Dirk J. Snyders

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.

Gambierol, a toxin produced by the dinoflagellate Gambierdiscus toxicus, is a potent blocker of voltage-gated potassium channels

In this study, we pharmacologically characterized gambierol, a marine polycyclic ether toxin which is produced by the dinoflagellate Gambierdiscus toxicus. Besides several other polycyclic ether toxins like ciguatoxins, this scarcely studied toxin is one of the compounds that may be responsible for ciguatera fish poisoning (CFP). Unfortunately, the biological target(s) that underlies CFP is still partly unknown. Today, ciguatoxins are described to specifically activate voltage-gated sodium channels by interacting with their receptor site 5. But some dispute about the role of gambierol in the CFP story shows up: some describe voltage-gated sodium channels as the target, while others pinpoint voltage-gated potassium channels as targets. Since gambierol was never tested on isolated ion channels before, it was subjected in this work to extensive screening on a panel of 17 ion channels: nine cloned voltage-gated ion channels (mammalian Na(v)1.1-Na(v)1.8 and insect Para) and eight cloned voltage-gated potassium channels (mammalian K(v)1.1-K(v)1.6, hERG and insect ShakerIR) expressed in Xenopus laevis oocytes using two-electrode voltage-clamp technique. All tested sodium channel subtypes are insensitive to gambierol concentrations up to 10 microM. In contrast, K(v)1.2 is the most sensitive voltage-gated potassium channel subtype with almost full block (>97%) and an half maximal inhibitory concentration (IC(50)) of 34.5 nM. To the best of our knowledge, this is the first study where the selectivity of gambierol is tested on isolated voltage-gated ion channels. Therefore, these results lead to a better understanding of gambierol and its possible role in CFP and they may also be useful in the development of more effective treatments.

Effects of 4-aminopyridine on inward rectifying and pacemaker currents of cardiac purkinje fibres

Abstract1.4-Aminopyridine (4-AP), in a concentration of 1–5mM, prolongs the action potential duration, induces spontaneous activity and depolarizes sheep cardiac Purkinje strands. These effects are different from those obtained with 0.1mM and are reversible.2.Voltage clamp experiments demonstrate that the higher drug concentrations affect membrane currents measured in the potential range between-100 and-40mV, in addition to the reduction of the transient outward current already described for small amounts of the drug (0.1–0.5mM).3.The analysis of membrane current modifications by 4-AP in the presence of cesium and barium ions indicates that 4-AP, in the higher concentration range, reduces the inward rectifying time independent potassium currentiK1 and modifies the voltage dependence of the time and voltage dependent pacemaker current. The steady-state activation curve of the pacemaker, current is shifted towards less negative potentials and is less steeply voltage dependent. The time constant (τ) curve has an increased maximum, displaced towards less negative potentials.4.The modifications by 4-AP of theiK1 and pacemaker currents explain the changes in resting potential, action potential duration and the induction of spontaneous activity. The latter effect is not the result of an indirect effect of 4-AP through increased release of neurotransmitters from sympathetic nerve endings. A possible action of 4-AP at the inside of the membrane, explaining the multiple actions, is discussed.

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.

Electrically Silent Kv Subunits: Their Molecular and Functional Characteristics

Electrically silent voltage-gated potassium (KvS) α-subunits do not form homotetramers but heterotetramerize with Kv2 subunits, generating functional Kv2/KvS channel complexes in which the KvS subunits modulate the Kv2 current. This poses intriguing questions into the molecular mechanisms by which these KvS subunits cannot form functional homotetramers, why they only interact with Kv2 subunits, and how they modulate the Kv2 current.

Kv2.1 and silent Kv subunits underlie the delayed rectifier K current in cultured small mouse DRG neurons

Silent voltage-gated K(+) (K(v)) subunits interact with K(v)2 subunits and primarily modulate the voltage dependence of inactivation of these heterotetrameric channels. Both K(v)2 and silent K(v) subunits are expressed in the mammalian nervous system, but little is known about their expression and function in sensory neurons. This study reports the presence of K(v)2.1, K(v)2.2, and silent subunit K(v)6.1, K(v)8.1, K(v)9.1, K(v)9.2, and K(v)9.3 mRNA in mouse dorsal root ganglia (DRG). Immunocytochemistry confirmed the protein expression of K(v)2.x and K(v)9.x subunits in cultured small DRG neurons. To investigate if K(v)2 and silent K(v) subunits are underlying the delayed rectifier K(+) current (I(K)) in these neurons, K(v)2-mediated currents were isolated by the extracellular application of rStromatoxin-1 (ScTx) or by the intracellular application of K(v)2 antibodies. Both ScTx- and anti-K(v)2.1-sensitive currents displayed two components in their voltage dependence of inactivation. Together, both components accounted for approximately two-thirds of I(K). A comparison with results obtained in heterologous expression systems suggests that one component reflects homotetrameric K(v)2.1 channels, whereas the other component represents heterotetrameric K(v)2.1/silent K(v) channels. These observations support a physiological role for silent K(v) subunits in small DRG neurons.

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.