Transcript (DHB)- Application to Voltage-Gated

Droplet Hydrogel Bilayer (DHB)- Application to Voltage-Gated
Lauriane Angué
1,2
Stephen J. Tucker
[email protected]
+
K
1/
Channels
Mark I. Wallace
2
Department of Physics, Clarendon Laboratory / Department of Chemistry, Chemistry Research Laboratory
Introduction:
Electrical recording
Droplet Hydrogel Bilayer method:
Artificial bilayers are a supplementary alternative to patchclamping for the study of ion channels. The components of
the system, such as the lipid compostion, the electrolyte
solution and the number of channels studied can be
controlled by the experimenter; something the in vivo
techniques do not allow.
An aqueous droplet is brought into contact with an
agarose surface immersed in a lipid/oil solution. A
droplet hydrogel bilayer (DHB) will form between
these two with a high stability. Two electrodes, one
inserted in the hydrogel and one in the droplet
detect the ion flux passing through the channel
embedded at the interface.
By directing a total internal reflection laser beam on
the bilayer, the fluorescence signal can be
recorded, simultaneously with electrical
measurements.
We have developed the droplet-hydrogel bilayer (DHB)
technique to study a variety of membrane proteins*. The
bilayer formed is quite stable and has a longer lifetime than
other artificial bilayers techniques. This technique combines
both electrical and fluorescence recordings. It is therefore a
usefull method if someone wants to investigate the structurefunction relationships within ion channels.
Figure 3: A bright-field image of a droplet forming a
bilayer with the lipid-coated agarose.
Image courtesy of Dr. Lydia Harriss Thesis “A
Single Molecule Investigation of Ion Channels and
Pore-Forming Peptides”
* Bayley H. et al, Mol. Biosyst. (2008) 4, 1191.
Electrical recordings of KvAP:
Figure 1: A
representation of
the PMMA device
used for DHB
formation.
Figure 2: A droplet hydrogel bilayer for simultaneous
TIRF microscopy and electrical measurements.
Image courtesy of Dr James Thompson
Fluorescence recording
Image courtesy of
Dr James
Thompson
KvAP-C247S
KvAP is a prokaryotic potassium channel whose X-ray structure of 3.2 Å
resolution was first reported in 2003. Each channel is formed by four
identical subununits, which form a pore at their intersection, where the K+
ions can go through the bilayer. The opening or closing of the pore
(« gating ») is controled in the case of voltage-gated K+ channels (Kv), by
the transmembrane voltage, which triggers a conformational change to
open the pore.
The sequence of this voltage-sensing domain is highly homologous to eukaryotic Kv channels, which makes it a good
model to understand how mammalian Kv channels respond to voltage changes. In the present investigation, a
fluorescent dye is covalently attached to the voltage sensing domain to probe the structure-function relationship
through activation by voltage.
KvAP-L121C-C247S labelled with
TMRM
Figure 4: Single channel traces recorded at 0 mV in 150 mM KCl, 10 mM HEPES, pH=7.0, by the DHB method.
Future work:
Fluorescence investigation of the labelled mutants: observation of a fluorescence change when the voltage is changed.
Scale the quantities down to single molecule experiment.
Simultaneous measurements of electrical and fluorescence recordings of the mutants.
Figure 5: Single channel traces recorded at 0 mV in 150 mM KCl, 10 mM HEPES, pH=7.0, by the DHB method.