The S4-S5 Linker of KCNQ1 Channels Forms a Structural Scaffold with the S6 Segment Controlling Gate Closure

Abstract

In vivo, KCNQ1 ␣-subunits associate with the ␤-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 ␣-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.KCNQ1 (KvLQT1) ␣-subunits tetramerize to create a voltage-gated K ϩ (Kv) channel. Like other Kv channels, each ␣-subunit contains six membrane spanning segments (S1-S6) with a pore loop between the fifth and sixth segment that forms the selectivity filter. The co-assembly with KCNE1 (minK) ␤-subunits generates the channel complex that underlies the native I Ks in the heart (1, 2). A fundamental property of all Kv channels is their ability to detect a change in membrane potential (V m ) 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). The channel activation gate is located in the C-terminal end of the S6 segment (S6 T ), 3 whereas the voltage-sensing domain (VSD) is formed by the S1-S4 segments with the charged S4 as the main component (5). Substitution of the S4 charges in KCNQ1 perturbed channel gating (6, 7) and gating currents that originate from the redistribution of these S4 charges have recently been recorded for the KCNQ family of Kv channels (8). These data indicate that as in Shaker-type channels, the S1-S4 segment in KCNQ1 forms the channel VSD that reorients upon a change in membrane potential. This VSD reorientation is then translated into channel gate opening or closure through an electromechanical coupling. This coupling mechanism remains poorly defined but sev...

KCNQ1 Channels Voltage Dependence through a Voltage-dependent Binding of the S4-S5 Linker to the Pore Domain

“…Rescue of L353A Channel Closure by V254L Peptide Further Supports Peptide Specificity-In the companion article (31), the partial open phenotype of the L353A mutant channel could be rescued by the V254L mutation in the channel S4S5 L. In parallel, in the experiments with peptides, introduction of the V254L mutation in the S4S5 L peptide restored its effect on the L353A mutant channel (Fig. 5, A and B).…”

“…To further address the specificity of the interaction between the peptides and the channel, we evaluated the effect of peptides bearing mutations known to decrease S4S5 L and S6 T interaction. In an accompanying paper, Snyders and co-workers (31) show that the V254A mutation in S4S5 L and the L353A mutation in S6 T generate an instantaneous current component, suggesting that S4S5 L /S6 T interaction is weakened by the mutations (see companion article (31)). Indeed, introduction of the V254A mutation into the S4S5 L peptide Leu 251 -Leu 266 abolished the peptide inhibition of the channel.…”

“…Black arrows in Fig. 1 show the high impact residues studied in KCNQ1, and red arrows show the interacting residues Val-254 and Leu-353 (12 and see accompanying article (31) . For that reason, one can suppose that these peptides will be the least efficient.…”

“…Scanning mutagenesis studies were performed in a cardiac voltage-gated potassium channel, KCNQ1 (10,11) to study the role of S4S5 L (see companion article (31)) and S6 T (12) in the voltage modulation of this channel. These studies identified residues implicated in the S4S5 L /S6 T interaction.…”

Homology Modeling: Generating Structural Models to Understand Protein Function and Mechanism

“…Homology-based structural models have already been used widely in in silico drug screening [11][12][13]. For biological experiments, structural models can be used to design mutations that lead to specific changes in the function or stability of the modeled protein [14,15]. Importantly, homology models can be used as starting models for molecular replacement in X-ray crystallography [16], leading to better experimental structures.…”