Alain J. Labro

Gating of Shaker-type channels requires the flexibility of S6 caused by prolines

The recent crystallization of a voltage-gated K+ channel has given insight into the structure of these channels but has not resolved the issues of the location and the operation of the gate. The conserved PXP motif in the S6 segment of Shaker channels has been proposed to contribute to the intracellular gating structure. To investigate the role of this motif in the destabilization of the α-helix, both prolines were replaced to promote an α-helix (alanine) or to allow a flexible configuration (glycine). These substitutions were nonfunctional or resulted in drastically altered channel gating, highlighting an important role of these prolines. Combining these mutations with a proline substitution scan demonstrated that proline residues in the midsection of S6 are required for functionality, but not necessarily at the positions conserved throughout evolution. These results indicate that the destabilization or bending of the S6 α-helix caused by the PXP motif apparently creates a flexible “hinge” that allows movement of the lower S6 segment during channel gating and opening.

Kv channel gating requires a compatible S4-S5 linker and bottom part of S6, constrained by non-interacting residues.

Voltage-dependent K+ channels transfer the voltage sensor movement into gate opening or closure through an electromechanical coupling. To test functionally whether an interaction between the S4-S5 linker (L45) and the cytoplasmic end of S6 (S6T) constitutes this coupling, the L45 in hKv1.5 was replaced by corresponding hKv2.1 sequence. This exchange was not tolerated but could be rescued by also swapping S6T. Exchanging both L45 and S6T transferred hKv2.1 kinetics to an hKv1.5 background while preserving the voltage dependence. A one-by-one residue substitution scan of L45 and S6T in hKv1.5 further shows that S6T needs to be α-helical and forms a “crevice” in which residues I422 and T426 of L45 reside. These residues transfer the mechanical energy onto the S6T crevice, whereas other residues in S6T and L45 that are not involved in the interaction maintain the correct structure of the coupling.

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

In vivo, KCNQ1 α-subunits associate with the β-subunit KCNE1 to generate the slowly activating cardiac potassium current (IKs). 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 (S4S5L) and the C-terminal end of S6 (S6T). Previously we reported that substitutions for Leu353 in S6T resulted in channels that failed to close completely. Closure could be incomplete because Leu353 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 S4S5L in KCNQ1 channel gating, we performed an alanine/tryptophan substitution scan of S4S5L. The residues with a “high impact” on channel gating (when mutated) clustered on one side of the S4S5L α-helix. Hence, this side of S4S5L most likely contributes to the electromechanical coupling and finds its residue counterparts in S6T. Accordingly, substitutions for Val254 resulted in channels that were partially constitutively open and the ability to close completely was rescued by combination with substitutions for Leu353 in S6T. Double mutant cycle analysis supported this cross-talk indicating that both residues come in close contact and stabilize the closed state of the channel.

A polyether biotoxin binding site on the lipid-exposed face of the pore domain of Kv channels revealed by the marine toxin gambierol.

Gambierol is a marine polycyclic ether toxin belonging to the group of ciguatera toxins. It does not activate voltage-gated sodium channels (VGSCs) but inhibits Kv1 potassium channels by an unknown mechanism. While testing whether Kv2, Kv3, and Kv4 channels also serve as targets, we found that Kv3.1 was inhibited with an IC50 of 1.2 ± 0.2 nM, whereas Kv2 and Kv4 channels were insensitive to 1 μM gambierol. Onset of block was similar from either side of the membrane, and gambierol did not compete with internal cavity blockers. The inhibition did not require channel opening and could not be reversed by strong depolarization. Using chimeric Kv3.1–Kv2.1 constructs, the toxin sensitivity was traced to S6, in which T427 was identified as a key determinant. In Kv3.1 homology models, T427 and other molecular determinants (L348, F351) reside in a space between S5 and S6 outside the permeation pathway. In conclusion, we propose that gambierol acts as a gating modifier that binds to the lipid-exposed surface of the pore domain, thereby stabilizing the closed state. This site may be the topological equivalent of the neurotoxin site 5 of VGSCs. Further elucidation of this previously undescribed binding site may explain why most ciguatoxins activate VGSCs, whereas others inhibit voltage-dependent potassium (Kv) channels. This previously undescribed Kv neurotoxin site may have wide implications not only for our understanding of channel function at the molecular level but for future development of drugs to alleviate ciguatera poisoning or to modulate electrical excitability in general.

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

Voltage-dependent potassium (Kv) channels are tetramers of six transmembrane domain (S1–S6) proteins. Crystallographic data demonstrate that the tetrameric pore (S5–S6) is surrounded by four voltage sensor domains (S1–S4). One key question remains: how do voltage sensors (S4) regulate pore gating? Previous mutagenesis data obtained on the Kv channel KCNQ1 highlighted the critical role of specific residues in both the S4-S5 linker (S4S5L) and S6 C terminus (S6T). From these data, we hypothesized that S4S5L behaves like a ligand specifically interacting with S6T and stabilizing the closed state. To test this hypothesis, we designed plasmid-encoded peptides corresponding to portions of S4S5L and S6T of the voltage-gated potassium channel KCNQ1 and evaluated their effects on the channel activity in the presence and absence of the ancillary subunit KCNE1. We showed that S4S5L peptides inhibit KCNQ1, in a reversible and state-dependent manner. S4S5L peptides also inhibited a voltage-independent KCNQ1 mutant. This inhibition was competitively prevented by a peptide mimicking S6T, consistent with S4S5L binding to S6T. Val254 in S4S5L is known to contact Leu353 in S6T when the channel is closed, and mutations of these residues alter the coupling between the two regions. The same mutations introduced in peptides altered their effects, further confirming S4S5L binding to S6T. Our results suggest a mechanistic model in which S4S5L acts as a voltage-dependent ligand bound to its receptor on S6 at rest. This interaction locks the channel in a closed state. Upon plasma membrane depolarization, S4 pulls S4S5L away from S6T, allowing channel opening.

Dual Effect of Phosphatidyl (4,5)-Bisphosphate PIP2 on Shaker K+ Channels

Background: Phosphatidylinositol (4,5)-bisphosphate (PIP2) regulates several voltage-gated K+ channels, but the molecular mechanism remains elusive. Results: PIP2 exerts on Shaker opposite effects on maximal current amplitude and activation voltage dependence. Conclusion: PIP2 stabilizes the gate in the open state and the voltage sensor in the resting state. Significance: This is the first description of an effect of PIP2 on voltage sensor movement. Phosphatidylinositol (4,5)-bisphosphate (PIP2) is a phospholipid of the plasma membrane that has been shown to be a key regulator of several ion channels. Functional studies and more recently structural studies of Kir channels have revealed the major impact of PIP2 on the open state stabilization. A similar effect of PIP2 on the delayed rectifiers Kv7.1 and Kv11.1, two voltage-gated K+ channels, has been suggested, but the molecular mechanism remains elusive and nothing is known on PIP2 effect on other Kv such as those of the Shaker family. By combining giant-patch ionic and gating current recordings in COS-7 cells, and voltage-clamp fluorimetry in Xenopus oocytes, both heterologously expressing the voltage-dependent Shaker channel, we show that PIP2 exerts 1) a gain-of-function effect on the maximal current amplitude, consistent with a stabilization of the open state and 2) a loss-of-function effect by positive-shifting the activation voltage dependence, most likely through a direct effect on the voltage sensor movement, as illustrated by molecular dynamics simulations.

Dual effect of PIP2 on Shaker potassium channels

Background: Phosphatidylinositol (4,5)-bisphosphate (PIP2) regulates several voltage-gated K+ channels, but the molecular mechanism remains elusive. Results: PIP2 exerts on Shaker opposite effects on maximal current amplitude and activation voltage dependence. Conclusion: PIP2 stabilizes the gate in the open state and the voltage sensor in the resting state. Significance: This is the first description of an effect of PIP2 on voltage sensor movement. Phosphatidylinositol (4,5)-bisphosphate (PIP2) is a phospholipid of the plasma membrane that has been shown to be a key regulator of several ion channels. Functional studies and more recently structural studies of Kir channels have revealed the major impact of PIP2 on the open state stabilization. A similar effect of PIP2 on the delayed rectifiers Kv7.1 and Kv11.1, two voltage-gated K+ channels, has been suggested, but the molecular mechanism remains elusive and nothing is known on PIP2 effect on other Kv such as those of the Shaker family. By combining giant-patch ionic and gating current recordings in COS-7 cells, and voltage-clamp fluorimetry in Xenopus oocytes, both heterologously expressing the voltage-dependent Shaker channel, we show that PIP2 exerts 1) a gain-of-function effect on the maximal current amplitude, consistent with a stabilization of the open state and 2) a loss-of-function effect by positive-shifting the activation voltage dependence, most likely through a direct effect on the voltage sensor movement, as illustrated by molecular dynamics simulations.

Role of the S6 C‐terminus in KCNQ1 channel gating

Co‐assembly of KCNQ1 α‐subunits with KCNE1 β‐subunits results in the channel complex underlying the cardiac IKs current in vivo. Like other voltage‐gated K+ channels, KCNQ1 has a tetrameric configuration. The S6 segment of each subunit lines the ion channel pore with the lower part forming the activation gate. To determine residues involved in protein–protein interactions in the C‐terminal part of S6 (S6T), alanine and tryptophan perturbation scans were performed from residue 348–362 in the KCNQ1 channel. Several residues were identified to be relevant in channel gating, as substitutions affected the activation and/or deactivation process. Some mutations (F351A and V355W) drastically altered the gating characteristics of the resultant KCNQ1 channel, to the point of mimicking the IKs current. Furthermore, mutagenesis of residue L353 to an alanine or a charged residue impaired normal channel closure upon hyperpolarization, generating a constitutively open phenotype. This indicates that the L353 residue is essential for stabilizing the closed conformation of the channel gate. These findings together with the identification of several LQT1 mutations in the S6 C‐terminus of KCNQ1 underscore the relevance of this region in KCNQ1 and IKs channel gating.

Being flexible: the voltage-controllable activation gate of kv channels.

Kv channels form voltage-dependent potassium selective pores in the outer cell membrane and are composed out of four α-subunits, each having six membrane-spanning α-helices (S1–S6). The α-subunits tetramerize such that the S5–S6 pore domains co-assemble into a centrally located K+ pore which is surrounded by four operational voltage-sensing domains (VSD) that are each formed by the S1–S4 segments. Consequently, each subunit is capable of responding to changes in membrane potential and dictates whether the pore should be conductive or not. K+ permeation through the pore can be sealed off by two separate gates in series: (a) at the inner S6 bundle crossing (BC gate) and (b) at the level of the selectivity filter (SF gate) located at the extracellular entrance of the pore. Within the last years a general consensus emerged that a direct communication between the S4S5-linker and the bottom part of S6 (S6c) constitutes the coupling with the VSD thus making the BC gate the main voltage-controllable activation gate. While the BC gate listens to the VSD, the SF changes its conformation depending on the status of the BC gate. Through the eyes of an entering K+ ion, the operation of the BC gate apparatus can be compared with the iris-like motion of the diaphragm from a camera whereby its diameter widens. Two main gating motions have been proposed to create this BC gate widening: (1) tilting of the helix whereby the S6 converts from a straight α-helix to a tilted one or (2) swiveling of the S6c whereby the S6 remains bent. Such motions require a flexible hinge that decouples the pre- and post-hinge segment. Roughly at the middle of the S6 there exists a highly conserved glycine residue and a tandem proline motif that seem to fulfill the role of a gating hinge which allows for tilting/swiveling/rotations of the post-hinge S6 segment. In this review we delineate our current view on the operation of the BC gate for controlling K+ permeation in Kv channels.

Phosphatidylinositol-4,5-Bisphosphate (PIP2) Stabilizes the Open Pore Conformation of the Kv11.1 (hERG) Channel

Phosphatidylinositol-4,5-bisphosphate (PIP(2)) is a phospholipid that has been shown to modulate several ion channels, including some voltage-gated channels like Kv11.1 (hERG). From a biophysical perspective, the mechanisms underlying this regulation are not well characterized. From a physiological perspective, it is critical to establish whether the PIP(2) effect is within the physiological concentration range. Using the giant-patch configuration of the patch-clamp technique on COS-7 cells expressing hERG, we confirmed the activating effect of PIP(2). PIP(2) increased the hERG maximal current and concomitantly slowed deactivation. Regarding the molecular mechanism, these increased amplitude and slowed deactivation suggest that PIP(2) stabilizes the channel open state, as it does in KCNE1-KCNQ1. We used kinetic models of hERG to simulate the effects of the phosphoinositide. Simulations strengthened the hypothesis that PIP(2) is more likely stabilizing the channel open state than affecting the voltage sensors. From the physiological aspect, we established that the sensitivity of hERG to PIP(2) comes close to that of KCNE1-KCNQ1 channels, which lies in the range of physiological PIP(2) variations.