Edited by: Mounir Tarek, Centre National de la Recherche Scientifique, France
Reviewed by: Nikita Gamper, University of Leeds, UK; David A. Brown, University College London, UK
*Correspondence: Gildas Loussouarn, L’Institut du Thorax, UMR 1087/CNRS UMR 6291, IRT-UN, 8 Quai Moncousu, BP 70721, 44007 Nantes Cedex 1, France. e-mail:
†Present address: Frank S. Choveau, UTHSCSA, San Antonio, TX, USA.
This article was submitted to Frontiers in Pharmacology of Ion Channels and Channelopathies, a specialty of Frontiers in Pharmacology.
This is an open-access article distributed under the terms of the
Voltage-gated potassium (Kv) channels are tetramers, each subunit presenting six transmembrane segments (S1–S6), with each S1–S4 segments forming a voltage-sensing domain (VSD) and the four S5–S6 forming both the conduction pathway and its gate. S4 segments control the opening of the intracellular activation gate in response to changes in membrane potential. Crystal structures of several voltage-gated ion channels in combination with biophysical and mutagenesis studies highlighted the critical role of the S4–S5 linker (S4S5L) and of the S6 C-terminal part (S6T) in the coupling between the VSD and the activation gate. Several mechanisms have been proposed to describe the coupling at a molecular scale. This review summarizes the mechanisms suggested for various voltage-gated ion channels, including a mechanism that we described for KCNQ1, in which S4S5L is acting like a ligand binding to S6T to stabilize the channel in a closed state. As discussed in this review, this mechanism may explain the reverse response to depolarization in HCN-like channels. As opposed to S4S5L, the phosphoinositide, phosphatidylinositol 4,5-bisphosphate (PIP2), stabilizes KCNQ1 channel in an open state. Many other ion channels (not only voltage-gated) require PIP2 to function properly, confirming its crucial importance as an ion channel cofactor. This is highlighted in cases in which an altered regulation of ion channels by PIP2 leads to channelopathies, as observed for KCNQ1. This review summarizes the state of the art on the two regulatory mechanisms that are critical for KCNQ1 and other voltage-gated channels function (PIP2 and S4S5L), and assesses their potential physiological and pathophysiological roles.
Voltage-gated ion channels are all designed according to a common pattern including six transmembrane segments (S1–S6), with S1–S4 forming the voltage-sensing domain (VSD), in which the positively charged S4 is the voltage sensor
If a general consensus started to emerge on the nature and the (nano-)metrics of the movement of the voltage sensor in voltage-gated channels, it is still not the case for the nature of the coupling between the voltage sensor movement and the gate opening. In this first part, we review most of the results obtained through various experimental approaches on various channels, that can give insights on the nature of the coupling, and we try to classify this coupling into two categories: a strong or a labile coupling between the main actors, namely, the S4–S5 linker (referred here as S4S5L) and the C-terminal part of the S6 transmembrane segment (S6T).
The ability of Kv channels to sense the membrane potential is conferred via the VSD. The S4 segment moves across the plasma membrane in response to changes in membrane potential, allowing the transition of the channel between a closed conformation and an open conformation. Many studies have investigated the nature of the S4 movement, and came up with three different models, with major differences in this movement.
* According to the crystal structure of KvAP, S4 coupled to S3 form a helical hairpin, or “paddle,” moving 15–20 Å across the lipid bilayer, as confirmed by avidin accessibility to different-length tethered biotin reagents (Jiang et al.,
* The “transporter” model, described in Shaker, involves a very small movement of S4 (2–3 Å) from a crevice in contact with the intracellular solution to another one in contact with the extracellular solution (Cha et al.,
* Finally, the helical screw model (Guy and Seetharamulu,
The variety of these models, and the fact that they predict a magnitude of the S4 movement across the membrane ranging from 2 Å (Cha et al.,
A structural model of Shaker, based on the crystal structure of Kv1.2, predicts an axial rotation and a translation of S4 (Yarov-Yarovoy et al.,
Which part of the channel links the voltage sensor movement to the gate opening? The physical interaction between S4S5L and S6T and the role of this interaction in translating the voltage sensor movement to the gate opening have been investigated in many voltage-gated channels by diverse techniques. The results obtained will be detailed below and in other reviews of the present Frontiers Research Topic, but it is important to note that many works stress the major role of this S4S5L–S6T interaction. Mutagenesis associated to functional studies using chimeras of Shaker and KcsA (Lu et al.,
These studies strongly indicate that the VSD-activation and gate coupling are associated through the S4S5L–S6T interaction. However, many questions remain to be elucidated. Are other regions of channels involved in this coupling? How exactly does this S4S5L–S6T interaction make the link between VSD-activation and gate coupling?
In addition to S4S5L and S6T interaction, other regions influence voltage-dependent channel activity. One of those regions is the N-terminus (Nter). In many signaling proteins, a PAS domain is present where it functions as a signal sensor and its name comes from the transcription factors in which it was first identified: period circadian protein (Per), aryl hydrocarbon receptor nuclear translocator protein (Arnt), and single-minded protein (Sim). The PAS domain is also present in the N-terminus of three Kv channel families, Kv10, Kv11, and Kv12. An interaction between the PAS domain and S4S5L has been postulated as underlying the slow deactivation process of hERG channels (Wang et al.,
The coupling between the VSD and the pore may also occur through interactions between transmembrane domains (TMD). Cross-linking studies in Shaker channel showed the proximity between the S4 and S5 segments, and suggested that interactions may be involved in the coupling between the VSD and the pore (Broomand et al.,
In summary, a network of interactions, including Nter, S1, S4S5L, and S6T, seems to be involved in the coupling between VSD and the pore.
The Shaker gene from
In an elegant work using chimeras in which Shaker pore module is replaced by the one of KcsA channel (Lu et al.,
In other functional studies, Shaker mutations in S4S5L and S6T were shown to have a dramatic effect on the slow component of the off-gating current. Together with the fact that closing the gate impacts on gating charge return, this has been interpreted as the S4S5L and S6T interaction allosterically keeping S4 in the “up” position and stabilizing the open state (Batulan et al.,
Altogether, these data support both the specificity and the strength of interaction between S4S5L and S6T, consistent with the mechanical lever mechanism, but in a more complex manner, with potentially state-dependent S4S5L and S6T interactions stabilizing the closed (Lu et al.,
The Kv1.2 channel is a Shaker-like voltage-gated potassium channel expressed in mammalian neurons and involved in the regulation of pre- and post-synaptic membrane excitability. The interaction between the S4–S5 linker and the S6 segment was observed in the crystal structure of Kv1.2 in the open state (Long et al.,
Only the combination of experimental and
It is now admitted that the VSD-pore coupling is mediated by the interaction between S4S5L and S6T. Several works on Shaker and Kv1.2 channels (above) suggest that the nature of this interaction is a strong coupling of the pore opening with voltage sensor movement. But in other channels, the interaction between S4S5L and S6T may be state-dependent, and leads to stabilization of the channel in the open or closed state. Forcing the interaction between S4S5L and S6T seems to stabilize hERG channels in a closed conformation (Ferrer et al.,
The hERG encodes the voltage-gated potassium channel underlying the cardiac delayed rectifier current,
The hyperpolarization-activated, cyclic-nucleotide-gated (HCN) channels represent a family of four members (HCN1-4) that carry
How can this difference in the gating behavior be explained? Two competing models have been proposed. The first model proposes that HCN channels are in an inactivated state when the membrane is depolarized and that its hyperpolarization induces channels to recover from inactivation and enter into an open state (Miller and Aldrich,
Alanine-scanning mutagenesis in HCN2 channel identified three S4S5L residues playing a major role in the S6 gate stabilization in the closed state (Chen et al.,
Kv4.2 channel belongs to the family of voltage-gated potassium channels related to the
Voltage-gated Na+ and Ca2+ channels (Nav and Cav, respectively), are fused tetrameric subunits with the same structural organization as proper tetrameric Kv channels. Indeed, Nav and Cav subunits contain four homologous but not identical domains, each including six transmembrane segments (S1–S6), a voltage sensor domain with a positively charged S4 segment and a pore region formed by the association of S5 and S6 segments.
Since the voltage-dependent activity of Na+ and Ca2+ channels is mediated by the S4 movements in response to membrane potential variation (Yang and Horn,
As developed earlier, it is broadly accepted that the interaction between S4S5L and S6T is extremely important for voltage-gated ion channels function (activation, deactivation, and inactivation). For that reason, disruption of such interaction may have dramatic physiological effects, and lead to certain forms of disease.
Both cardiac and neurological disorders have been linked to impaired S4–S5L and S6T interactions in Kv channels. For instance, many mutations of the KCNQ1 channels lead to the LQT, a cardiac disease characterized by prolonged ventricular repolarization, arrhythmias, and sudden death. Interestingly, looking specifically at the S4S5L, it was shown that LQT1 mutations (type 1 LQT, associated with mutations in KCNQ1) are clustered on the one side of the S4S5L α-helix structure, that is putatively responsible for interactions with the S6T region (Boulet et al.,
On the other hand, the pathological effect of a Kv1.1 channel mutation is consistent with the mechanical lever model of Kv1 channels (Figure
Recently, it was proven that S4S5L and S6 regions of the voltage-gated calcium channel Cav2.3 are coupled during the activation process (Wall-Lacelle et al.,
Phosphatidylinositol 4,5-bisphosphate (PIP2) is a minor acidic membrane lipid found primarily in the inner leaflet of the plasma membrane. PIP2 was first described as the precursor of the second messengers inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) when cleaved by receptor-activated phospholipase C (PLC; Berridge,
The KCNQ1/KCNE1 kinetic model shares similarities with the one of Kir6.2/SUR1 channel (Enkvetchakul et al.,
Although KCNQ1 is a voltage-gated channel on its own, KCNE1 leads to changes in the current properties: it increases the amplitude, shifts the voltage dependence of activation toward more positive potentials, slows activation and deactivation kinetics, and suppresses inactivation (Barhanin et al.,
Neurotransmitter and hormone receptor stimulations activate different signaling pathways that adjust the protein phosphorylation status. Among others, Gq/G11-protein coupled receptors, like muscarinic acetylcholine (ACh) receptors (M1), stimulate the PLC which hydrolyzes PIP2 (Berridge,
Protein kinase A (PKA) is another well-characterized kinase that regulates
More recently, Matavel et al. (
Five members have been identified in the KCNQ channel family (KCNQ1–5), each with a specific tissue distribution. In the heart, intestine, and inner ear, KCNQ1 subunits, assembling with auxiliary KCNE subunits, are important for repolarization and K+ transport (Barhanin et al.,
Similar to KCNQ1 (Loussouarn et al.,
In addition to PIP2, several kinds of phosphoinositides but also other phospholipids are present in the plasma membrane and are capable of regulating the “M-type” K+ current (Telezhkin et al.,
The hERG or KCNH2 encodes the pore-forming subunit of the channel that is responsible for the rapid delayed rectifier K+ current,
In addition to the delayed rectifiers KCNQ1 and hERG, other voltage-gated channels are regulated by PIP2: the voltage-gated Ca2+ channels (Cav) channels (Wu et al.,
Many studies have investigated the role of PIP2 in the regulation of voltage-gated KCNQ channels activity. Recovery of KCNQ2/KCNQ3 current following muscarinic stimulation requires re-synthesis of PIP2 (Suh and Hille,
A decrease in PIP2 may be the major determinant for a decrease in a KCNQ current upon activation of some Gq/11-coupled receptors, but the mechanism may also be more complex for other Gq/11-coupled receptors. Regarding regulation of the M-current, two distinct pathways following PLC activation and IP3 and DAG production have been described (Figure
The first pathway, for which the decrease in PIP2 is the major determinant of M-current depression, is induced by the activation of M1 muscarinic ACh and AT1 angiotensin II receptors (Zaika et al.,
The second pathway, activated by bradykinin B2 and purinergic P2Y receptors (Figure
The location of presumed PIP2-binding sites and the characteristic of their motifs have been investigated in several channels. For KCNQ channels, evidence support the idea that the PIP2-binding site(s) is (are) located mainly within the C-terminus. For instance, the H328C mutation in helix A within the C-terminus of KCNQ2 (residue in green in Figure
Because all KCNQ channels share a common structure and are up-regulated by PIP2 (Loussouarn et al.,
In hERG also, a PIP2-binding site seems to be located in the C-terminus. Deletion of a segment (883–894) in the C-terminus of hERG abolished the effects of PIP2 on channel amplitude and voltage dependence of activation (Bian et al.,
We described above that activation of Gq/11 signaling pathways leads to PIP2 depletion and consequently to decreased channel current. However, several works suggest that unbinding of PIP2 due to decreased affinity for KCNQ channels, rather than PIP2 depletion, can underlie Gq/11-mediated depression of KCNQ current (Delmas and Brown,
As previously described in this review, mutagenesis studies have identified clusters of positively charged residues, mainly located in the cytosolic C-terminus of channels that may interact with the negatively charged PIP2. The S4 segment possesses several positively charged residues, suggesting that PIP2 might also affect its movement by interacting with some of these residues.
Several studies are consistent with the idea that lipids can interact with the voltage sensor and modulate its motion; although most of these studies focus on interactions in the outer leaflet (PIP2 is situated in the inner leaflet). Structural studies on KvAP and on a Kv1.2-Kv2.1 chimeric channel show that some residues of S4 are exposed to lipids (Lee et al.,
According to those studies, S4 is stabilized in the activated position by interaction with outer-leaflet phospholipids. The structure of the Kv1.2-Kv2.1 chimeric channel suggests that an inner-leaflet phospholipid may also interact with the S4–S5 linker (Long et al.,
As mentioned above, the importance of PIP2 regulation of voltage-gated ion channels is now proven and clear. Thus, one might ask how far this crucial factor affects the physiological functions of these channels. Is it limited to a biophysical/regulatory effect, or does it have major impact; for instance, can an impaired interaction with PIP2 lead to human disease? While this issue was partly answered for non-voltage-gated ion channels (Logothetis et al.,
The KCNQ1-KCNE1 potassium channel complex underlies the
The KCNE1 beta-subunit is critical for a proper activity of KCNQ1 in the heart, and KCNE1 mutations are also associated with a LQT (type 5 LQT syndrome, LQT5). It was shown that neutralization of positive charges located in KCNE1 C-terminus is associated with LQT5 (Lai et al.,
Regulation of hERG channels by PIP2 has been described in Section “Part 2: Human Ether-a-go-go Related Gene.” PIP2 stabilizes hERG open state changing the amplitude and deactivation kinetics (Bian et al.,
The importance of PIP2 regulation for proper voltage-gated ion channels function deserves thus all our attention. Although no direct connection between the phospholipid and channelopathies has been proven, apart from the LQT studies, the data obtained so far open a wide range of possibilities. An impressive list of phosphoinositide-sensitive channels has been presented in a recent review (Logothetis et al.,
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Fayal Abderemane-Ali and Fabien C. Coyan are recipients of a grant from the French Ministère de la Recherche. Zeineb Es-Salah-Lamoureux is supported by fellowships from the Lefoulon Delalande foundation and the French Foundation for Medical Research (FRM).