Biomedical Engineering Reference
In-Depth Information
CaCl
2
. The alkali cations K
+
(best conducting cation) and Li
+
(worst conducting cation) were chosen for this set of experiments.
In the presence of Ca
2+
, ǻ
G
also changes linearly with increasing
cation concentration and the slope is steeper for K
+
than for Li
+
(
Fig. 14 A
, open symbols). Linear regressions to the data in the
presence of CaCl
2
reveal a slope of
m
Li
,
Ca
= (0.585 ± 0.008) μS
mM
-1
and
m
K
,
Ca
(2.76 ± 0.09) μS mM
-1
. The observed conductance
in the presence of Ca
2+
is approximately 2-fold lower than in its
absence, independent of the conducting alkali cation. Bamberg and
Läuger
50
studied the influence of Ca
2+
in detail and showed that the
gramicidin channel is not partially blocked by divalent ions, but its
open probability is reduced. Interestingly, the open probability is a
function of the lipid composition of the membrane. For DPhPC-
bilayers, they observed a reduction in conductance of approximate-
ly 70 %, while we monitored a decrease of about 45 % in pore-
spanning membranes composed of DPhPC/DOPC.
We raised the question of how many active gramicidin chan-
nels contribute to the observed change in conductance. Thus, the
conductance for one gramicidin A channel at 10 mM KCl was tak-
en into account (
G
= 2.15 pS), which was obtained from planar
lipid bilayer experiments.
78
The number of active ion channels in-
corporated into the pore-suspending membrane was calculated
from the obtained ǻ
G
value at 10 mM KCl. According to this cal-
culation, roughly 3·10
7
conducting gramicidin dimers are inserted
into the pore-suspending membrane. With the area of one gramici-
din D channel of 0.4 nm
2
, and the total substrate area of 0.0104
cm
2
, an area fraction of 0.001 % is occupied by active ion chan-
nels. A theoretical maximum area fraction of 0.6 % of gramicidin
D in the pore-spanning membrane would be possible, assuming a
100 % peptide transfer from the vesicles into the pore-suspending
membranes. However, as only conducting dimers are electrically
monitored, the number is significantly lower. This result shows
that a large number of peptides can be transferred into the pore-
suspending membranes, rendering the protocol promising for stud-
ies that involve the activity of transporters with small turnover
numbers of one molecule or less per second.
There are several publications dealing with the electrical re-
cording of gramicidin in freestanding BLMs as well as SSMs or
tethered lipid membranes (tBLMs). Hence, the question arises
what is the advantage of using pore-spanning membranes. One ob-
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