Chemistry Reference
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bottomof the chamber. The upper chamber was thenmoved in a downward direction
until the membrane came into contact with the agarose-coated coverslip. By slightly
increasing the pressure in the upper chamber, the membrane began to thin out and
finally the center of the membrane became a bilayer (Figure 4.2b). This process of
thinning was facilitated by applying membrane voltages (
100mV) and the bilayer
was then observed using a normal bright
field microscope.
A bilayer membrane can also be formed at the tip of a glass pipette and single
fluorophores in the membrane can be detected optically with TIRF. Because glass
pipettes generatemuch lower auto-
uorescence and scatter excitation lightmuch less
than plastic apertures, they are suitable for single channel imaging. However, they are
not convenient for measuring drug binding to channels since perfusion inside the
pipettes presents dif
culties.
4.3
Simultaneous Optical and Electrical Recording of the Single BK-Channels
The techniques described above were applied to achieve an image of a channel
protein [9]. Figure 4.3 shows an example of simultaneous optical and electrical
single channel recordings with a self-standing bilayer in which a Ca-activated
K-channel was
fluorescently labeled and incorporated into a bilayer by the vesicle
fusion technique. Figure 4.3A illustrates the strategy used to incorporate the
fluorescently labeled channel into the arti
cial membrane. The vesicles prepared
from bovine trachea were incubated with monoclonal anti-BK-channel antibodies,
which bind speci
cally to the BK-channel (Figure 4.3B). After removal of unreacted
antibodies by ultracentrifugation, the channel in the vesicular membrane was
transferred into the bilayer by vesicle-fusion. In order to induce rapid fusion of the
vesicle into the limited area of the bilayer, vesicles that had been osmotically loaded
were puffed through a
fine glass pipette.
Figure 4.4 shows the results of the experiments. The channel was labeled with
Cy5-dye molecules and applied to the upper side of the bilayer. Channel proteins
cannot be reconstituted into bilayer membranes simply by adding them to the
aqueous solution. This is in direct contrast to small amphipathic peptides that
spontaneously become incorporated into bilayers. We utilized vesicle-fusion tech-
niques to reconstitute channel proteins into the arti
cial bilayers. In a speci
ed area
of themembrane andwithin a short time-frame, only one channel protein is expected
to be incorporated into the membrane. However, to observe the moment the channel
becomes incorporated into the arti
cial membrane, more sophisticated techniques
are required. The vesicles were added directly to the bilayers through a
fine capillary
tube. This was immediately followed by an instantaneous increase in ionic concen-
tration in the vicinity of themembrane by injecting a small volume of solution of high
salt concentration. The optimum conditions for successfully incorporating the
channel into the membrane were not the same for all channel types. In fact, even
for proteins of the same type, the conditions changed fromone protein preparation to
the next. Thus, the experimental conditions such as the amount of protein to be used
