85enables the modification of endogenous channels; however, it can be challenging
to unequivocally demonstrate whether and where the modification occurs given
the electrophile’s lack of chemoselectivity (Banghart et al. 2009 ). The second al-
ternative approach utilizes glycocalyx re-engineering to metabolically incorporate
thiol groups into the cell surface sialic acid residues on N- and O-linked glycans
(Mahal et al. 1997 ). This straightforward approach entails incubating cells with an
unnatural peracetylated mannosamine derivative for 2–3 days (Hua et al. 2011 ).
Similar to tethers with non-specific electrophiles, selective labeling arises from the
effective molarity generated by the ion channel-targeting moiety because the entire
cell surface is coated with reactive thiol groups. Expression of an unglycosylated
version of the ion channel complex provides a means to determine whether the
bioreactive tether specifically modified the unnatural N-glycans on the ion channel
subunit(s).
The vast majority of bioreactive tethers have been designed to target the ex-
tracellular surface of the channel. To move into the cytoplasm, we have replaced
thiol-specific chemistries with calmodulin (Mruk et al. 2012 ). In this intracellular
version, a chemically-derivatized calmodulin acts as the anchoring moiety by bind-
ing to its site(s) on the ion channel, obviating the need for a covalent bond (Fig. 1 ,
cytoplasmic reagent). In theory, any high affinity protein-peptide interaction could
be converted into a cytoplasmic bioreactive tether. Furthermore, robust calmodulin
binding sites can be designed and engineered into ion channels that do not bind
calmodulin (Mruk et al. 2014 ). In addition to reversible binding strategies, future
bioreactive tethers that utilize bioorthogonal chemistries (e.g. azides, alkynes, alde-
hydes and ketones) via unnatural amino acid incorporation could facilitate covalent
modifications to ion channel surfaces that have been protected by the lipid bilayer
and reducing environment of the cytoplasm.
- Electrophysiology with bioreactive tethers: The advantage of probing ion chan-
nel function with bioreactive tethers is being able to monitor changes in channel
function using electrophysiology. Oocytes extracted from Xenopus laevis are a
versatile expression system for the structural and functional investigation of ion
channels and transporters. In addition, their large size makes them amendable
to introduction of exogenous molecules, during recordings or during biogenesis
via microinjection (Maduke et al. 1998 ; Marsal et al. 1995 ; Mruk et al. 2012 ;
Nowak et al. 1998 ). For experiments using different macromolecules (e.g. RNA
and protein), sequential injection into the oocyte gives the most reproducible
results. For channels containing an extracellular cysteine, oocytes are stored in
glutathione to prevent oxidation of the engineered cysteine. Before experiments,
oocytes are quickly rinsed in solution containing a reducing agent to expose the
free cysteines. Electrophysiological recordings are taken 24–96 h after injection
when the current is ~ 10x over background current (> 1 μA). For experiments
using purified protein, the recording window is adjusted to the turnover rate of
the protein injected. The bioreactive tether can be directly dissolved in the perfu-
sion media, allowing for constant exposure to the reagent. Due to their opacity,
the oocyte is non-ideal for studies with azobenzene linkers.
Bioreactive Tethers