Lipid Bilayer-Membrane Protein Coupling - Membrane Biophysics, Biological and Medical Physics, Biomedical Engineering - page 98

After peptide addition, the aqueous phases were stirred for about 5 min before the
measurements resumed. The total amount of added dimethyl sulfoxide was less than
0.5 % of the volume of the electrolyte solution, a concentration that has no effect on
Alm or gA channel function. The lipid bilayer membrane containing n -decane was
3-4 h stable whereas the membrane containing squalene was very unstable, especially
when a potential was applied across the membrane.
Single-channel experiments were performed using the bilayer-punch method [ 4 ]
and a Dagan 3900A patch-clamp amplifier (Dagan Corp., Minneapolis, MN) with a
3910 bilayer-expander module. The current signal in experiments with Alm chan-
nels was filtered at 20 kHz, and digitally filtered at 8 kHz while the current signal in
experiments with gA channels was filtered at 2 kHz, digitized at 20 kHz, and digi-
tally filtered at 500 Hz before the single-channel transitions were detected using the
algorithm described by Andersen [ 4 ] and implemented in Visual Basic (Microsoft
Corp., Redmond, WA). Relative total times spent by different open (conducting)
states and the closed (non-conducting) state of Alm channels were determined by
using frequency counts (in Origin 6.1 from OriginLab Corp., Northampton, MA) of
the recorded current traces of about 1-3 min. The frequency counts were plotted as
functions of the conductance of Alm channels for each recorded current trace, and
peaks were found at the non-conducting and all conducting levels.
Single-channel lifetimes for gA channels were determined as described by Sawyer
et al. [ 78 ] and Durkin et al. [ 25 ], a procedure that allows for separate determination
of the lifetimes of different channel types.

5.4.2 Results

Gramicidin A Channel Results

Figure 5.12 a shows representative current traces obtained in the DC 18 : 1 PC / n -decane
bilayer in the presence of gA − (13) and AgA(15). Figure 5.12 b shows how gA chan-
nels formed from different gA monomers appear with different current transition
amplitudes, namely gA −

(13) channels at 1 . 95 ± 0 . 12 pA and AgA(15) channels at

3 11 pA, respectively. The average gA channel lifetimes ( τ ) were estimated

by fitting a single-exponential distribution (see Fig. 5.12 c)

. 05 ± 0 .

exp t
τ

N ( t )

) =

, (5.28)

N ( 0

where N is the number of channels lasting longer than time t (for details see [ 13 ]).

Figure 5.13 summarizes the average lifetimes τ AgA ( 15 )

( t )

and τ gA − ( 13 )

of gA chan-
nels formed by dimerization of two gA monomers AgA(15) and gA − (13), respec-
tively, in lipid bilayers of different thickness (see Table 5.2 ). In a DC 18 : 1 PC
bilayer we observe that by increasing the gA channel length by only about 3 Å