Biomedical Engineering Reference
In-Depth Information
(
ii
)
Reconstitution of Channel Forming Peptides
As nano-BLMs exhibit very similar membrane resistances as
classical BLMs and are accessible from both sides, they are well
suited for the reconstitution of channel forming peptides. In the
next paragraphs, we describe several examples of peptide reconsti-
tution from the aqueous phase, resulting in the insertion of single
peptides, which allow the investigation of their channel conduct-
ance.
(
a
)
Gramicidin
One of the most well-known model systems for ion transport
through peptide-based channels is gramicidin A. This linear penta-
decapeptide is synthesized by
Bacillus brevis
48,49
forming a chan-
nel in lipid bilayers consisting of two antiparallel oriented mono-
mers bound to each other by six hydrogen bonds. The resulting
dimer has a length of only 26 Å, which is sufficient to span the hy-
drophobic part of a membrane. Owing to the given length, a func-
tional gramicidin channel is only formed in a single lipid bilayer
and this fact can be used to verify that nano-BLMs are indeed sin-
gle lipid bilayers. The transmembrane ion channel selectively fa-
cilitates the permeation of alkali cations. The ion selectivity fol-
lowing K
+
> Na
+
> Li
+
as well as the partial blockade by Ca
2+
are
well understood.
50,51
Thus, it has been widely used together with
novel lipid bilayer systems such as solid supported membranes to
investigate their integrity for membrane transport functions.
52,53
For single channel recordings, nano-BLMs were bathed in 500
mM KCl solution. Application of a holding potential of
V
h
= +70
mV across the bilayer allowed the voltage-driven ion current in-
duced by gramicidin upon its bilayer partition to be measured.
Gramicidin dissolved in ethanol was added to the nano-BLM in a
10 nM concentration. After insertion of two half channels, single
conductance states as well as multiples of those with distinct con-
ductance states were recorded.
23
A single open state exhibits a cur-
rent flow of (4.2 ± 0.2) pA, which translates in a gated conduct-
ance state of (60 ± 2) pS. Voltage-clamp experiments were also
carried out in 500 mM CsCl solution, resulting in a conductance of
(76 ± 5) pS. In 500 mM LiCl, only single channel openings were
observed, which occurred rather rarely with a mean conductance
Search WWH ::
Custom Search