Application Note

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

Lipid bilayer recordings of amechanosensitive channel, MscL,
using Nanion’s pressure clamp
Theelectrophysiology

teamatNanion

Technologies

GmbH,Munich.

The MscL protein samples were kindly provided by Dr. Chris Gandhi, CalTech, California

Summary
Solvent-free planar lipid bilayers were formed in an automated manner using suction to attract a giant unilamellar vesicle (GUV) to the patch clamp chip which
subsequently bursts across the aperture. Incubation ofGUVs with purified MscL channel protein yielded proteoliposomes. 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 MscL as shown here and also other ion
channel proteins of interest.
In order to study the effect of pressure, the functional
MscL purified was reconstituted in our system. The reconstitution was done in GUVs and then bilayers were formed
on a chip (Kreir, Farre et al. 2008). The Port-a-Patch system
has a pump controlled by a computer and could apply
from +300 to -300 mBar and is controlled via software allowing accurate pressure control. All pressure applications could be visualized and recorded at the same timeas the recordings.

Introduction
Mechanosensitive (MS) ion channels were first describedin gram positive bacteria using either giant protoplasts ofbacteria and patch clamping, or the fusion of membranewith liposomes. The MS channels have then been foundin organisms of different phylogenetic origin including
mammalians, plants, fungi, bacteria (Gram-positive and-negative) and recently archaebacteria (Martinac et al.,1992) (Pivetti, Yen et al. 2003).
Download more Application Notes from www.nanion.de

In response to mechanical stimuli, the MS ion channels open
and convert the mechanical signal into electrical and/or biochemical signals.
This suggests their role in physiological functions in different
types of cells such as osmoregulation in bacteria, turgor
control in plant cells as well as mechanosensation like touch,hearing, proprioception and cell division in eukaryotic cells.
The MS channels have been extensively studied in bacteria(gram negative and positive) where three types of MS channels were found: MscL (large conductance), MscS (smallconductance, known as being 50 % of the conductance ofMscL), and MscM (mini conductance). For example, MscL,
from E. coli, is a large non selective channel with a conductance of 2.5 to 3 nS (Sukharev 1999). The structure of MscL
has been resolved to high resolution. The alpha helices span
the membrane twice and contain an inter-TMS-loop thatconnects the two transmembrane domains. The channel has
also an amino-terminal alpha helix and a short carboxyterminal helix. MscL from Mycobacterium tuberculosis has been
resolved to 3.5 Ǻ resolution giving the residues information ofMscL (Chang, Spencer et al. 1998).

Application NoteFigure 2: MscL EC gating recorded at 30mV and the indicated negativehydrostatic pressure.

When the suction was turn off, from -25 to 0 mBar thecurrent was reduced, indicated that the channel is
only responding to the suction. The voltage was applied.
Some channels were still activated at 0 mBar,Figure 1: MscL EC trace. MscL EC gating recorded at 30mV and theindicated negative hydrostatic pressure, in symmetrical recording
solution (200mM KCl, 40mM MgCl2, 5mM Hepes, pH 7.2/KOH).
The histogram shows the conductances of each opening level(100 s recording). The conductance of a single channel is ~2.45 nS

with a low open probability. When the suction was onagain, the current was increasing right away (figure 3).

It forms a homopentameric channel and it is proposedthat the carboxy-terminal helix form a bundle when
the channel is closed. The phenomenon leading to theopen state after mechanical stimuli (pressure) is theconformational change involving the amino-terminal
helix. Despite the structural knowledge, the gating mechanisms via forces applied to membrane lipid bilayers
are still poorly understood. Electrophysiology recordings
shows that MscL, despite its large conductance and its
non selective pore, have different subconductance

Figure 3: MscL EC gating recorded at 30mV and the indicated negativehydrostatic pressure.

states, as well and the studies of Sukharev (1999) which
proposed a model of gating (Sukharev, Sigurdson etal. 1999). Recently, the structure of MscL in an expanded
intermediate state has been resolved by X-ray diffraction(3.82 Ǻ) (Liu, Gandhi et al. 2009) in comparison to the previous crystal structure in the closed state.

Results
For formation of a planar lipid bilayer, 1 to 3 µl of the proteoliposomes solution is pipetted onto the patch clamp
chip. The microstructured chip, which is commonly usedin patch clamp experiments with cells, contains an aperture approximately 1 micron in diameter. The GUVs arepositioned onto the aperture in the chip by applicationof a slight negative pressure. Typically, (−)10 to (−)40mbars are sufficient for reliable positioning within a few
seconds after GUV addition. When the GUVs touch theglass surface of the chip, they burst and form planar bilayers with a seal resistance of tens to hundreds of GΩ.
Current in response of pressure and voltage is shownin Figure 1. The current was elicited using a continuous
voltage of 30 mV and a suction of -25 mBar.
Download more Application Notes from www.nanion.deFigure 4: MscL EC gating recorded at 30mV and the indicated negativehydrostatic pressureApplication NoteSummary
In summary, we used the Port-a-Patch and Vesicle
Prep Pro to study MscL channel and successfully recorded and produced high quality single-multi channel data. The patch clamp method offers the possibility to investigate ion channels and their effectors, inreal time, and with unparalleled sensitivity comparedto other methods.

References

Proteoliposome preparation

1. Chang, G., R. H. Spencer, et al. (1998). „Structure of theMscL homolog from Mycobacterium tuberculosis: a gated mechanosensitive ion channel.“ Science 282(5397):2220-2226.
2. Kreir, M., C. Farre, et al. (2008). „Rapid screening ofmembrane protein activity: electrophysiological analysisof OmpF reconstituted in proteoliposomes.“ Lab Chip8(4): 587-595.
3. Liu, Z., C. S. Gandhi, et al. (2009). „Structure of a tetrameric MscL in an expanded intermediate state.“ Nature461(7260): 120-124.
4. Pivetti, C. D., M. R. Yen, et al. (2003). „Two families ofmechanosensitive channel proteins.“ Microbiol Mol Biol
Rev 67(1): 66-85, table of contents
5. Sukharev, S. (1999). „Mechanosensitive channels inbacteria as membrane tension reporters.“ FASEB J 13
Suppl: S55-61.
6.Sukharev, S. I., W. J. Sigurdson, et al. (1999). „Energeticand spatial parameters for gating of the bacterial large
conductance mechanosensitive channel, MscL.“ J Gen
Physiol 113(4): 525-540.

MscL solubilized in detergent was added to the solution
containing GUVs in 1 M sorbitol. The mixture ofGUVs and protein was incubated for 1 hour at room
temperature, followed by the addition of Bio-Beads®
SM-2 (Bio-Rad) at 40 mg/ml in GUVs solution. The mixture
was incubated with the Bio-Beads for 2 hours
at room temperature to remove the detergent. BioBeads
were discarded after centrifugation and theprotein containing GUVs could be used immediately.
Electrophysiology
Patch clamp experiments were performed with thePorta-Patch®, using borosilicate glass chips with anaperture diameter of approximately 1 μm. Based on theaperture diameter of the chip and a specific capacitanceof DPhPC of 0.5 μF/cm2, the membrane capacitancecould be estimated to be in the order of a few fF.
Experiments were done in 200 mM KCl, 40mM MgCl2,5mM Hepes, pH 7.2/ KOH. The data were filtered at 3 kHz
or 10 kHz (Bessel filter, HEKA amplifier) digitized at asampling rate of 50 kHz and analyzed with Clampfit (Axoninstruments). The bilayer formation process was computer

Methods

controlled by the PatchControl software (Nanion).

GUVs
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 Vesicle Prep Pro setup.
Nanion Technologies GmbHphone + 49 89 219 095-0

Ganghoferstr. 70A

fax80339 Munich, Germany

www.nanion.de • info@nanion.de+49 89 218997960