92 K. Mruk and W. R. Kobertz
revealed that the top of the S3 transmembrane domain is farthest away from the
center of the pore, followed by S1 and that the extracellular loop between the S3
and S4 transmembrane domains are the closet to the channel pore. The subsequently
solved crystal structures of different Kv channels (Lee et al. 2005 ; Long et al. 2005 )
indicated that although these distances were systematically shorter than the atomic
distances, the arrangement of transmembrane helices was correct. Reexamination
of the data revealed that using 50 % block yield similar distances as seen in the crys-
tal structure (Morin and Kobertz 2008b), demonstrating that these molecular tape
measures provide a straightforward approach to determine the radial arrangement
of helices around the pore of K+ channels.
As the number of high-resolution structures of ion channel partners has in-
creased, so has the utility of the panel of channel blockers. Using an extended
panel of the polyglycine blockers, we developed an intracellular tethered blocker
approach to measure distances between the cytoplasmic protein, calmodulin, and
pore of KCNQ2/KCNQ3 K+ channels (Mruk et al. 2012 ). In this intracellular ver-
sion, calmodulin acts as the targeting moiety, increasing the effective concentration
of a low affinity K+ channel blocker, tetraethylammonium (TEA). To reach the cy-
toplasm, the TEA-derivatized calmodulin tethers were injected into Xenopus oo-
cytes expressing heteromeric KCNQ2/KCNQ3 channels and the resultant currents
were measured for each tether length. Unlike previously described extracellular
tethers, this intracellular approach utilized wild type channel subunits, obviating
the need for cysteine mutants and the general caveats associated with site-specific
mutagenesis. After generating distances between calmodulin residues and the qua-
ternary-ammonium binding site (Fig. 4b), we generated quaternary models of the
calmodulin-KCNQ2/KCNQ3 complex in the open state using previously crystal-
lized calmodulin and Kv1.2 structures (Fig. 4c). These models placed calmodulin
very close to the gate of a conducting KCNQ2/KCNQ3 channel. In addition to iden-
tifying the location of calmodulin binding, these chemically-derivatized calmodu-
lin proteins also indicated that unlike previously thought, calmodulin can associate
with channels at the cell surface, which was later corroborated by the Villarroel
Group (Gomez-Posada et al. 2011 ).
In addition to tethered blockers, ion channel biophysicists have used fluores-
cence and luminescence resonance energy transfer (FRET/LRET) as spectroscopic
rulers to map distances and channel movements (Chanda et al. 2005 ; Posson et al.
2005 ). These methods are advantageous in that they do not require the channel to
be actively conducting, but for several technical reasons (Bosmans 2013 ), are chal-
lenging to employ for distances that are shorter than 20 Å. Combining energy trans-
fer and tethered blockers, the Chandra lab generated a library of “tethered quench-
ers”: fluorescence quenchers (nitroxy radicals and dibromo groups) tethered to TEA
via PEG linkers to measure distances between multiple sites within the Shaker K+
channel (Jarecki et al. 2013 ). The advantage of this hybrid approach is that it does
not rely on the channel being in the open state; thus, tethered quenchers can provide
structural information about an ion channel in its closed, inactivated or desensitized
state. Using voltage-clamp fluorometry, they determined distance constraints for
four different sites on the channel and detected distance changes as small as 4 Å.