Bioreactive Tethers 79
firing. The first bioreactive tether (Bartels et al. 1971 ) used the acetylcholine receptor
agonist, trans-3-(a-bromomethyl)-3’-[a-(trimethylammonium)methyl]azobenzene
( trans-Q-Br), to probe the kinetics of the nicotinic acetylcholine receptor (Lester
et al. 1979 ; Chabala and Lester 1986 ). trans-Q-BR had two functionalities—(1)
the ability to selectively alkylate a cysteine in the acetylcholine binding pocket;
(2) photoisomerize between inactive and active configurations when bound to the
receptors—permitting the measuring of agonist-induced conductance upon treat-
ment with light. Similarly, the Karpen lab also utilized modified agonists to probe
the ligand binding domains of cyclic nucleotide-gated channels and later study the
multiple site regulation of channel gating (Brown et al. 1993 ; Karpen and Brown
1996 ; He and Karpen 2001 ). The photoaffinity probe, 8-p-azidophenacylthio-
cGMP (APT-cGMP) labeled the smaller subunit of the cGMP-activated channel
in the presence of UV light leading to channel activation. This irreversible activa-
tion allowed them to measure the functional consequences of ligand binding to
the remaining binding sites on the channel without the tethered ligand dissociating
and rebinding, providing insight into the individual binding sites that could not be
extracted from studies using free ligand alone.
In addition to tethered agonists, tethered blockers have also been used to probe
ion channel structure. The first tethered blocker study utilized a panel of quaternary
ammoniums (QAs) linked to a maleimide with variable tether lengths to measure
distances from the Shaker K+ channel voltage-sensing domain to the external tet-
raethylammonium (TEA) blocking site within the channel pore (Blaustein et al.
2000 ). Because QAs have low affinity for K+ channels, inhibition is highly depen-
dent on the effective concentration generated by the length of the linker between
the tether site and the QA. Therefore, this panel of tethered blockers behaved as
molecular calipers to measure radial distances from the Shaker K+ conducting pore
to extracellular residues in the voltage-sensing domain. This tethered blocker strat-
egy revealed that the top of the S3 transmembrane domain is the farthest away from
the pore, followed by S1 and that the S3-S4 loop is the closest to the pore. This ar-
rangement of transmembrane helices has been confirmed by several high-resolution
structures of voltage-gated potassium channels (Long et al. 2005 , 2007 ). As the
number of high-resolution structures of ion channels increased, so have the func-
tionalities of these tethered blockers, enabling nanometer resolution of ion channel
movements, quaternary structural determinations, and spatiotemporal control of ac-
tion potential firing in neurons and live animals.
In this chapter, we cover three classes of tethers. The first are photoswitchable
tethers, which change conformation in response to light allowing for spatiotemporal
control of ion channel function. The second are molecular rulers that take advantage
of changes in localized concentration that occur with tether length. Panels of these
tethers have not only provided structural information about the ion channel itself,
but also about its associated partner proteins. Lastly, we discuss biochemically re-
active tethers. These tethers rely on basic chemistry in combination with a cell’s
normal biosynthetic pathways to manipulate channel function.