91At the time, the PLD tethered approach was particularly remarkable because
there was no high-resolution structural information about the channels. But as
with photoswitchable tethers, the solution of the crystal structure of the bacterial
K+ channel, KcsA (Doyle et al. 1998 ), prompted investigators to use this strategy
to probe the voltage-sensing domain of Kv channels. Little was known about the
arrangement of the four sets of four transmembrane helices surrounding the pore
domain of the channel. To answer this question, Blaustein et al. synthesized a panel
of quaternary ammoniums tethered to maleimides with varying length polyglycine
linkers (Blaustein et al. 2000 ). Oocytes expressing extracellular cysteine-mutants of
the Shaker K+ channel were labeled and the current was measured for each length
tether. Using the fully extended tether length and onset of inhibition, this panel
Fig. 4 Tethered blockers as tape measures. a Cartoon of a channel with a quaternary ammonium
tethered to an extracellular cysteine ( red) or intracellular regulatory protein ( gold). The trans-
lucent blockers indicate that the tether is too short to reach the quaternary ammonium binding
site. b Distance calibration for three calmodulin residues (T35, T45, and T111). Curve fits to
generate the midpoint of inhibition ( dotted lines) are represented. c Quaternary models of the
KCNQ2/KCNQ3-calmodulin complex (membrane view), showing the channel subunits in grey
and calmodulin in pastels. The front subunits are not shown for clarity. Interrogated residues are
shown in spacefill, colors are same as in b. Potassium ions are in red, the purple sphere denotes the
quaternary ammonium binding site, and the dark blue sphere is used to approximate the trajectory
of the tethered blocker for the measured distances
Bioreactive Tethers