Abstract
In vivo, KCNQ1 α-subunits associate with the β-subunit KCNE1 to generate the slowly activating cardiac potassium current (I(Ks)). Structurally, they share their topology with other Kv channels and consist out of six transmembrane helices (S1-S6) with the S1-S4 segments forming the voltage-sensing domain (VSD). The opening or closure of the intracellular channel gate, which localizes at the bottom of the S6 segment, is directly controlled by the movement of the VSD via an electromechanical coupling. In other Kv channels, this electromechanical coupling is realized by an interaction between the S4-S5 linker (S4S5(L)) and the C-terminal end of S6 (S6(T)). Previously we reported that substitutions for Leu(353) in S6(T) resulted in channels that failed to close completely. Closure could be incomplete because Leu(353) itself is the pore-occluding residue of the channel gate or because of a distorted electromechanical coupling. To resolve this and to address the role of S4S5(L) in KCNQ1 channel gating, we performed an alanine/tryptophan substitution scan of S4S5(L). The residues with a "high impact" on channel gating (when mutated) clustered on one side of the S4S5(L) α-helix. Hence, this side of S4S5(L) most likely contributes to the electromechanical coupling and finds its residue counterparts in S6(T). Accordingly, substitutions for Val(254) resulted in channels that were partially constitutively open and the ability to close completely was rescued by combination with substitutions for Leu(353) in S6(T). Double mutant cycle analysis supported this cross-talk indicating that both residues come in close contact and stabilize the closed state of the channel.
Highlights
A fundamental property of all Kv channels is their ability to detect a change in membrane potential (Vm) and to respond to this change by opening or closing their activation gate that seals off the ion permeation pathway in a closed configuration [3, 4]
An alanine or a charged residue substitution for Leu353 precluded normal channel closure and resulted in a constitutively partial open channel. This impaired channel closure could be explained in two ways: 1) the side chain of residue Leu353 itself forms the cytoplasmic activation gate that seals off the ion permeation pathway or 2) Leu353 interacts with residues of S4-S5 linker (S4S5L) in the closed channel configuration and mutating Leu353 results in a loosened electromechanical coupling with a failure to close completely
Alanine/Tryptophan Scanning Mutagenesis of S4S5L in KCNQ1—To determine which residues in S4S5L are involved in KCNQ1 channel gating, we performed an alanine substitution scan combined with selected tryptophan mutations
Summary
S6T, C-terminal end of the S6 segment; S4S5L, S4-S5 linker; VSD, voltage-sensing domain. S4S5L in KCNQ1 Activation and Inactivation Processes in the S4S5L of KCNQ1 that participate in the gating machinery. These residues group together on one side of S4S5L according to a homology model based on the Kv1.2 structure. By combining specific S4S5L substitutions with Leu353 mutants, we could restore normal channel closure, suggesting that these residue pairs interact or come in close contact in the closed channel configuration and are part of the electromechanical coupling in KCNQ1 channels
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