94 K. Mruk and W. R. Kobertz
linker (Fig. 5b). Because CTX binds to the redesigned KCNQ1 channels with high
affinity, at nanomolar concentrations, CTX-MAL behaves as affinity labeling re-
agent—binding to the channel first, and then the maleimide reacts with a cysteine
that is within reach of the tether arm. These covalently bound, tethered toxins ir-
reversibly inhibit channel function and preclude additional blockers from binding
to the channel. By engineering a cysteine into the N-terminus of a specific KCNE β
subunit, CTX-MAL was used to selectively and irreversibly block KCNQ1-KCNE
complexes that contained a specific KCNE subunit. This tethered blocker approach
unequivocally demonstrated that KCNQ1 channels can assemble with two different
Fig. 5 Delivery of molecular probes via chemically-reactive tethers. a Cartoon of selective label-
ing of a functioning ion channel using a cleavable bioreactive tether. Star and halo depict the
chemically reactive group and the effective molarity created by ligand binding. Cleavage (scis-
sors) of the tether releases the channel binding ligand, restoring function and leaving behind the
molecular probe on a subunit of the ion channel complex. b Structures of the various bioreactive
probes. CTX: charybdotoxin. Note the CTX-Mal tether is not cleavable by either reductant or light