Ion Channels - Single Molecule Dynamics in Life Science - page 95

Figure 4.4 were consistent with the results obtained in patch-clamp or conventional
planar bilayer experiments [10], showing that it is possible to record the natural
properties of channel proteins with the method described herein.
We have developed a novel method for simultaneously recording the optical and
electrical properties of single ion-channels. This method is so sensitive that we can
image single fluorphores in the membrane and should prove applicable to a wide
variety of channel proteins. It is also possible, with this method, to observe single

fluorescent molecules in solution when they are very close to the membrane.
This means that we can directly observe the interaction between a single channel
protein and its ligand molecules labeled with a RyR

coupling using solid supported bilayers. Such observations will greatly increase our
understanding of the dynamics of ligand - receptor interaction and the activation
mechanisms at the single-molecule level in ligand-activated receptor channels such
as nACh and glutamate receptor channels.

fluorescent dye as we did in Ry -

4.4
Detection of Channel Conformational Change

Brisenko et al. observed conformational change in single gramicidin channels using
the same type of membranes [11]. They reported an approach for simultaneous
fluorescence imaging and electrical recording of single gramicidin channels.
Fluorescently labeled (Cy3 and Cy5) gramicidin derivatives were imaged at the
single-molecule level using far- eld illumination and cooled CCD camera detection.
Simultaneous electrical recording detected gramicidin homodimer (Cy3/Cy3,
Cy5/Cy5) andheterodimer (Cy3/Cy5) channels.Heterodimer formationwasobserved
optically by the appearance of a uorescence resonance energy transfer (FRET) signal
(irradiation of Cy3, detection of Cy5). The number of FRET signals increased with
increasing channel activity. In numerous cases the appearance of a FRETsignal was
observed to correlate with a channel opening event that could be detected electrically.
Lu and his colleagues reported their studies on the conformational changes in the
gramicidin channel using patch-clamp fluorescence microscopy, which simulta-
neously combines single-molecule fluorescence spectroscopy and single-channel
current recordings using arti cial membranes formed at the tip of glass pipettes [12].
By measuring single-pair fluorescence resonance energy transfer and uorescence
self-quenching from dye-labeled gramicidin channels, they showed that the
ef ciency of single-pair fluorescence resonance energy transfer and self-quenching
is widely distributed, which re ects a broad distribution of conformations.

4.5
Optical Patch-Clamping

Demuro and Parker recently described an optical technique known as optical
patch-clamping which uses total internal re ection fluorescence (TIRF) microscopy