Biomedical Engineering Reference
In-Depth Information
2.
Analysis of Gramicidin D Activity
We aimed to reconstitute ion channels in pore-spanning mem-
branes by fusion of proteoliposomes on functionalized porous sub-
strates. Gramicidin D turned out to be a good test case for the de-
velopment of the principle strategy.
( i )
Channel Activity of Gramicidin D Reconstituted into
Pore-Spanning Membranes
Large unilamellar vesicles composed of DOPC doped with 2
mol% gramicidin D were prepared and the conformation of grami-
cidin D was analyzed by circular dichroism (CD) spectroscopy,
verifying that it forms the conducting conformation. 29 To generate
pore-suspending membranes with reconstituted active gramicidin
channels, we followed the strategy of first generating a pore-
suspending bilayer composed of DPhPC/DOPC (6:4) from vesicle
spreading before adding gramicidin D-doped DOPC vesicles to the
system for 8-12 hours. Porous alumina substrates with partially
opened pore bottoms were used, as the lipid membranes form con-
siderably faster on these substrates (see Section II). If the alumina
oxide resistance R ox is known, model (2') can be applied for analy-
sis. For large R ox , the overall resistance R a resembles R m,o (Eq. 3).
This allows to directly link the change in overall resistance to a
change in R m,o generated by a conducting ion channel. Throughout
the preparation steps, a buffer solution containing TMA was used.
TMA is too large to diffuse through the gramicidin channel 74 and,
thus, the membrane resistance is supposed to remain unaffected
upon insertion of the channel active peptide. To verify the func-
tional transfer of gramicidin D into the pore-spanning membranes,
its channel activity was monitored by adding monovalent cations
(LiCl, NaCl, and KCl) to the TMA-containing buffer solution into
the upper cis compartment of the liquid cell ( Fig. 1 B ). The distinct
selectivity for alkali cations and its concentration dependence are
the main characteristics of the gramicidin channel. Impedance
spectra were recorded before and after increasing the ion concen-
tration to follow the electrical response of the membrane system.
The entire experiment with various cations was performed on one
membrane preparation, as the electrical properties of pore-
spanning membranes depend on the underlying alumina substrate,
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