93These distances were consistent with computational resting-state models proposed
by Jensen et al., solidifying the understanding of Kv channel’s movements between
functional states (Jensen et al. 2012 ).
The molecular ruler approach has provided detailed structural information (at
the Ångstrom level) for ion channels and their partner proteins, which has often
been confirmed through high-resolution structures and molecular modeling. The
approach is incredibly modular, allowing for the probing of different channels by
simply varying the agonist, blocker, quencher or different accessory subunit. How-
ever, the challenge with the approach is that it is cumbersome because an entire
panel of compounds must be synthesized, purified and tested. The applicability of
this method would increase dramatically if there was an approach to systematically
elongate (or shorten) tether length during electrophysiological recordings.
3.3 High Affinity Biochemically-Reactive Tethers
Tethering small ligands close to their binding sites provides another surgical ap-
proach to study ion channel structure and function. In addition, designing tethers
with chemically cleavable linkers affords the opportunity to deliver molecular
probes to specific ion channel subunits (Fig. 5a). During the last decade, many
unique peptide and small molecule toxins with selective channel affinities have
been identified. Many of the peptide toxins are well folded, cysteine-disulfide bond-
ed small proteins that can be expressed in bacteria with a “spinster” cysteine, pro-
viding a handle for chemical modification. The identification of new types of high
affinity ion channel ligands expands the repertoire of molecules available to probe
ion channel function and increases the possibilities for teasing apart the contribu-
tions of specific channels in neuronal networks all in the context of wild-type cells.
Using peptide toxins to probe ion channel structure and function has a long
and storied use, including determining the α-subunit stoichiometry of K+ channels
(MacKinnon 1991 ), mapping the outer channel vestibule of ion channels (Chen
et al. 2003 ), and aiding in the synthesis of therapeutic compounds to target par-
ticular classes of ion channels (Lewis and Garcia 2003 ). In addition, toxins have
been genetically encoded to be tethered to the plasma membrane to control channel
function (Ibanez-Tallon and Nitabach 2012 ). Traditional structure-function studies
have determined the residues required for toxin binding to specific ion channels,
allowing toxin sensitivity to be engineered into insensitive ion channels further ex-
panding the usefulness of this technique.
Morin and Kobertz took advantage of these versatile toxin properties to cre-
ate a chemically-reactive toxin to probe the cardiac IKs complex made up of the
KCNQ1 K+ channel and its regulatory KCNE β subunits (Morin and Kobertz
2007 ). Because high affinity ligands did not exist for the extracellular vestibule of
wild type KCNQ1, charybdotoxin sensitivity was engineered into KCNQ1 chan-
nels (Chen et al. 2003 ). The first derivatized toxin synthesized was a maleimido-
charybdotoxin (CTX-MAL), which contained a non-cleavable triethylene glycol
Bioreactive Tethers