TOPOGRAPHY AND RECOGNITION IMAGING OF CELLS - Life at the Nanoscale: Atomic Force Microscopy of Live Cells - page 155

living and ixed cells lead to similar results (force distributions and binding
probabilities). The data obtained with living cells are presented in Fig.
7.8 . The anti-K v 11.1 (hERG)-extracellular antibody is known to bind to the
voltage-sensor domain (S1-S2 region) of HERG K + channel ( Fig. 7.7a ) . The
speciic binding of the antibody to the extracellular part of hERG channel
was characterized by a unique unbinding force. To conirm the speciicity of
this binding, blocking experiments were carried out by injecting either free
antibodies or free peptide antigen. In both cases, almost no unbinding events
were observed. Binding probabilities (probability to record an unbinding
event in force-distance cycles) from several experiments were also calculated
( Fig. 7.8a ) . The binding probability of ~30% was calculated for the interaction
between anti-Kv11.1-extracellular antibody and hERG HEK-293 cells. When
free anti-Kv11.1 antibodies or free peptide antigens were present in solution,
the binding probability drastically decreased to the level of ~2% ( Fig. 7.8a ) .
By constructing an empirical probability density function of the unbinding
forces ( Fig. 7.8b ) , the maximum of the distribution was found to be 45

p

9 pN.
Another indicator of the speciicity, a very low binding probability (~1.5%)
with a force peak of ~25 pN ( Fig. 7.8b ) , was found for the parent HEK-293 cells
not expressing hERG K + channels. These results illustrate that the extracellular
part of hERG K + channel expressed in living cells can be speciically detected
at the molecular level by using epitope-speciic antibodies.
The possible effects of ErgTx1 on antibody binding were further
investigated. Force curves were accumulated before and after ErgTx1
multiple injections in the same scan area with the same functionalized tip. In
the presence of ErgTx1 at different concentrations, the peak force for force
distributions ( Fig. 7.8b ) remains at the same position, whereas the binding
probability between antibody and living hERG HEK 293 cells dramatically
decreased following multiple ErgTx1 injections ( Fig. 7.8 ) . These results
provide support about a possible new binding site of ErgTx1 in the voltage-
sensor domain of hERG K + channel.
Thus, it has been demonstrated that the combination of dynamic molecular
recognition imaging (TREC) with single-molecule force spectroscopy is a
suitable method to obtain information about the structure and function of
hERG K + channels. Both techniques exploit AFM tips with a very low surface
density of ligands (~400 molecules per μm 2 ) and thus allow the detection
of single molecular events. Functionalization of AFM tips with anti-Kv11.1
(hERG)-extracellular antibody enabled them to detect binding sites of hERG
on the cell surface expressed hERG channels. The main outcome of this study
reveals that the voltage-sensing domain (S1-S4) of hERG K + channel might be
one of the binding sites of ErgTx1.