Application Note

Channel:Cells:
Tools:KcsA
GUVs
Port-a-Patch
Vesicle Prep Pro

Lipid bilayer recordings of KcsA channels
reconstituted in proteliposomesTheelectrophysiology

teamatNanion

Technologies

GmbH,Munich.

The KCsA protein samples were kindly provided by Prof. O. Pongs, Hamburg.

Summary
Solvent-free planar lipid bilayers were formed in anafter GUV addition. When the GUVs touch the glass sur-automated manner using suction to attract a giant

face of the chip, they burst and form planar bilayers (1)unilamellar vesicle (GUV) to the patch clamp chip whichwith a seal resistance of tens to hundreds of GΩ (Table 1).

subsequently bursts across the aperture. Incubation ofA schematic of the KcsA protein and the bilayer forma-GUVs with purified KcsA channel protein yielded proteoli-tion process is shown in Figure 1,posomes. These proteoliposomes allow for immediate recording of channel activity after GUV sealing. The rapid
formation of protein-containing planar lipid bilayers is ofpotential use for the efficient electrophysiological characterization of KcsA as shown here (Fig. 2) and also otherion channel proteins of interest.

Results
For formation of a planar lipid bilayer containing the KcsA
protein, 1 to 3 µl of the proteoliposome solution was pipetted onto the patch clamp chip. The microstructured chip,
which is commonly used in patch clamp experimentswith cells, contains an aperture approximately 1 micronin diameter. 5 µl of a sterile-filtered solution on each sideis adequate to wet the glass chip and liposomes or compounds can be applied directly from the accessible top
side of the chip. The GUVs were positioned onto the aperture of the chip by application of a negative pressure.
Typically, (-) 10 to (-) 40 mbar were sufficient for reliable
positioning of a vesicle within a few seconds

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Figure 1: Top: Schematic of the KcsA potassium channel, which has 4
identical subunits and each subunits contains two alpha-helices connectedby an approximately 30 amino acids long loop proof-reading into the poreregion. Bottom: The formation of a planar lipid bilayer by vesicle fusion onto
a microperforated glass chip is schematically shown.

Application NoteTable 1:
Distribution of seal resistances of planar lipid bilayers formed with DPhPC in
10% cholesterol GUVs. (n=161)

In contrast to conventional bilayer recordings, where reconstituion of proteins is achieved by adding the protein
after bilayer formation in the presence of detergent, weinsert the protein into the GUVs directly after the electroformation step. When kept at 4oC, storage of the proteoliposomes was possible for weeks in a standard Eppendorf
tube in 1 M sorbitol.
Figure 1: Measurements of KcsA conductance in 100 mM KCI. Top:
Representative current trace of the KcsA chennel at a transmembranepotential of +150 mV. Bottom: Ramp -200 mV to 200 mV.

Referencesat room temperature and then overnight at 4oC to remove the detergent. Bio-Beads were discarded after

1. M. Kreier, C. Farre, M. Beckler, M.George and N. Fertig.
2008 Rapid Screening of Membrane Protein Activity:
Electrophysiological Analysis of OmpF Reconstituted inProteoliposomes. Lab Chip, 2008, DOI: 10.1039/b713982a.

centrifugation and the protein containing GUVs could
be used immediately.
Electrophysiology
Patch clamp experiments were performed with the Port-Methods

a-Patch, using borosilicate glass chips with an aperture

GUVs

diameter of the chip and a specific capacitance of DPh-diameter of approximately 1 µm. Based on the aperture

Planar lipid bilayers were obtained from Giant Unilamellar
Vesicles (GUVs) prepared by using the electroformation
method in an indium tin oxide (ITO) coated glass chamberconnected to the Nanion Vescile Prep Pro setup.

PC of 0.5 µF/cm2, the membrane capacitance could be
estimated to be in the order of a few fF. Experiments weredone in internal solution: 100 mM KCI, 10mM HEPES pH 7and external solution: 100 mM KCI, 10 mM MES pH 4. The
data were filtered at 3 kHz or 10 kHz (Bessel filter, HEKAamplifier) digitized at a sampling rate of 50 kHz andProteoliposomes Preparation
KcsA solubilized in 400 nM Imidazol, 200 mM NaPO4, 150

mM KCI, pH 7.8 was added to the solution containing
GUVs at concenctration of 1-1.5mg/ml in 100 mM sorbi-analyzed with Clampfit (Axon instruments). The bilayer
formation process was computer controlled by thePatchControl software (Nanion).

tol. The mixture of GUVs and protein was incubated for 1hour at room temperature, followed by the addition ofBio-Beads SM-2 (Bio-Rad) at 40 mg/ml in GUVs solution.
The mixture was incubated with the Bio-Beads for 1 hour

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