Elsevier

Bioelectrochemistry

Volume 78, Issue 2, June 2010, Pages 176-180
Bioelectrochemistry

Short communication
A metal-supported biomimetic micromembrane allowing the recording of single-channel activity and of impedance spectra of membrane proteins

https://doi.org/10.1016/j.bioelechem.2009.08.007Get rights and content

Abstract

A novel tethered bilayer lipid micromembrane (tBLµM) was prepared and characterized. It consists of a mercury cap electrodeposited on a platinum microelectrode, about 20 µm in diameter. The micromembrane was prepared by tethering to the mercury cap a thiolipid monolayer and by then self-assembling a lipid monolayer on top of it. The thiolipid consisted of a disulfidated tetraoxyethylene hydrophilic spacer covalently linked to two phytanyl chains. Upon incorporating OmpF porin in the tBLµM, its single-channel activity was recorded by the patch-clamp technique, and its particular features described. An electrochemical impedance spectrum of the tBLµM incorporating OmpF porin is also reported. To the best of our knowledge, this tBLµM is the first metal-supported biomimetic micromembrane capable of incorporating non-engineered channel proteins in a functionally active state from their detergent solutions, and of allowing the recording of single-channel activity and of impedance spectra of these proteins via ion translocation into the hydrophilic spacer. The limited spaciousness of the spacer prevents a statistical analysis based on current–amplitude or blockage–time histograms. Nonetheless, the robustness, stability, ease of preparation and disposability of the present tBLµM may open the way to the realization of a channel-protein microarray platform allowing a high throughput drug screening.

Introduction

The preparation of rugged lipid bilayers capable of incorporating bulky membrane proteins has been the subject of extensive research. The possibility of using these “biomimetic membranes” for the investigation of the function of membrane proteins and for biosensor applications is of paramount importance. Ion channels are usually investigated in lipid bilayers suspended in a small hole of a Teflon septum (bilayer lipid membranes: BLMs) interposed between two aqueous solutions, or in a high number of holes of a microporous substrate [1], [2]; alternatively, they are investigated in bilayers tethered to a metal support via a hydrophilic “spacer” (tethered bilayer lipid membranes: tBLMs) (Ref. 3 and references therein). Thanks to their particular robustness, tBLMs have potential for biosensor applications. They are obtained by anchoring to the metal surface a “thiolipid” monolayer and by then self-assembling a lipid monolayer on top of it. A particularly convenient thiolipid, first employed by Naumann et al. [4], [5], [6] on a smooth gold substrate and called DPTL, consists of a tetraethyleneoxy (TEO) hydrophilic chain covalently linked at one end to a lipoic acid residue, for anchoring to the metal via a disulfide group, and bound via ether linkages to two phytanyl chains at the other end.

Valinomycin [5] and the channel-forming peptides gramicidin and melittin [6] have been incorporated in Au-supported DPTL|phospholipid tBLMs and their functional activity has been verified. The binding constant of the short L24 peptide on top of these tBLMs was also estimated [7]. However, solid metals such as Au block the lateral movement of the thiolipid molecules, which are linked to the surface atoms of the metal by covalent bonds. It is, therefore, quite difficult to succeed in incorporating bulky channel-forming proteins in Au-supported tBLMs. Moreover, channel-forming proteins must span the whole tBLM, including the thiolipid monolayer, in order to translocate ions into the hydrophilic TEO moiety and to give rise to a resulting current. On the other hand, thanks to its liquid nature, mercury provides a defect-free surface to the self-assembling film, and imparts a high fluidity to the tBLM by allowing the lateral movement of the thiolipid molecules anchored to its surface.

The incorporation of proteins with extramembrane domains requires a significant hydration of the spacer. In this respect, the hydration of the TEO moiety of a DPTL monolayer is definitely higher on an Hg support than on an Au support. A structural and functional characterization of a DPTL monolayer tethered to Au was recently reported by Vockenroth et al. [8] using neutron reflectivity (NR) and electrochemical impedance spectroscopy (EIS). The TEO moiety was found to be only partly hydrated at the more positive potentials. However, at − 0.600 V vs. Ag|AgCl (0.1 M KCl) a pronounced increase in the neutron scattering length density of the spacer was observed, denoting an increased amount of water transferred into this region. Leitch et al. [9] drew similar conclusions using polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS). Thus, the fraction of nonhydrated C6-point double bondO of the lipoic acid ester group was found to be ~ 50% at the more positive potentials and to attain a value of ~ 30% at − 0.600 V, which indicates increasing hydration of the spacer at these negative potentials. Analogous conclusions where also drawn by McGillivray et al. [10] by using NR, EIS and Fourier-transform IRRAS to investigate an Au-supported thiolipid monolayer similar to DPTL. To increase the hydration of the monolayer, this thiolipid was diluted with short β-mercaptoethanol (βME) molecules. This permitted water molecules to be accommodated in the more spacious thiolipid–βME mixture. Gold-supported tBLMs with spacers consisting of mixtures of thiolipids with short-chain thiols have been frequently employed in the literature to incorporate channel-forming peptides [3], also using the DPTL spacer [6].

As distinct from Au-supported tBLMs, Hg-supported tBLMs do not require the use of mixed thiolipid–thiol spacers to incorporate channel-forming peptides and proteins. Thanks to the fluidity imparted to the thiolipid monolayer by the liquid mercury surface, these tBLMs may incorporate bulky proteins such as OmpF porin from Escherichia coli [11] and the HERG potassium channel [12] in a functionally active state. Moreover, upon incorporating gramicidin or valinomicin, the TEO moiety of DPTL in aqueous KCl solution undergoes a conformational change ascribable to its elongation, as the applied potential is stepped from a fixed initial value of − 0.200 V/SCE to a final value of − 0.500 V/SCE [13]. As the final value of the potential step becomes progressively more negative, the charge of K+ ions accommodated in the TEO spacer increases rapidly, attaining a maximum limiting value of about 4.5 × 10 9 C m2 (45 µC cm 2) at − 0.8 V/SCE. This corresponds to three potassium ions per DPTL molecule, denoting an appreciable hydration of the spacer. When comparing interfacial phenomena on different metals, rational potentials should be used, namely potentials referred to the potential of zero charge (pzc) of the given metal in contact with a nonspecifically adsorbed 1,1-valent electrolyte. The pzc equals − 0.435 V/SCE for Hg [14] and − 0.040 V/SCE for polycrystalline Au [15]. Therefore, an appreciable hydration of the TEO moiety, possibly accompanied by its elongation, takes place in the proximity of a rational potential of about zero on Hg, but at a much more negative rational potential of about − 0.600 V on Au, close to the DPTL desorption from this metal.

Besides OpmF porin [11] and the HERG potassium channel [12], the ion carrier valinomycin [16], the channel-forming peptides melittin [17], [18] and gramicidin [19], and the small integral proteins sarcolipin [20] and phospholamban [21] have been incorporated and investigated in DPTL|lipid tBLMs supported by a hanging mercury drop electrode (HMDE). The unique ability of this mercury-supported tBLM to incorporate membrane proteins in a functionally active state led us to fabricate and characterize a solid-supported tethered bilayer lipid microelectrode (tBLµM) exhibiting the same advantageous features. This system allows the recording of single-channel activity of peptides and proteins incorporated in the tBLM and translocating ions into the hydrophilic spacer. This is the subject of the present short communication.

Section snippets

Experimental

The water used was obtained from water produced by an inverted osmosis unit, upon distilling it once and then distilling the water so obtained from alkaline permanganate. Merck (Darmstadt, Germany) Suprapur® KCl was baked at 500 °C before use to remove any organic impurities. Diphytanoylphosphatidylcholine (DPhyPC) was purchased in chloroform solution from Avanti Polar Lipids (Birmingham, AL, U.S.A.). The 2,3,di-O-phytanyl-sn-glycerol-1-tetraethylene-glycol-D,L-α lipoic acid ester lipid (DPTL)

Results and discussion

Fig. 1 shows the current recorded as a function of time at different applied potentials on a tBLµM immersed in aqueous 0.1 M KCl, after incorporating OmpF porin. To this end, 2 µL of the OmpF porin stock solution was added to 2.5 mL of the working solution, thus decreasing the POE concentration to 0.8 ppm. This dilution brings the detergent concentration well below its critical micelle value (~ 30 ppm), thus causing the protein to be released by the detergent and incorporated into the tBLµM.

Conclusions

To the best of our knowledge, the present tBLµM is the first metal-supported biomimetic micromembrane capable of incorporating non-engineered channel proteins in a functionally active state from their detergent solutions, and of allowing the recording of single-channel activity and of impedance spectra of these proteins via ion translocation into the hydrophilic spacer. It can monitor the functional activity of channel proteins and their possible voltage dependence. It can also monitor the

Acknowledgements

Thanks are due to Ente Cassa di Risparmio di Firenze for financial support and to Dr. Arnaud Basle, University of Basel (Basel, Switzerland), for the generous gift of an OmpF porin sample.

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