Abstract
The IKs current has an established role in cardiac action potential repolarization, and provides a repolarization reserve at times of stress. The underlying channels are formed from tetramers of KCNQ1 along with one to four KCNE1 accessory subunits, but how these components together gate the IKs complex to open the pore is controversial. Currently, either a concerted movement involving all four subunits of the tetramer or allosteric regulation of open probability through voltage-dependent subunit activation is thought to precede opening. Here, by using the E160R mutation in KCNQ1 or the F57W mutation in KCNE1 to prevent or impede, respectively, voltage sensors from moving into activated conformations, we demonstrate that a concerted transition of all four subunits after voltage sensor activation is not required for the opening of IKs channels. Tracking voltage sensor movement, via [2-(trimethylammonium)ethyl]methanethiosulfonate bromide (MTSET) modification and fluorescence recordings, shows that E160R-containing voltage sensors do not translocate upon depolarization. E160R, when expressed in all four KCNQ1 subunits, is nonconducting, but if one, two, or three voltage sensors contain the E160R mutation, whole-cell and single-channel currents are still observed in both the presence and absence of KCNE1, and average conductance is reduced proportional to the number of E160R voltage sensors. The data suggest that KCNQ1 + KCNE1 channels gate like KCNQ1 alone. A model of independent voltage sensors directly coupled to open states can simulate experimental changes in IKs current kinetics, including the nonlinear depolarization of the conductance-voltage (G-V) relationship, and tail current acceleration as the number of nonactivatable E160R subunits is increased.
Highlights
The IKs current has an established role in cardiac action potential repolarization, and provides a repolarization reserve at times of stress
Similar observations of fluorescence–voltage (F–V) and G–V nonconcordance have been made in the IKs channel complex [22, 23], and led these authors to incorporate a concerted opening step in channel gating into their IKs models, with the implication that the voltage sensor (VS) in all four subunits must be activated before pore opening can occur
Our results suggest that activation of all four VSs is not required for the IKs channel to conduct current, and that in IKs channels a final concerted transition is not obligatory for the pore to open but rather channels are better represented by a model where the pore can open from multiple closed-channel conformations, once one or more VS domains have entered an activated conformation
Summary
Channels with the E160R Mutation in All Four Q1 Subunits Are Nonfunctional. In wild-type (WT) channels, the VS domains of Q1 respond to changes in the membrane potential in a manner that eventually increases channel pore open probability. Dose–response relationships for V319Y EQQ and E160R/V319Y EQ*Q overlay one another, with IC50s for external TEA+ of 5.3 mM for both (SI Appendix, Fig. S5D) This result indicates that subunits containing the E160R mutation are not excluded from IKs channel complexes during assembly, and shows an identical TEA+ responsiveness, which suggests a uniform population of expressed channels. The second construct had both the E160R and G229C mutations in the first Q1 (G229C Q*-Q) When both MTSET fusion constructs were expressed in the presence of E1, they produced currents with a similar V1/2 of activation, 40 to 42 mV (SI Appendix, Fig. S10 A–D and Table S1). Significant change in the t1/2 before (sweep 5) or after (sweep 20) addition of 2 mM MTSET (Fig. 7D) This result shows that the E160R mutation in the same subunits as G229C prevents the G229C from being modified by MTSET when channels are activated. For G219C Q*-Q + E1, fluorescence changes were not observed, indicating that the presence of the E160R mutation was impeding VS movement (Fig. 8D, Lower)
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