Chemistry Reference
In-Depth Information
Table 4.1
Binding duration of ryanodine to RyR.
pCa 5
Liposome
pCa 3
Ryanodine concentration
10 nM
10 nM
1nM
10nM
100nM
1M
1
254
468
464
445
370
t
2
5690
3294
3657
120 000
t
solution only when they came very close to the membrane and bound to the
receptors.
Figure 4.2 shows the horizontal bilayer apparatus we developed for single channel
imaging [8]. Figure 4.2a shows the bilayer apparatus consisting of two chambers.
The upper chamber is made from a glass tube which can be moved using a piezo
micromanipulator. A thin plastic
filmwith a small pore in the center is attached to the
bottom of the upper chamber. The pore on the
film was made on a projection by the
same method used to make an aperture for a conventional vertical bilayer. The lower
chamber consists of a glass dish with a hole in the bottom over which a coverslip is
fixed with adhesive just prior to the experiment. The upper and lower solution
chambers are connected to a patch-clamp ampli
er through Ag-AgCl electrodes. The
coverslips were washed thoroughly, coated with agarose by painting with a warmed
solution of 0.2
-
0.5% agarose in water, and then air-dried at room temperature. There
was no special attachment between the agarose and the glass surface. The dried
coverslip was
fixed over the hole in the bottom of the chamber with adhesive.
The membrane was formed as follows: a thick membrane was formed across
the hole by adding a small amount of lipid solution (20mg lipid/ml n-decane) to the
Figure 4.2 (a) The bilayer apparatus. This
apparatus consists of two chambers; the upper
chamber is made from a glass tube. A thin
(0.1
membrane formed on the agarose consisted of
two parts: the bilayer membrane in the center
and the surrounding thick annulus. (b) An aerial
view of the membrane taken under a bright field
microscope. The contrast was slightly enhanced
with a CCD camera.
0.2mm thick) polypropylene film is
attached to the bottom of the upper chamber.
This film has a small pore in the center across
which the bilayers were formed. The bilayer
-
