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. 2005 Jun;88(6):4000-7.
doi: 10.1529/biophysj.104.058255. Epub 2005 Mar 4.

Nanopore unitary permeability measured by electrochemical and optical single transporter recording

Roland Hemmler  1 , Guido Böse, Richard Wagner, Reiner Peters
Affiliations

Nanopore unitary permeability measured by electrochemical and optical single transporter recording

Roland Hemmler et al. Biophys J. 2005 Jun.

Abstract

For the analysis of membrane transport processes two single molecule methods are available that differ profoundly in data acquisition principle, achievable information, and application range: the widely employed electrical single channel recording and the more recently established optical single transporter recording. In this study dense arrays of microscopic horizontal bilayer membranes between 0.8 microm and 50 microm in diameter were created in transparent foils containing either microholes or microcavities. Prototypic protein nanopores were formed in bilayer membranes by addition of Staphylococcus aureus alpha-hemolysin (alpha-HL). Microhole arrays were used to monitor the formation of bilayer membranes and single alpha-HL pores by confocal microscopy and electrical recording. Microcavity arrays were used to characterize the formation of bilayer membranes and the flux of fluorescent substrates and inorganic ions through single transporters by confocal microscopy. Thus, the unitary permeability of the alpha-HL pore was determined for calcein and Ca(2+) ions. The study paves the way for an amalgamation of electrical and optical single transporter recording. Electro-optical single transporter recording could provide so far unresolved kinetic data of a large number of cellular transporters, leading to an extension of the nanopore sensor approach to the single molecule analysis of peptide transport by translocases.

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Figures

FIGURE 1
FIGURE 1
Scheme of the experimental setup used for the electrical and optical recording of single nanopores in bilayer microarrays.
FIGURE 2
FIGURE 2
Creation of bilayer membranes spanning 50 μm holes and electrical recording of nanopore formation in those bilayer membranes. (A) Horizontal (left) and vertical (right) confocal scans of a bilayer membrane doped with a fluorescent lipid analog. The lipid annulus displays a strong, the bilayer region a dim fluorescence. Scale bars, 50 μm. (B) Formation of α-HL pores in the bilayer membrane as indicated by current steps (mean step size 7.2 pA at 30 mV, 250 mM KCl).
FIGURE 3
FIGURE 3
Creation of bilayer membranes spanning test compartments of 70 μm diameter and optical recording of transport through nanopores in those bilayer membranes. (A) Horizontal (left) and vertical (right) confocal scans of a bilayer membrane doped with a fluorescent lipid analog. Scale bars, 50 μm. (B) Transport of the small hydrophilic fluorescent tracer calcein through a bilayer membrane containing α-HL pores. Scale bar, 50 μm. (C) Transport kinetics obtained by normalizing the fluorescence in the test compartment (FTC) by the fluorescence in the cis-compartment (FCC) and plotting versus time. The lag between addition of α-HL and the onset of calcein transport is due to the time needed for α-HL molecules to diffuse to the TC array, to insert into bilayer membranes and to form pores by oligomerization. Full symbols: experimental data derived from B. Open symbols, control experiment of bilayer membranes without α-HL pores.
FIGURE 4
FIGURE 4
Formation of bilayer membranes spanning pores of track-etched membrane filters and electrical recording of nanopore formation in those bilayer membranes. (A) Horizontal (left) and vertical (right) confocal scans of bilayer membranes spanning filter pores of 5 m diameter. Most of the filter pores are spanned by bilayer membranes. Occasionally, pores occluded by lipid droplets are seen. Scale bars, 20 μm (left) and 10 μm (right). (B) Formation of α-HL pores in 5 μm bilayer membranes as indicated by current steps (mean step size 34.5 pA at 30 mV, 1 M KCl). (C) Formation of α-HL pores in 0.8 μm bilayer membranes as indicated by current steps. (Inset) Resolution of a large step into three unitary steps.
FIGURE 5
FIGURE 5
Creation of bilayer membranes spanning test compartments of 5 μm diameter and optical recording of transport through nanopores in those bilayer membranes. (A) Horizontal (left) and vertical (right) confocal scans of bilayer membranes. Scale bars, 5 μm. (B) Transport of calcein through bilayer membranes containing α-HL pores. (Left) In horizontal sections with the focal plane set to cut through the test compartments ∼3–5 μm below the surface of the chip test, compartments lacking bilayer membranes are seen to be filled with calcein. (Right) Shortly after addition of α-HL to the cis-chamber, calcein appears in those test compartments covered by a bilayer membrane and having acquired one or a few α-HL pores. (C) Normalized fluorescence of two of the test compartments shown in (B). Normalization excludes fluctuations of image intensities caused by variations of z position or laser intensities.
FIGURE 6
FIGURE 6
Unitary permeability of the α-HL pore for calcein and Ca2+. Arrays of 5 μm diameter bilayer membranes containing <1 α-HL pore on average were used to measure the permeation of calcein or Ca2+ into test compartments. Plots of the frequency of the transport rate constant k versus k display distinct peaks representing bilayer membranes with 1, 2, 3,… α-HL pores. For calcein and Ca2+, the unitary rate constant k1 (largest peak) was found to be ∼0.03 min−1 (A) and ∼0.35 min−1 (B), respectively. The histogram in A was derived from 238 k values obtained in 14 experiments, that in (B) from 63 k values obtained in nine experiments.
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