82 K. Mruk and W. R. Kobertz
45 Å (7 glycines) are both much harder to synthesize and purify; however, contract
research organizations (CROs) that specialize in peptide synthesis are able to gener-
ate longer polyglycine linkers. Indeed, we have utilized this approach to generate
bioreactive tethers as long as 66 Å (12 glycines) (Mruk et al. 2012 ). For longer
linkers, polyethylene glycol (PEG) monomers can be used. Several companies cur-
rently sell Boc- and Fmoc-protected PEGylated amino acids. Alternatively, the PEG
monomers can be synthesized (Jarecki et al. 2013 ).
Photoisomerizable linkers offer the ability to instantaneously alter tether length
on a functioning channel. Azobenzene has been used as the linker because excitation
with different wavelengths of light leads to cis-trans and trans-cis isomerization
(Fig. 2c) without any long-lived excited states, reactive intermediates, or competing
reactions (Bartels et al. 1971 ). Azobenzene absorbs long-wave UV and visible light,
which can be easily generated by flash lamps and pulsed lasers. Figure 2c depicts
the simplest two-step synthesis using diaminoazobenene. After purification, the
second amino group is modified, yielding a bifunctional photoisomerizable linker.
Several different derivatives of azobenzene are commercially available, allowing
for the addition of different spacers and electrophiles.
Tethers with orthogonally cleavable linkers provide another approach to manipu-
late channel function as well as deliver small molecule probes to functioning ion
channels. Three functional groups have been utilized to create a cleavable linker:
disulfide bonds, dithiocarbmates, and nitroindolines (Table 1 ). Nitroindoline con-
taining linkers are cleavable with UV light whereas disulfide bonds and dithio-
carbmates are cleaved by cell compatible reductants (TCEP and DTT); however,
the latter functional group does not result in the regeneration of a thiol group, but
rather a secondary amine. The original references of these cleavable linkers provide
sufficient detail of these multi-step syntheses (Morin and Kobertz 2007 , 2008a;
Vytla et al. 2011 ; Hua and Kobertz 2013 ). Once synthesized, these linkers can be
appended to ion targeting moieties as described above.
- Identifying an attachment site: For experiments that do not involve distance
measurements, the ion channel attachment site of the bioreactive tether is criti-
cal for efficient labeling and precise control of channel function. For extracel-
lular tethering, site-directed cysteine mutagenesis has been predominately used
to generate a specific attachment site. Maleimide is the most commonly used
electrophile because it is relatively stable and forms an irreversible thioether
bond with cysteine at physiological pH. Because the reaction between a bioreac-
tive tether and the target cysteine is strongly dependent on tether length, several
different target cysteines should be tested to identify an ideal attachment site.
Alternatively, the linker of the bioreactive tether may be elongated (or short-
ened); however in practice, optimizing the attachment site on the ion channel is
more facile and effective (Morin and Kobertz 2007 ).
There are two attachment site alternatives to mutating a specific residue to cyste-
ine. The first is to utilize a non-specific electrophile (e.g. acrylamide, chloroacet-
amide, epoxide) and exploit the effective molarity of the bioreactive tether to target
a specific ion channel (Fortin et al. 2008 ). An advantage of this approach is that it