Research
Synthetic pores and channels
Our goals are to design and build systems that will insert into
bilayer membranes and open passages for the transport of ions and small
molecules through bilayer membranes. This functional goal does not
compel any particular structure, and we have made a wide variety of
active compounds based on cyclodextrins, crown ethers, palladium
complexes, linear and cyclic esters, etc. What these compounds have in
common is their ability to interact with themselves and with the
components of a bilayer membrane to create larger structures that span
the thickness of the bilayer. Within or around these active structures
there is a pool of water that allows ions to move through the membrane.
Although this structure is tenuous when compared to natural channels,
these compounds function as well as channels that have had billions of
years to evolve. And even though very primitive, the
conductvity-time behaviour of the channel can be very regular as shown
below. Each of these steps corresponds to a few molecules (3-5?)
opening a small defect through which millions of ions can flow over
many seconds. The abrupt on-off behavior is inditingishable
fomr some natirally-occuring ion channels.
Current projects in this area are focused on efficient syntheses of
channel-forming compounds, on
probing the mechanistic details of the channels we have developed, on
channels with defined “portals”, and
on refining how we document and analyze the conductance-time data we
generate.
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Probing channel mechanisms
Ion channels are catalysts for the relocation of ions and
small molecules across bilayer membranes. Like other catalysts,
the details
of the active structures are tough to figure out and require a large
number of
different techniques to probe what is occurring. The voltage-clamp
experiment that generates the conductance-time records is one
technique we
use as it tells us what single molecules are doing. We also use
bilayer
vesicles or liposomes to probe how large numbers of channels act on
average. In these experiments a dye is entrapped inside a 100 nm
diameter
cell-like container and the transport through the membrane is reported
by
changes in the dye emission. Some compounds we prepare are
inherently
fluorescent (below) so that they can report on their environment as
they move
through the system. In the end all we can do is rule out some
types of
mechanisms as inconsistent with the experimental results or too simple
for the
results we observe.
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Cyclodextrin channels
Cyclodextrins are cyclic oligomers of glucose linked to have a
central cavity with the sugar hydroxyl groups pointing above and below
the ring. Supramolecular chemists have explored these hosts for
decades and the first synthetic ion channel was built on this
structure. Our work uses this scaffold to build "walls" for the
channel in a quick process in which an azide derivative of the
cyclodextrin is "clicked" with an acetylene to produce a tetrazole with
the wall appended. The central hole is a large cavity and
should give high conductance channels. We do not find that except
when we provide a non-polar guest for the cyclodextrin host.
Under these conditions the conductance "flickers" between a large
opening and one that is partially blocked by the guest.
Other cyclodextrin channels we have made show much more erratic
activity involving "spikes" of short duration, bursts that appear to be
multileveled, and some truly chaotic openings. The compound shown
below has almost exclusively this very irregular behavior and it really
does not matter on which time scale we look - it looks equally
ugly. In fact this is a beautiful example of "self-similarity"
and we have been able to show that these compounds follow a power-law
relationship in the durations that the current exceeds a given
threshold. So there is obviously some underlying order in this
system, even if it does not very irregular.
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Voltage-clamp experiment
The conductance of a single channel is readily detected in
the conductance-time records shown on this page. These arise from
a small (0.2mm) patch of bilayer between two pools of electrolyte
solution. The bilayer is formed by painting lipid across the
hole. The experiment is called "voltage-clamp" as a potential is held
at a constant value across the membrane and the current fluctuations
are measured. The bilayer is an insulator, so without a channel
there is no current; only when a channel inserts can any current
flow. The simple
on-off "square-top"
openings can be analyzed easily to provide clues about the size and
stability
of the active structures. But what of the other types of
activities we
see? We know they reflect underlying structures, but they contain
so much
information that we have a hard time even recognizing patterns and
similarities
within a single record, let alone over many different experiments,
involving
different conditions and compounds. Part of the problem is that
the time
varies from milliseconds to hours and the conductance from pico- to
tens of
nano-Siemens.
We have developed an "activity grid" that records
conductance and duration on a log-log grid with color patches showing
the types
of behaviors observed. For example green is the square-top type
of
openings, red are for spikes, blue is for multi-level openings, yellow
for
flickers, and purple of the erratic openings. Anything we cannot
observe
due to how the experiment is conducted is shown in grey. These
grids can
be summed over many experiments to build up a picture for a single
compound,
and then the composite pictures can be compared with other compounds to
uncover
structure-activity relationships.
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Supported liquid membranes
Bilayer membranes are close mimics of nature, but pose severe
problems in many practical applications. A much simpler and robust
membrane can be made by mixing a solvent into a supporting polymer.
Such “supported liquid membranes” are much thicker than a bilayer, but
allow transport via carriers to occur. There have been many practical
applications of this type of membrane; almost all have used a
concentration gradient as the driving force. Our current goals in this
area are to use electrochemical techniques to probe the mechanisms of
such carrier-based systems, and to develop new applications in which
concentration gradients can be manipulated by electrochemical means.
Printed electrochemical membrane sensors is one practical spin-off our
work in this area. A spin-off of that spin-off are antifouling coatings
for use in the marine environment.
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