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i.e. a fusion of single molecule imaging and single channel current recording.
Edelstein et al. showed that ligand binding events to ligand-gated ion-channels are
more complex than ionic events, due to multiple interconversions between different
conformational states at the same degree of ligation [3]. Compared to single channel
current recording, which directly measures current through a channel pore,
single ligand binding events have been inferred indirectly from multi-molecule
experiments. There has been a strong need for technology that allows simultaneous
measurement of single binding events and single channel current
fluctuations,
which would enable us to establish a new
eld of single molecule pharmacology.
In this chapter, we will introduce some technologies developed for this purpose.
4.2
Artificial Bilayers
Lipid bilayers are indispensable to maintain channel activity and measure the ionic
current. So far, most single channel imaging experiments have made use of arti cial
bilayer membranes into which
fluorescently labeled channels are incorporated. In the
1990s single
first time.
Thermal diffusion of individual lipid molecules was directly observed in solid
supported bilayers [4] and self-standing bilayers [5, 6]. In both types of membrane,
these results indicate a rapid lateral diffusion of lipids, which is thought to relate to the
function of channel proteins. Supported bilayers are durable and suitable for long-
termobservationbut not for single channel current recording because to date it has not
been possible to block leakage from the edge of themembrane and fromdefects in the
membranes which are caused mainly by unevenness along the surface of the solid
supports. In contrast, self-standing bilayers in an aqueous environment or on a
hydrophilic gel are suitable for single channel recording because they have little
leakage current although they are very fragile and dif cult to handle.
fluorescent particles in arti
cial bilayers were detected for the
4.2.1
Solid Supported Bilayers
As described above, we can directly see lateral motion of single fluorescent particles
in themembranes if themembranes are suf ciently large. Furthermore, interactions
between receptor molecules and ligands can be detected at the single molecule level
using a TIRF microscope. However, individual
fluorophores in solution cannot be
visualized by video rate TIRF recordings but do increase background intensity
because of their rapid three-dimensional thermal diffusion. They are detected as
single spots only when bound to the receptor in membranes. Using this technique,
we studied interaction between a single cardiac ryanodine receptor channel (RyR2)
and single ryanodine (Ry) molecules. RyR2 is the calcium release channel from the
cardiac sarcoplasmic reticulum membrane, which is named after its agonist,
ryanodine. Several facts regarding Ry binding to RyR are known: (1) Ry binds to
an open form of RyR from the cytoplasmic side; (2) RyR is a homotetramer, but
 
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