86 K. Mruk and W. R. Kobertz
Channels can also be expressed in cell culture and channel function monitored by
whole cell patch clamp recordings. Human embryonic kidney (HEK293) kidney
cells are often used, as they are easy to maintain and produce small background
current from endogenous channels. Similar to oocytes, the tether is added to the ex-
tracellular recording solution and continuously perfused for both the whole cell and
inside-out configuration. Alternatively, primary cells can be isolated from animal
models. Slices must be pre-treated with the tether before recording and slices are
exposed to a constant stream of perfusion solution, which includes CO 2 to maintain
the slice.
For studies using photoreactive or photoswitchable tethers, a light source is
required. Compatible light sources include a monochromator, a xenon lamp, or a
high-power microscope with a mercury lamp or LED with corresponding beam
splitters. A continuous-wave or ultrafast-pulsed laser is particularly advantageous
for patterned illumination and two-photon excitation experiments.
3 Applications
Bioreactive tethers have been utilized to determine subunit and ligand stoichiom-
etry, control ion channel function, elucidate currents from heteromeric complexes,
develop potent and irreversible inhibitors, and measure radial distances of ancillary
subunits and voltage sensors from the ion conduction pathway (Table 1 ). Because
bioreactive tethers have been used to solve unique functional, structural and stoi-
chiometric conundrums for a wide variety of ion channels, we discuss the applica-
tions of bioreactive tethers not by the individual applications, but organized into
three classes of tethers: photoswitchable, molecular rulers, and chemically reactive.
3.1 Photoswitchable Tethers
Among the different techniques to control ion channel function, optical methods
outshine the competition because light can be focused onto a small area sparing
neighboring cells and activated quickly allowing for rapid onset or termination of
activity. Optical control over protein function can be divided into two methods:
phototriggers and photoswitches. Photoswitches offer the advantage of being re-
versible and can be switched repeatedly providing exquisite spatiotemporal control
over protein function. The most commonly used photoswitches to study ion channel
function are azobenzenes. Azobenzene undergoes reversible cis/trans isomerization
around a nitrogen-nitrogen double bond that connects two benzene rings (Demsel-
ben 1834 ; Hartley 1937 ). Illumination with long wavelength ultraviolet light leads
to trans to cis photoisomerization whereas illumination with visible light induces
cis to trans photoisomerization creating a switch between the two conformations
(Fig. 3a). As the switch toggles between the two states, the change in geometry
alters the ligand-binding efficacy, leading to modulation of channel function.