Chemistry Reference
In-Depth Information
contains only one site with a high af
nity for Ry; (3) there is no high af
nity binding at
high pCa (intracellular Ca
2 þ
concentration less than 10
7
M); (4) the binding time
constant of Ry to RyR has been obtained using biochemical methods (161min).
The cardiac sarcoplasmic reticulum vesicles containing RyR2 were incubated
with anti-RyR antibodies labeled with a Cy5-dye (dye/protein
1). A single RyR2
was labeled with at most four
fluorophores because it contains four subunits. Ry was
labeled with Bodipy-FL. Glass supported bilayer membranes were made by the
method of vesicle fusion. Vesicle suspension containing
¼
fluorescently labeled
channels was placed on the glass support. When the calcium concentration in the
solution was adjusted to 1mM, the vesicles fused and lipid bilayers were formed on
the glass surface. By controlling vesicle concentration, we were able to produce
bilayers in a very small area withinwhich channel proteins were con
ned. Thus it was
possible to neglect the effect of lateral diffusion of channels.
As shown in Figure 4.1a, we were able to directly observe the interaction
between
fluorescent Ry (BodipyFL-Ry) and RyR (Cy5-RyR2) at the single molecule
level by adding a solution that contained
fluorescent Rys to RyR immobilized on
a glass. Figures 4.1b
-
e show the images of Cy5
-
RyRs immobilized on glass and
Bodipy FL
-
Rys bound to the channels. Figure 4.1b and Figure 4.1d are images of
Cy5
-
RyRs, and Figure 4.1c and Figure 4.1e show BodipyFL
-
Rys. Figure 4.1b and
Figure 4.1c are
fluorescence images excited by a red laser (633 nm) while Figure 4.1d
and Figure 4.1e were taken during excitation with both a green (532 nm) and a red
(633 nm) laser. These
figures show that there were three RyRs on the glass
(Figures 4.1b and 4.1d) and at least two Rys bound to two out of three channels
(Figure 4.1e). We determined binding durations of individual Rys. Figure 4.1f shows
the binding duration histogram measured in the presence of 10 nM Ry and 10
M
Ca
2 þ
, which highly activates the channel. The histogram can be
tted by two
exponential functions with the time constants of
m
3294ms.
Table 4.1 summarizes the binding durations at pCa5 and pCa3, which shows the
single RyR2 channel and ryanodine binding dynamics for the
first time by using
single molecule imaging techniques. In the active state (10
5M calcium) ryanodine
showed both long (3
t
¼
445ms and
t
¼
1
2
s) binding durations to RyR while it
only showed short durations at pCa3. These bindings might correspond to high and
low af
nity binding of ryanodine. Thismethod is very simple and has a high temporal
resolution, allowing sub-millisecond resolution using faster detection systems, such
as an avalanche photo-diode (APD).
6 s) and short (300
500
m
-
-
4.2.2
Self-Standing Bilayers
The arti
cial planar bilayer technique has been used to study many types of
channels and is a potential tool to study ionic channels having revealed both
pharmacological and dynamic behaviors [7]. For our purposes, bilayers were formed
horizontally so single
fluorescent particles in the membrane could be imaged using
a TIRF microscope [6, 8]. To prevent the vertical movement of the membrane and
possible breakage of the membrane by touching the glass bottom of the chamber,
