Biomedical Engineering Reference
In-Depth Information
concentration of 0.7-2.7 ng mL
-1
. Since the insertion of a Cx26 ol-
igomer is a random process, activity is observed sometimes al-
ready after several minutes, but in other cases only after hours. By
means of voltage clamp experiments, information about the con-
ductance states of Cx26 can then be gathered. If a hemichannel
was inserted, current traces were recorded and different holding
potentials ranging between
V
h
= +150 mV and -150 mV were ap-
plied. It turned out that the hemichannels exhibit a pure Ohmic be-
haviour with a mean conductance of (33 ± 3) pS (
Fig. 11 A
).
This result is in good agreement with conductance values ob-
tained from the same hemichannel inserted into planar bilayers on
microstructured glass supports, a method provided by the company
Nanion (Munich, Germany).
69
A statistical analysis of all single
channel events reveals one prominent conductance state with a
mean conductance of
G
1
= (34 ± 8) pS. In addition, but less fre-
quently, larger conductance states were observed with two distri-
butions at
G
2
= (70 ± 8) pS and
G
3
= (165 ± 19) pS. Other groups
found similar single channel conductance values for reconstituted
hemichannels, being in the range of 35-316 pS.
70,71
This result in-
dicates that even Cx26 hemichannels can be inserted into nano-
BLMs in a functional manner.
IV. IMPEDANCE ANALYSES ON PORE-SPANNING
MEMBRANES
Nano-BLMs with membrane resistances in the range of gigaohms
prove to be a suitable model system for the investigation of pep-
tides and large transmembrane proteins on a single channel level,
as outlined in Section III. In contrast, pore-spanning lipids bilayers
generated by vesicle fusion on porous alumina exhibit resistances
two to three orders of magnitude lower than those of nano-BLMs.
Still, ion channel activity can be measured by using EIS. This
technique allows detecting conducting channel proteins in pore-
spanning bilayers in an integral manner and, thus, overcomes the
need for gigaohm seals. Here, we present the monitoring of ion
channel activity, namely of OmpF and gramicidin, by means of
EIS.
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