TOPOGRAPHY AND RECOGNITION IMAGING OF CELLS - Life at the Nanoscale: Atomic Force Microscopy of Live Cells - page 152

In addition, standard single-molecule force spectroscopy measurements
were conducted on the same scanned surface area with the AFM tip
functionalized with VE-cadherin-Fc. Force curves were accumulated ( ~

500) before and after the blocking experiment. The force distribution of
cadherin-cadherin dissociation illustrates multiple force peaks of one-,
two- and threefold binding with a force quantum of ~40 pN ( Fig. 7.6b ) . This
characteristic force ingerprint is very similar to an isolated VE-cadherin
system. 35 The speciic unbinding events were abolished in free Ca 2+ conditions
(addition of 5 mM EDTA) accompanied by a reduction in binding probability
(from ~30% to ~1%). Therefore, force spectroscopy data explicitly conirm
that speciic domains contain active VE-cadherin

n

cis -dimers.

7.4.2 Localizaon of Ergtoxin-1 Receptors on the Voltage-Sensing
Domain of hERG K + Channel

TREC and single-molecule force spectroscopy have been recently introduced
as a novel way to investigate the properties of voltage-gated channels in cells. 43
Usually, the information about the structure and function of different voltage-
gated channels in living cells was gained from patch-clamp investigations.
The single-molecule AFM techniques have been exploited to detect a new
receptor site(s) for ergtoxin-1 (ErgTx1) in the voltage-sensing domain of the
human ether-à-go-go-related gene (hERG) potassium (K with the

aim of expanding an understanding about the microscopic mechanism of the
hERG K

+

) channel,

44

channel plays an important
role in the heart, 44 peripheral sympathetic ganglia, brain and tumour cells.
hERG channels are largely involved in myocardial repolarization

+

channel blockade with ErgTx1. hERG K

+

and are
associated with both the inherited and the acquired (drug induced) long QT
syndromes that may be responsible for fatal cardiac arrhythmias. ErgTx1
belongs to scorpion toxins, 43 which are K + channel blockers, and which
binds to the hERG channel with 1:1 stoichiometry and high afinity (Kd ~ 10
nM). Peptide toxins usually block the pore of the channel, either directly by
occupying the selective ilter or by binding to an electrostatic ring surrounding
the pore. Previously, it has been identiied that ErgTx1 binds to the outer
vestibule of the hERG channel.

45,46

Nevertheless, a characteristic feature of the
action of ErgTx1 on hERG is an incomplete block of macroscopic current
events at concentrations orders of magnitude higher than the

47

K d value. Such
results suggest that ErgTx1 is a gating modiier rather than a pore blocker. 48,49
In addition, it binds near the pore and cannot fully occlude the permeation
pathway. 50,51 The binding site for ErgTx1 on hERG is thought to be formed, at
least in part, by the extracellular linker between S5 transmembrane helix and
the pore helix (S5P linker), 48 which is critically involved in voltage-dependent
inactivation in hERG. 52