pumps (active in the membrane at numbers exceeding 2 × 10(6) mm(-2)) are able to sustain a short-circuit current of 32.6 pA mm(-2) and an open-circuit voltage of 78 mV, providing for a maximum power transfer of 1.27 pW mm(-2) from a single bilayer. Two series-stacked bilayers provide a voltage sufficient to operate an integrated circuit with a conversion efficiency of chemical to electrical energy of 14.9%.

An artificial molecular pump.

Carrier proteins consume fuel in order to pump ions or molecules across cell membranes, creating concentration gradients. Their control over diffusion pathways, effected entirely through noncovalent bonding interactions, has inspired chemists to devise artificial systems that mimic their function. Here, we report a wholly artificial compound that acts on small molecules to create a gradient in their local concentration. It does so by using redox energy and precisely organized noncovalent bonding interactions to pump positively charged rings from solution and ensnare them around an oligomethylene chain, as part of a kinetically trapped entanglement. A redox-active viologen unit at the heart of a dumbbell-shaped molecular pump plays a dual role, first attracting and then repelling the rings during redox cycling, thereby enacting a flashing energy ratchet mechanism with a minimalistic design. Our artificial molecular pump performs work repetitively for two cycles of operation and drives rings away from equilibrium toward a higher local concentration.

A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters.

The last 15 years have been characterized by an explosion in the ability to overexpress and purify membrane proteins from prokaryotic organisms as well as from eukaryotes. This increase has been largely driven by the successful push to obtain structural information on membrane proteins. However, the ability to functionally interrogate these proteins has not advanced at the same rate and is often limited to qualitative assays of limited quantitative value, thereby limiting the mechanistic insights that they can provide. An assay to quantitatively investigate the transport activity of reconstituted Cl(-) channels or transporters is described. The assay is based on the measure of the efflux rate of Cl(-) from proteoliposomes following the addition of the K(+) ionophore valinomycin to shunt the membrane potential. An ion sensitive electrode is used to follow the time-course of ion efflux from proteoliposomes reconstituted with the desired protein. The method is highly suited for mechanistic studies, as it allows for the quantitative determination of key properties of the reconstituted protein, such as its unitary transport rate, the fraction of active protein and the molecular mass of the functional unit. The assay can also be utilized to determine the effect of small molecule compounds that directly inhibit/activate the reconstituted protein, as well as to test the modulatory effects of the membrane composition or lipid-modifying reagents. Where possible, direct comparison between results obtained using this method were found to be in good agreement with those obtained using electrophysiological approaches. The technique is illustrated using CLC-ec1, a CLC-type H(+)/Cl(-) exchanger, as a model system. The efflux assay can be utilized to study any Cl(-) conducting channel/transporter and, with minimal changes, can be adapted to study any ion-transporting protein.

An organic electronic biomimetic neuron enables auto-regulated neuromodulation.

Current therapies for neurological disorders are based on traditional medication and electric stimulation. Here, we present an organic electronic biomimetic neuron, with the capacity to precisely intervene with the underlying malfunctioning signalling pathway using endogenous substances. The fundamental function of neurons, defined as chemical-to-electrical-to-chemical signal transduction, is achieved by connecting enzyme-based amperometric biosensors and organic electronic ion pumps. Selective biosensors transduce chemical signals into an electric current, which regulates electrophoretic delivery of chemical substances without necessitating liquid flow. Biosensors detected neurotransmitters in physiologically relevant ranges of 5-80 µM, showing linear response above 20 µm with approx. 0.1 nA/µM slope. When exceeding defined threshold concentrations, biosensor output signals, connected via custom hardware/software, activated local or distant neurotransmitter delivery from the organic electronic ion pump. Changes of 20 µM glutamate or acetylcholine triggered diffusive delivery of acetylcholine, which activated cells via receptor-mediated signalling. This was observed in real-time by single-cell ratiometric Ca(2+) imaging. The results demonstrate the potential of the organic electronic biomimetic neuron in therapies involving long-range neuronal signalling by mimicking the function of projection neurons. Alternatively, conversion of glutamate-induced descending neuromuscular signals into acetylcholine-mediated muscular activation signals may be obtained, applicable for bridging injured sites and active prosthetics.

Structural basis for Na(+) transport mechanism by a light-driven Na(+) pump.

Krokinobacter eikastus rhodopsin 2 (KR2) is the first light-driven Na(+) pump discovered, and is viewed as a potential next-generation optogenetics tool. Since the positively charged Schiff base proton, located within the ion-conducting pathway of all light-driven ion pumps, was thought to prohibit the transport of a non-proton cation, the discovery of KR2 raised the question of how it achieves Na(+) transport. Here we present crystal structures of KR2 under neutral and acidic conditions, which represent the resting and M-like intermediate states, respectively. Structural and spectroscopic analyses revealed the gating mechanism, whereby the flipping of Asp116 sequesters the Schiff base proton from the conducting pathway to facilitate Na(+) transport. Together with the structure-based engineering of the first light-driven K(+) pumps, electrophysiological assays in mammalian neurons and behavioural assays in a nematode, our studies reveal the molecular basis for light-driven non-proton cation pumps and thus provide a framework that may advance the development of next-generation optogenetics.

Light-driven ion-translocating rhodopsins in marine bacteria.

Microbial rhodopsins are the photoreceptive membrane proteins found in diverse microorganisms from within Archaea, Eubacteria, and eukaryotes. They have a hep-tahelical transmembrane structure that binds to an all-trans retinal chromophore. Since 2000, thousands of proteorhodopsins, genes of light-driven proton pump rhodopsins, have been identified from various species of marine bacteria. This suggests that they are used for the conversion of light into chemical energy, contribut-ing to carbon circulation related to ATP synthesis in the ocean. Furthermore, novel types of rhodopsin (sodium and chloride pumps) have recently been discovered. Here, we review recent progress in our understanding of ion-transporting rhodopsins of marine bacteria, based mainly on biophysical and biochemical research.