Biomedical Engineering Reference
In-Depth Information
strate the applicability of nano-BLMs to monitor the activity of ion
channel peptides even down to the single channel level by voltage
clamp experiments.
1.
Peptidic Carriers and Ion Channels
To electrically monitor the activity of carriers and ion channel
forming peptides, the setup depicted in
Fig. 1 A
is routinely used.
For single channel recordings, two Ag/AgCl electrodes in the
cis
and
trans
compartment were connected to an Axopatch 200B
patch clamp amplifier (Axon Instruments, Foster City, CA, USA)
in capacitive or resistive feedback configuration. The
trans
com-
partment was connected to ground and all potentials in the
cis
compartment are given relative to ground. Data were collected
with a sampling rate of 10 kHz and filtered with a 4-pole low pass
Bessel filter with a cut-off frequency of 500 Hz-1 kHz.
(
i
)
Reconstitution of the Ion Carrier Valinomycin
As a first example, the ion carrier valinomycin was investigat-
ed in nano-BLMs. Valinomycin is a cyclic depsipeptide from
Streptomyces fulvissimus
consisting of three identical units. It me-
diates the transport of many alkali and alkaline earth cations in or-
ganic films or solvents of low polarity. It is highly selective for K
+
and Rb
+
over Li
+
, Na
+
, and alkaline earth cations by forming a
three-dimensional complex around the cation in order to increase
its solubility in nonpolar organic solvents or the interior of a bio-
logical membrane. In solid supported membranes, the transport
characteristics of valinomycin has been investigated in an integral
manner by means of impedance spectroscopy.
41,42
However, the
ion transport is always hampered by the underlying capacitance of
the substrate. Impedance analysis of nano-BLMs doped with
valinomycin should, however, allow an immediate extraction of
the change in membrane resistance as a result of the valinomycin
transport activity. To reconstitute a nano-BLM system with
valinomycin, first a 1,2-diphythanoyl-
sn
-glycero-3-phospho-
choline (DPhPC) nano-BLM on an octadecanethiol submonolayer
was prepared and then valinomycin dissolved in dimethylsulfoxide
was added in front of the membrane, which results in the sponta-
neous insertion of the depsipeptide. Impedance spectra were rec-
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