Biomedical Engineering Reference
In-Depth Information
4.4 Characterization of Phi29 Connector Channels by Single
Channel Electrophysiological Assays
4.4.1 Experimental Setup
A standard horizontal BLM chamber (Eastern Sci LLC) was utilized in these
experiments. A thin Teflon film with an aperture of 100-200
m (Easter Sci
LLC) in diameter was used as a partition to separate the chamber into
cis-
(working
volume 250
m
L) and
trans-
(working volume 2.5 mL) compartments. For connector
insertions, diluted liposome stock solution was directly added to the
cis-
compart-
ment. A final molar ratio of lipid:connector was set to 4,000:1 to 16,000:1 for
single channel conduction assays.
The thickness of the BLM is critical for efficient insertion of connector reconsti-
tuted liposomes with the BLM. The capacitance of the membrane produced from
a voltage ramp is first obtained using the equation
C ¼ IdV=dt
m
Þ
1
,where
I
is
ð
the current measured, and
dV=dt
ð
Þ
is the applied voltage ramp. The thickness can
A C
1
then be expressed as
d ¼ e
0
e
s
ð
Þ
e
0
and
e
s
are permittivity of free
, where
m
1
), respectively. The optimum
lipid thickness is ~5 nm, which corresponds to the thickness of a single lipid bilayer.
A pair of Ag/AgCl electrodes connected directly to the head-stage of a current
amplifier was used to measure the current traces across the bilayer lipid membrane.
The trace was recorded using an Axopatch 200B patch clamp amplifier coupled
with the Axon DigiData 1322A or Axon DigiData 1440 analog-digital converter
(Axon Instruments). All the voltages reported are those of the
trans
-compartment.
Data was low band-pass filtered at a frequency of 1 kHz and acquired at 500
10
12
F
m
1
) and lipid (~3 F
space (8.854
s
time intervals, if not specified. The PClamp 9.1 software (Axon Instruments) was
used to collect the data, and the software Clampfit was used for data analysis.
m
4.4.2
Insertion of the Connector into Planar Bilayer
Lipid Membrane
Direct incubation of the connector with liposomes or a planar lipid bilayer did not lead
to channel formation in the bilayer membrane (Fig.
4.4a
). Connector insertions into
the bilayer were only observed upon vesicle fusion of connector reconstituted proteo-
liposomes with the planar bilayer (see schematic Fig.
4.3e
). The channel insertion
events were observed as discrete step-wise increase in conductance (each step repre-
sents the insertion of a single connector channel) as shown in a continuous current
trace (Fig.
4.4b
), under either positive or negative transmembrane voltage (Fig.
4.4c
).
The number of connector channels inserted into the membrane can be obtained
by simply counting the steps in the current trace during the course of insertion.
A series of extensive investigations under a wide range of solution conditions
revealed that the step size of the current jump per single connector insertion were
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