Electrochemical Analysis of Ion Channels and Transporters in Pore-Suspending Membranes - Applications of Electrochemistry and Nanotechnology in Biology and Medicine - page 291

the bipyridine residues with Ni 2+ . The peptide sequence itself 59 had
already been shown to insert into lipid bilayers as an amphipathic
Į-helix, forming cation selective ion channels with an effective
diameter of 0.8 nm under voltage control. 59-61 From the effective
diameter and computer simulations it was concluded that a hex-
americ bundle is formed in lipid bilayers. 60,62 The synthetic peptide
was inserted into nano-BLMs from an ethanolic solution and its
channel properties were characterized by single channel recordings
under voltage clamp conditions in the absence and presence of
NiCl 2 . Characteristic rectangular current traces were observed ei-
ther in the absence or in the presence of Ni 2+ . 58 We attribute this
step-like current increase and decrease to the assembly and disas-
sembly of a helix bundle composed of a defined number of amphi-
pathic peptide helices. Altogether, four different opening levels
were discernable in the current range of around 25 pA (O 1 ), 35 pA
(O 2 ), 45 pA (O 3 ) and 70 pA (O 4 ). Channel events of one opening
level were often observed in bursts, which lasted several seconds.
Event histograms were used to determine the conductance states
( Fig. 10 B ), resulting in four Gaussian distributions with G 1 = (131
± 19) pS, G 2 = (181 ± 20) pS, G 3 = (234 ± 20) pS, and G 4 = (374 ±
70) pS. Twenty one percent of all events did not fall into these
conductance levels but were larger than 600 pS. Taking the fact
that the lowest conductance state is attributed to six peptide mon-
omers forming a helix bundle, as proposed by DeGrado and
coworkers, 59,60 the higher conductance states would be a result of a
continuous increase in the number of helices participating in the
bundle, up to nine helices.
In the presence of Ni 2+ , the analysis of the conductance states
indeed revealed a considerable change. NiCl 2 was added in a con-
centration of 2.5-5 μM to both sides of the membrane and voltage
clamp experiments were again performed. The majority of events
were found in a current range of 15-110 pA. A histogram analysis
demonstrated that the conductance states are in the same range as
those observed in the absence of Ni 2+ ( Fig. 10C ). However, their
distribution is significantly different. Four Gaussian functions
were fitted to the data with the assumption that the mean conduct-
ance states do not change in the presence of Ni 2+ , thus keeping the-
se four parameters constant. It is obvious that the first and second
conductance levels are very prominent, while the third and fourth
ones are greatly diminished. The relative area of the first and se-