Location of KCNE1 relative to KCNQ1 in the I _KS potassium channel by disulfide cross-linking of substituted cysteines

Abstract

The cardiac-delayed rectifier K ؉ current (IKS) is carried by a complex of KCNQ1 (Q1) subunits, containing the voltage-sensor domains and the pore, and auxiliary KCNE1 (E1) subunits, required for the characteristic IKS voltage dependence and kinetics. To locate the transmembrane helix of E1 (E1-TM) relative to the Q1 TM helices (S1-S6), we mutated, one at a time, the first four residues flanking the extracellular ends of S1-S6 and E1-TM to Cys, coexpressed all combinations of Q1 and E1 Cys-substituted mutants in CHO cells, and determined the extents of spontaneous disulfide-bond formation. Cys-flanking E1-TM readily formed disulfides with Cysflanking S1 and S6, much less so with the S3-S4 linker, and not at all with S2 or S5. These results imply that the extracellular flank of the E1-TM is located between S1 and S6 on different subunits of Q1. The salient functional effects of selected cross-links were as follows. A disulfide from E1 K41C to S1 I145C strongly slowed deactivation, and one from E1 L42C to S6 V324C eliminated deactivation. Given that E1-TM is between S1 and S6 and that K41C and L42C are likely to point approximately oppositely, these two cross-links are likely to favor similar axial rotations of E1-TM. In the opposite orientation, a disulfide from E1 K41C to S6 V324C slightly slowed activation, and one from E1 L42C to S1 I145C slightly speeded deactivation. Thus, the first E1 orientation strongly favors the open state, while the approximately opposite orientation favors the closed state.arrhythmias ͉ cardiac repolarization ͉ electrophysiology ͉ atrial fibrillation ͉ S1T he slow, outwardly rectifying K ϩ current (I KS ) is one of two delayed rectifier K ϩ currents critical for repolarization of the heart, particularly during sympathetic nervous system stimulation (1, 2). The I KS channel is composed of four pore-forming KCNQ1 (Q1) subunits and two auxiliary KCNE1 (E1) subunits (3-5). Several human mutations in Q1 and E1 cause variants of long QT syndrome (6), short QT syndrome (7), or atrial fibrillation (8, 9).Although a tetramer of Q1 subunits alone forms a voltagegated channel, only Q1 and E1 together form a channel with the slow activation and deactivation kinetics and the minimal inactivation characteristics of I KS (10, 11). Furthermore, E1 is necessary for sympathetic modulation of I KS (12). How E1 exerts its effect on Q1 is not yet fully understood.There have been a number of conclusions about Q1-E1 interactions in the I KS channel, not all of which are compatible. There is evidence for (13) and against (14, 15) the contribution of E1 to the pore wall and its accessibility from the pore. There is also evidence that E1 interacts with the pore domain, although not necessarily exposed in the pore (16,17), that the E1 TM helix (E1-TM) interacts directly with Q1 S4 helix (18), that E1 modulates Q1 through its C terminus (19-21), and that E1 interacts with the cytoplasmic Q1 S4-S5 linker (22).More recently, a site of possible Q1-E1 interaction was suggested by the association of mutations in Q1 S...

KCNE1 alters the voltage sensor movements necessary to open the KCNQ1 channel gate

“…To avoid fluorophore labeling of endogenous cysteines on the I Ks channel, we first mutated two endogenous cysteine residues that are predicted to be accessible to the extracellular milieu, C214 and C331, to alanine, as in Chung et al (11). These two mutations have limited functional consequences on the channel expressed either alone or with KCNE1, and thus we used this KCNQ1 construct (with C214A; C331A) as a background for subsequent experiments.…”

“…Two broad possibilities seem likely: (i) KCNE1 restricts the KCNQ1 voltage sensor, delaying its movement in response to changes in membrane voltage, and/or (ii) KCNE1 restricts the movement of the KCNQ1 gate or changes the coupling between movement of the voltage sensor and opening of the channel gate so that the KCNQ1/KCNE1 channel opens more slowly in response to voltage sensor movement. Recent disulfide-crosslinking experiments have established that KCNE1 is likely located between the peripheral voltage-sensing domain and the pore domain of KCNQ1, in a unique position to affect voltage-sensing movements, coupling between the voltage sensor and pore, or both (11,12).…”

Steric hindrance between S4 and S5 of the KCNQ1/KCNE1 channel hampers pore opening

“…Binding of KCNE1 may reduce the flexibility and lower the mobility 44 , so that Phe232 may have a narrower space to avoid Phe279 during depolarization. KCNE1 is known to bind to the PD 21,22 and the voltage sensor domain 23,25 . However, the precise location of KCNE1 still remains to be elucidated 26,[44][45][46] .…”

“…KCNE1 has been reported to bind to the PD of KCNQ1 21,22 , but growing evidence suggests the possibility that the VSD could be an interaction site as well [23][24][25][26] . By using the substituted cysteine accessibility method (SCAM) 27,28 , two groups including ours previously showed that KCNE1 slows the reaction rate of extracellular MTS reagents with the S4 segment, indicating that KCNE1 stabilizes the downstate of the VSD 23,29 .…”

KCNE3 acts by promoting voltage sensor activation in KCNQ1

“…However, disulfide crosslinking studies suggest that KCNE1 localizes laterally of the central pore domain in the otherwise lipid-filled crevices between voltage sensor domains of KCNQ1 channels (28)(29)(30), such that KCNE1 could act on voltage sensors, the pore, or the coupling between voltage sensors and pore. Because of the high sequence similarity within the KCNE family, KCNE3 is likely positioned in the KCNQ1 channel structure much like KCNE1.…”