78 K. Mruk and W. R. Kobertz
These reagents have been traditionally bifunctional: containing two “targeting”
moieties that associate with an ion channel of interest linked by a flexible tether,
which bestows these molecules enhanced avidity (Fig. 1 ). The non-covalent target-
ing moieties can be small molecules, peptides, or even small proteins that bind to
an ion channel of interest whereas the chemically-reactive targeting groups have
primarily relied on the chemoselectivity of the thiol group, though other functional
groups have been employed. In contrast, the tethers can be simple strings of small
molecules with defined lengths or customized with additional functionalities such
as photochromicity or cleavability. The modular nature of the bioreactive tether
enables the simple design of a molecular probe that predictably manipulates ion
channel function, providing instrumental tools for studying ion channel complexes.
Given the abundance of specific ligands for ion channels, the only limitation is the
imagination and patience to synthesize the desired set of bioreactive tethers.
Bioreactive tethers provide unique advantages over simple ion channel agonists,
antagonists, and blockers. Bioreactive tethers that reversibly bind and then cova-
lently modify an ion channel via the reagent’s electrophile act as selective affin-
ity labeling reagents. An ideal length tether can increase the local concentration
into the millimolar range, accelerating the reaction between the bioreactive moiety
and the ion channel. This effective molarity also yields electrophilic reagents with
greater specificity compared to traditional electrophiles, which randomly react with
any cell surface protein. Another advantage of bioreactive tethers is that the cova-
lently attached modifier’s (i.e. blocker or agonist) efficacy is directly proportional
to tether length. This correlation can be exploited to make molecular measurements
as well as manipulate ion channel function.
Bioreactive tethers have evolved over the course of ion channel studies from
probing the biophysical properties of ion channels to manipulating action potential
Fig. 1 Cartoon depiction of cell-based tethered blocker approaches. Cells expressing an ion chan-
nel complex of interest are either bathed in the reagent ( wavy lines) or the bioreactive tether can
be injected into cells. Electrophysiological recordings (shown) or fluorescence is used to monitor
signal changes caused by application of the bioreactive tether