Chemistry Reference
In-Depth Information
7.5.5
Combined Electrical and Fluorescence Measurements
Pioneering work on combined optical and electrical recordings originated from
Yanagidas laboratories [110, 111, 186]. The activity of single ryanodine receptor
channels reconstituted in planar lipid bilayers [112, 187] and of single nAChRs
expressed in Xenopusoocytes has been imaged using a
fluorescent indicator of
calcium
flux [113]. The formation of single gramicidin dimers was monitored by
combined electrical and FRETmeasurements [114]. Planar patch-clamp instruments
based on microstructured devices are used in the pharmaceutical industry for high-
throughput screening of compounds targeted to ion channels. Great bene
ts can be
derived from the planar design of the chips used to perform single-molecule
fluorescence measurements [115]. With further improvements, simultaneous elec-
trical and
fluorescence recordings will enable the correlation between local structural
fluctuations or ligand binding and channel activity at the single-molecule level to
be elucidated. Good candidates for this procedure would be ligand-gated ion
channels [116].
7.6
Forces of Ligand
-
Receptor Interactions in Living Cells
7.6.1
Principles of Single-molecule Dynamic Force Spectroscopy and Applications to Cell
Surface Receptors
Various techniques have been developed to measure molecular forces. Most
prominent force transducers are optical and magnetic tweezers, atomic force
microscopy (AFM) cantilevers, and biomembrane force probes. In recent years,
atomic force microscopy has become a powerful technique to visualize and
manipulate single biomolecules in native-like environments, making it possible to
study the structure of complex biological systems at sub-nanometer resolu-
tion [117, 118], the folding/unfolding trajectories of single proteins [119
-
121],
and the dynamics of chemical bond rupture/formation between a ligand and
its receptor [122, 123]. In the following section, particular emphasis will be given
to the analysis of ligand
-
receptor interactions in living cells by dynamic force
spectroscopy. For recent reviews on the principles of single molecule force measure-
ments, preparation of tips, and recognition imaging, the reader can refer to
Kienberger et al. [124], and Hinterdorfer and Dufrene [125]. (For reviews on the
use of AFM in living cells, see [126
-
130]).
Different con
gurations for the immobilization of living cells have been used in
single-molecule force measurements (Figure 7.4A): (i) the AFM tip is functionalized
with ligand molecules and living cells expressing speci
c membrane receptors
are immobilized on the supporting surface, (ii) a cell is mounted on a modi
ed
(tipless) AFM cantilever (Figure 7.4B) and ligands are attached on the supporting
