Biomedical Engineering Reference
In-Depth Information
pore.
26
Point histogram analysis of the current traces allowed for
the determination of the three different conductance levels with
G
1
= (1700
r
80) pS,
G
2
= (3360
r
80) pS and
G
3
= (5060
r
50) pS. In
addition, we investigated whether we are also able to monitor the
reported fast kinetics and subconductance states. Also, these char-
acteristics of the porin pore were found in nano-BLMs, indicating
that the close proximity of immobilized lipid bilayers on the pore
rims does not influence the ion channel activity.
26
Nestorovich et
al.
67
reported that the
E
-lactam antibiotic ampicillin is capable of
blocking the OmpF channel by moving in and out of the channel.
This process takes place on a rather fast time scale. It turned out
that the time resolution of our system is not sufficient to fully re-
solve every single ampicillin blockade as was demonstrated in the
work of Nestorovich et al.
67
However, blockades of ampicillin
were clearly detected as downward spikes of the ion flow during
the opening of one monomer.
26
These results indicate that the po-
rous substrate underneath the membrane does not prevent the full
functionality of a large transmembrane protein, even though one
membrane, spanning a 60 nm pore of the porous alumina substrate,
is composed of only roughly 4000 lipids.
(
ii
)
Connexon 26
Connexins are members of a multigene family of membrane-
spanning proteins that form intercellular channels, which are com-
posed of two hexameric hemichannels, called connexons (Cx).
These intercellular channels organize into gap junctional plaques
and span the extracellular space/matrix of adjacent cells, thus al-
lowing a passive exchange of small molecules up to about 1 kDa.
68
By the formation of gap junctions, two membranes can be electri-
cally connected. As a prerequisite for the electrical coupling, con-
nexons need to be reconstituted into a membrane system that is
accessible from both sides. Nano-BLMs can provide the required
accessibility and, at the same time, the stability to connect a mem-
brane such as the plasma membrane of a cell via the formation of
gap junctions. To elucidate the functionality of a connexon in
nano-BLMs, connexon 26 (Cx26) isolated from baculovirus-
infected
Sf9 cells was reconstituted. This was achieved by adding
Cx26 dissolved in a 1 % (
v
/
v)
octyl-polyoxyethylene (
o
-POE) de-
tergent solution to the
cis
side of the Teflon cell, leading to a final
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