A Biological Porin Engineered into a Molecular, Nanofluidic Diode

Miedema, Harald S.; Vrouenraets, Maarten; Wierenga, Jenny; Meijberg, Wim; Robillard, George T.; Eisenberg, Robert S.

doi:10.1021/nl0716808

Cited by 82 publications

(92 citation statements)

References 41 publications

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“…The rectification ratio at 50 mV (the conductance at þ50 mV divided by the conductance at -50 mV) is g þ50 /g -50 ¼ 60 + 10 (n ¼ 6, 1 M KCl, 25 mM Tris HCl, pH 8.0, where the value is likely to be higher than that quoted because of the uncertainty in the leak current associated with the lipid bilayer), which is an order of magnitude higher than the rectification ratio for previously engineered biological diodes 17,18 . An additional advantage of the 7R-aHL pore is that it works over a range of ionic strengths (from 0.2 to 3 M KCl), whereas all previous engineered biological and abiological diodes operate at relatively low ionic strengths (typically, less than 100 mM) [17][18][19][20] . Recent 'nanofluidic diodes' have been prepared from protein 17,18 or abiological [19][20][21] pores by introducing static positive charges at one end of the inner surface of the pore and negative charges at the opposite end.…”

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confidence: 91%

“…An additional advantage of the 7R-aHL pore is that it works over a range of ionic strengths (from 0.2 to 3 M KCl), whereas all previous engineered biological and abiological diodes operate at relatively low ionic strengths (typically, less than 100 mM) [17][18][19][20] . Recent 'nanofluidic diodes' have been prepared from protein 17,18 or abiological [19][20][21] pores by introducing static positive charges at one end of the inner surface of the pore and negative charges at the opposite end. When the pores are filled with a dilute electrolyte, they rectify ionic current by a mechanism that is thought to resemble the charge depletion that occurs at np junctions in semiconductor diodes 22 .…”

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Droplet networks with incorporated protein diodes show collective properties

et al. 2009

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Recently, we demonstrated that submicrolitre aqueous droplets submerged in an apolar liquid containing lipid can be tightly connected by means of lipid bilayers [1][2][3][4][5] to form networks [4][5][6] . Droplet interface bilayers have been used for rapid screening of membrane proteins 7,8 and to form asymmetric bilayers with which to examine the fundamental properties of channels and pores 9 . Networks, meanwhile, have been used to form microscale batteries and to detect light 4 . Here, we develop an engineered protein pore with diode-like properties that can be incorporated into droplet interface bilayers in droplet networks to form devices with electrical properties including those of a current limiter, a half-wave rectifier and a full-wave rectifier. The droplet approach, which uses unsophisticated components (oil, lipid, salt water and a simple pore), can therefore be used to create multidroplet networks with collective properties that cannot be produced by droplet pairs.To obtain directional ionic current flows in droplet networks ( Fig. 1), we constructed a diode-like pore from staphylococcal a-haemolysin (aHL). aHL forms a heptameric protein pore 10 that inserts vectorially into lipid bilayers 11 . The crystal structure of the pore reveals a 14-stranded transmembrane b barrel capped by an extramembraneous domain, which contains a roughly spherical cavity 10 ( Fig. 2a, left). The wild-type (WT) pore is a 'blank slate' for protein engineering with properties similar to those of an electrolyte-filled tube; it is weakly rectifying and weakly anion selective and gates only at extreme applied potentials of either polarity 12 .aHL has been modified by mutagenesis or targeted chemical modification to form pores with a wide range of properties [13][14][15][16] , but none has exhibited sufficient rectification for our purpose. We had, however, noticed that aHL pores with positively charged side chains projecting into the lumen of the transmembrane b barrel tended to gate (open and close) at negative potentials. Therefore, in an attempt to obtain a fully rectifying pore, we tested an extreme version of aHL in which seven residues were replaced with arginines (7R-aHL) to yield a heptameric pore in which 49 additional positively charged side chains were located within the barrel (Fig. 2a, right). In 1 M KCl, 25 mM Tris HCl at pH 8.0, 100 mM, in planar lipid bilayers, the 7R-aHL pore has a unitary conductance of 0.95 + 0.01 nS (þ50 mV, n ¼ 8). The conductance of the WT pore under the same conditions is similar (0.99 + 0.02 nS, n ¼ 4), which suggests, surprisingly, that the drastically altered 7R-aHL pore is properly formed. The current-voltage (I-V) characteristics of 7R-aHL in 1 M KCl, however, showed virtually complete current rectification (Fig. 2b,c). At positive applied potentials, 7R-aHL remained in an open form with a stable steady-state current and infrequent short-lived closures of less than 10 ms. By contrast, at negative applied potentials, the pore was closed, with occasional brief current spikes ascriba...

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confidence: 91%

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Droplet networks with incorporated protein diodes show collective properties

et al. 2009

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“…Several nanofluidic platforms based on nanopores and nanochannels were reported to produce ionic current rectification by symmetry breaking 15 in geometries [16][17][18][19] , surface charge distributions (either intrinsic material properties 20,21 or chemically modified properties 22,23 ), bath concentrations 24 , or a combination of them, for example, by positively and negatively patterning charged regions in conical nanopores 25 . Nevertheless, it has not been possible to change the predefined rectifying properties obtained by these approaches once the devices are made.…”

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Field-effect reconfigurable nanofluidic ionic diodes

2011

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several types of nanofluidic devices based on nanopores and nanochannels have been reported to yield ionic current rectification, with the aim to control the delivery of chemical species in integrated systems. However, the rectifying properties obtained by existing approaches cannot be altered once the devices are made. It would be desirable to have the ability to modulate the predefined properties in situ without introducing external chemical stimuli. Here we report a field-effect reconfigurable nanofluidic diode, with a single asymmetrically placed gate or dual split-gate on top of the nanochannel. The forward/reverse directions of the diode as well as the degrees of rectification can be regulated by the application of gate voltages. Compared with the stimuli-responsive tuning of the rectification properties, the electrostatic modulation offers a fully independent and digitally programmable approach for controlling the preferential conduction of ions and molecules in fluids. This device would serve as a building block for largescale integration of reconfigurable ionic circuits.

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“…The results are of relevance for the mechanisms regulating the transport through ion channels, [17][18][19] for electric signal transduction in cell cycle and embryogenesis, 14,16 and for building ionic circuits in the emerging field of nanofluidics. 6,[20][21][22][23][24][25] The set-up used in the experiments is shown schematically in Fig. 1.…”

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Electrical pumping of potassium ions against an external concentration gradient in a biological ion channel

Queralt-Martín

García-Giménez

Aguilella

et al. 2013

Applied Physics Letters

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We show experimentally and theoretically that significant currents can be obtained with a biological ion channel, the OmpF porin of Escherichia coli, using zero-average potentials as driving forces. The channel rectifying properties can be used to pump potassium ions against an external concentration gradient under asymmetric pH conditions. The results are discussed in terms of the ionic selectivity and rectification ratio of the channel. The physical concepts involved may be applied to separation processes with synthetic nanopores and to bioelectrical phenomena. Ratchet systems have the ability of rectifying unbiased fluctuations into directed transport. 1-3 Both synthetic and biological nanochannels may show non-zero electrical currents driven by zero-average time dependent forces. [4][5][6][7] The electrical rectification is on the basis of the observed phenomena 4,5,8,9 and the potential applications concern separation processes and energy conversion. 10-12 Electrical rectification and ratchet phenomena can also be of relevance for signal averaging of weak electric fields and cell plasticity because ion channels, together with ion pumps and gap junction complexes, play a crucial role in cell communication and control. [13][14][15][16] In this Letter, we present measurements and model calculations of the rectifying properties of a biological ion channel (the outer membrane protein F, OmpF, a porin expressed in Escherichia coli bacteria 17 ). We consider the pH-controlled net current induced by zero-average oscillating potentials and the electrical pumping of potassium ions against an external concentration gradient, a phenomenon previously reported in synthetic nanopores. 4,5 We show that the complex interplay between the current rectification and the channel ionic selectivity is crucial for understanding the uphill transport of ions. The results are of relevance for the mechanisms regulating the transport through ion channels, [17][18][19] for electric signal transduction in cell cycle and embryogenesis, 14,16 and for building ionic circuits in the emerging field of nanofluidics. 6,[20][21][22][23][24][25] The set-up used in the experiments is shown schematically in Fig. 1. A single OmpF ion channel is reconstituted on a planar lipid bilayer and communicates the two halves of a conductivity cell filled with KCl solutions of concentrations c cis (side of protein addition) and c trans . pH cis and pH trans denote the corresponding pH values. A detailed description of the reconstitution procedure can be found elsewhere. 17,26 The electric potential V is positive when it is higher at the trans side of the membrane cell. An Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA) in the voltage-clamp mode is used for measuring both V and the electric current (I) through the channel. I is positive when it flows from solution trans to solution cis in Fig. 1.The OmpF porin contains both basic (positive) and acidic (negative) residues, 27 and thus, the net charge of the channel depends on the ionization state of th...

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A Biological Porin Engineered into a Molecular, Nanofluidic Diode

Cited by 82 publications

References 41 publications

Droplet networks with incorporated protein diodes show collective properties

Droplet networks with incorporated protein diodes show collective properties

Field-effect reconfigurable nanofluidic ionic diodes

Electrical pumping of potassium ions against an external concentration gradient in a biological ion channel

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