TEA(+)-sensitive KCNQ1 constructs reveal pore-independent access to KCNE1 in assembled I-Ks channels

I-Ks, a slowly activating delayed rectifier K+ current through channels for med by the assembly of two subunits KCNQ1 (KvLQT1) and KCNE1 (minK), contri butes to the control of the cardiac action potential duration. Coassembly o f the two subunits is essential in producing the characteristic and physiol ogically critical kinetics of assembled channels, but it is not yet clear w here or how these subunits interact. Previous investigations of external ac cess to the KCNE1 protein in assembled I-Ks channels relied on occlusion of the pore by extracellular application of TEA(+), despite the very low TEA( +) sensitivity (estimated EC50 > 100 mM) of channels encoded by coassembly of wild-type KCNQ1 with the wild type (WT) or a series of cysteine-mutated KCNE1 constructs. We have engineered a high affinity TEA(+) binding site in to the h-KCNQ1 channel by either a single (V319Y) or double (K318I, V319Y) mutation, and retested it for pore-delimited access to specific sites on co assembled KCNE1 subunits. Coexpression of either KCNQ1 construct with WT KC NE1 in Chinese hamster ovary cells does not alter the TEA(+) sensitivity of the homomeric channels (IC50 approximate to 0.4 mM [TEA(+)](out)), providi ng evidence that KCNE1 coassembly does not markedly alter the structure of the outer pore of the KCNQ1 channel. Coexpression of a cysteine-substituted KCNE1 (F54C) with V319Y significantly increases the sensitivity of channel s to external Cd2+, but neither the extent of nor the kinetics of the onset of (or the recovery from) Cd2+ block was affected by [TEA(+)](o) at 10X th e IC50 for channel block. These data strongly suggest that access of Cd2+ t o the cysteine-mutated site on KCNE1 is independent of pore occlusion cause d by TEA(+) binding to the outer region of the KCNE1/V319Y pore, and that K CNE1 does not reside within the pore region of the assembled channels.