Virucidal Properties of Photocatalytic Coating on Glass against a Model Human Coronavirus.

The antimicrobial properties of photocatalysts have long been studied. However, most of the available literature describes their antibacterial properties, while knowledge of their antiviral activity is rather scarce. Since the outset of the coronavirus disease 2019 (COVID-19) pandemic, an increasing body of research has suggested their antiviral potential and highlighted the need for further research in this area. In this study, we investigated the virucidal properties of a commercial TiO2-coated photocatalytic glass against a model human coronavirus. Our findings demonstrate that the TiO2-coated glass consistently inactivates coronaviruses upon contact under daylight illumination, in a time-dependent manner. A 99% drop in virus titer was achieved after 3.9 h. The electron micrographs of virus-covered TiO2-glass showed a reduced number of virions compared to control glass. Morphological alterations of TiO2-exposed viruses included deformation, disruption of the viral envelope, and virion ghosts, endorsing the application of this material in the construction of protective elements to mitigate the transmission of viruses. To the best of our knowledge, this is the first report showing direct visual evidence of human coronaviruses being damaged and morphologically altered following exposure to this photocatalyst. IMPORTANCE Surface contamination is an important contributor to SARS-CoV-2 spread. The use of personal protective elements and physical barriers (i.e., masks, gloves, and indoor glass separators) increases safety and has proven invaluable in preventing contagion. Redesigning these barriers so that the virus cannot remain infectious on them could make a difference in COVID-19 epidemiology. The introduction of additives with virucidal activity could potentiate the protective effects of these barriers to serve not only as physical containment but also as virus killers, reducing surface contamination after hand touch or aerosol deposition. We performed in-depth analysis of the kinetics of photocatalysis-triggered coronavirus inactivation on building glass coated with TiO2. This is the first report showing direct visual evidence (electron microscopy) of coronaviruses being morphologically damaged following exposure to this photocatalyst, demonstrating the high potential of this material to be incorporated into daily-life high-touch surfaces, giving them an added value in decelerating the virus spread.

Acute Brain Stroke Evolution Detected by 18F-FDG PET/CT and MRI Justifies the Discordance of Lesions in a Patient With Lymphoproliferative Syndrome.

ABSTRACT: A 50-year-old man with angioimmunoblastic T-cell lymphoma in complete response to treatment presented new hypermetabolic brain lesions on 18F-FDG PET/CT suggestive of malignancy. These findings were correlated by MRI that showed cortical-subcortical peripheral lesions typical of acute ischemic infarction. A restaging 18F-FDG PET/CT showed that hypermetabolic lesions were replaced by ametabolic areas, supporting chronic infarction. Early ischemia presents transitory FDG increase. Brain lymphomas are highly FDG avid and difficult to differentiate from acute cerebral infarction. In view of the discordance of abnormal areas of intracranial uptake on PET FDG, MRI confirmation is required to avoid misinterpretation.

Influence of Kv11.1 (hERG1) K+ channel expression on DNA damage induced by the genotoxic agent methyl methanesulfonate.

Besides their crucial role in cell electrogenesis and maintenance of basal membrane potential, the voltage-dependent K+ channel Kv11.1/hERG1 shows an essential impact in cell proliferation and other processes linked to the maintenance of tumour phenotype. To check the possible influence of channel expression on DNA damage responses, HEK293 cells, treated with the genotoxic agent methyl methanesulfonate (MMS), were compared with those of a HEK-derived cell line (H36), permanently transfected with the Kv11.1-encoding gene, and with a third cell line (T2) obtained under identical conditions as H36, by permanent transfection of another unrelated plasma membrane protein encoding gene. In addition, to gain some insights about the canonical/conduction-dependent channel mechanisms that might be involved, the specific erg channel inhibitor E4031 was used as a tool. Our results indicate that the expression of Kv11.1 does not influence MMS-induced changes in cell cycle progression, because no differences were found between H36 and T2 cells. However, the canonical ion conduction function of the channel appeared to be associated with decreased cell viability at low/medium MMS concentrations. Moreover, direct DNA damage measurements, using the comet assay, demonstrated for the first time that Kv11.1 conduction activity was able to modify MMS-induced DNA damage, decreasing it particularly at high MMS concentration, in a way related to PARP1 gene expression. Finally, our data suggest that the canonical Kv11.1 effects may be relevant for tumour cell responses to anti-tumour therapies.

The EAG Voltage-Dependent K+ Channel Subfamily: Similarities and Differences in Structural Organization and Gating.

EAG (ether-à-go-go or KCNH) are a subfamily of the voltage-gated potassium (Kv) channels. Like for all potassium channels, opening of EAG channels drives the membrane potential toward its equilibrium value for potassium, thus setting the resting potential and repolarizing action potentials. As voltage-dependent channels, they switch between open and closed conformations (gating) when changes in membrane potential are sensed by a voltage sensing domain (VSD) which is functionally coupled to a pore domain (PD) containing the permeation pathway, the potassium selectivity filter, and the channel gate. All Kv channels are tetrameric, with four VSDs formed by the S1-S4 transmembrane segments of each subunit, surrounding a central PD with the four S5-S6 sections arranged in a square-shaped structure. Structural information, mutagenesis, and functional experiments, indicated that in "classical/Shaker-type" Kv channels voltage-triggered VSD reorganizations are transmitted to PD gating via the α-helical S4-S5 sequence that links both modules. Importantly, these Shaker-type channels share a domain-swapped VSD/PD organization, with each VSD contacting the PD of the adjacent subunit. In this case, the S4-S5 linker, acting as a rigid mechanical lever (electromechanical lever coupling), would lead to channel gate opening at the cytoplasmic S6 helices bundle. However, new functional data with EAG channels split between the VSD and PD modules indicate that, in some Kv channels, alternative VSD/PD coupling mechanisms do exist. Noticeably, recent elucidation of the architecture of some EAG channels, and other relatives, showed that their VSDs are non-domain swapped. Despite similarities in primary sequence and predicted structural organization for all EAG channels, they show marked kinetic differences whose molecular basis is not completely understood. Thus, while a common general architecture may establish the gating system used by the EAG channels and the physicochemical coupling of voltage sensing to gating, subtle changes in that common structure, and/or allosteric influences of protein domains relatively distant from the central gating machinery, can crucially influence the gating process. We consider here the latest advances on these issues provided by the elucidation of eag1 and erg1 three-dimensional structures, and by both classical and more recent functional studies with different members of the EAG subfamily.

New Structures and Gating of Voltage-Dependent Potassium (Kv) Channels and Their Relatives: A Multi-Domain and Dynamic Question.

Voltage-dependent potassium channels (Kv channels) are crucial regulators of cell excitability that participate in a range of physiological and pathophysiological processes. These channels are molecular machines that display a mechanism (known as gating) for opening and closing a gate located in a pore domain (PD). In Kv channels, this mechanism is triggered and controlled by changes in the magnitude of the transmembrane voltage sensed by a voltage-sensing domain (VSD). In this review, we consider several aspects of the VSD⁻PD coupling in Kv channels, and in some relatives, that share a common general structure characterized by a single square-shaped ion conduction pore in the center, surrounded by four VSDs located at the periphery. We compile some recent advances in the knowledge of their architecture, based in cryo-electron microscopy (cryo-EM) data for high-resolution determination of their structure, plus some new functional data obtained with channel variants in which the covalent continuity between the VSD and PD modules has been interrupted. These advances and new data bring about some reconsiderations about the use of exclusively a classical electromechanical lever model of VSD⁻PD coupling by some Kv channels, and open a view of the Kv-type channels as allosteric machines in which gating may be dynamically influenced by some long-range interactional/allosteric mechanisms.

Relative positioning of Kv11.1 (hERG) K+ channel cytoplasmic domain-located fluorescent tags toward the plasma membrane.

Recent cryo-EM data have provided a view of the KCNH potassium channels molecular structures. However, some details about the cytoplasmic domains organization and specially their rearrangements associated to channel functionality are still lacking. Here we used the voltage-dependent dipicrylamine (DPA)-induced quench of fluorescent proteins (FPS) linked to different positions at the cytoplasmic domains of KCNH2 (hERG) to gain some insights about the coarse structure of these channel parts. Fast voltage-clamp fluorometry with HEK293 cells expressing membrane-anchored FPs under conditions in which only the plasma membrane potential is modified, demonstrated DPA voltage-dependent translocation and subsequent FRET-triggered FP quenching. Our data demonstrate for the first time that the distance between an amino-terminal FP tag and the intracellular plasma membrane surface is shorter than that between the membrane and a C-terminally-located tag. The distances varied when the FPs were attached to other positions along the channel cytoplasmic domains. In some cases, we also detected slower fluorometric responses following the fast voltage-dependent dye translocation, indicating subsequent label movements orthogonal to the plasma membrane. This finding suggests the existence of additional conformational rearrangements in the hERG cytoplasmic domains, although their association with specific aspects of channel operation remains to be established.

Functional characterization of Kv11.1 (hERG) potassium channels split in the voltage-sensing domain.

Voltage-dependent KCNH family potassium channel functionality can be reconstructed using non-covalently linked voltage-sensing domain (VSD) and pore modules (split channels). However, the necessity of a covalent continuity for channel function has not been evaluated at other points within the two functionally independent channel modules. We find here that by cutting Kv11.1 (hERG, KCNH2) channels at the different loops linking the transmembrane spans of the channel core, not only channels split at the S4-S5 linker level, but also those split at the intracellular S2-S3 and the extracellular S3-S4 loops, yield fully functional channel proteins. Our data indicate that albeit less markedly, channels split after residue 482 in the S2-S3 linker resemble the uncoupled gating phenotype of those split at the C-terminal end of the VSD S4 transmembrane segment. Channels split after residues 514 and 518 in the S3-S4 linker show gating characteristics similar to those of the continuous wild-type channel. However, breaking the covalent link at this level strongly accelerates the voltage-dependent accessibility of a membrane impermeable methanethiosulfonate reagent to an engineered cysteine at the N-terminal region of the S4 transmembrane helix. Thus, besides that of the S4-S5 linker, structural integrity of the intracellular S2-S3 linker seems to constitute an important factor for proper transduction of VSD rearrangements to opening and closing the cytoplasmic gate. Furthermore, our data suggest that the short and probably rigid characteristics of the extracellular S3-S4 linker are not an essential component of the Kv11.1 voltage sensing machinery.

Gating mechanism of Kv11.1 (hERG) K+ channels without covalent connection between voltage sensor and pore domains.

Kv11.1 (hERG, KCNH2) is a voltage-gated potassium channel crucial in setting the cardiac rhythm and the electrical behaviour of several non-cardiac cell types. Voltage-dependent gating of Kv11.1 can be reconstructed from non-covalently linked voltage sensing and pore modules (split channels), challenging classical views of voltage-dependent channel activation based on a S4-S5 linker acting as a rigid mechanical lever to open the gate. Progressive displacement of the split position from the end to the beginning of the S4-S5 linker induces an increasing negative shift in activation voltage dependence, a reduced z g value and a more negative ΔG 0 for current activation, an almost complete abolition of the activation time course sigmoid shape and a slowing of the voltage-dependent deactivation. Channels disconnected at the S4-S5 linker near the S4 helix show a destabilization of the closed state(s). Furthermore, the isochronal ion current mode shift magnitude is clearly reduced in the different splits. Interestingly, the progressive modifications of voltage dependence activation gating by changing the split position are accompanied by a shift in the voltage-dependent availability to a methanethiosulfonate reagent of a Cys introduced at the upper S4 helix. Our data demonstrate for the first time that alterations in the covalent connection between the voltage sensor and the pore domains impact on the structural reorganizations of the voltage sensor domain. Also, they support the hypothesis that the S4-S5 linker integrates signals coming from other cytoplasmic domains that constitute either an important component or a crucial regulator of the gating machinery in Kv11.1 and other KCNH channels.

ERK and RSK are necessary for TRH-induced inhibition of r-ERG potassium currents in rat pituitary GH3 cells.

The transduction pathway mediating the inhibitory effect that TRH exerts on r-ERG channels has been thoroughly studied in GH3 rat pituitary cells but some elements have yet to be discovered, including those involved in a phosphorylation event(s). Using a quantitative phosphoproteomic approach we studied the changes in phosphorylation caused by treatment with 1µM TRH for 5min in GH3 cells. The activating residues of Erk2 and Erk1 undergo phosphorylation increases of 5.26 and 4.87 fold, respectively, in agreement with previous reports of ERK activation by TRH in GH3 cells. Thus, we studied the possible involvement of ERK pathway in the signal transduction from TRH receptor to r-ERG channels. The MEK inhibitor U0126 at 0.5µM caused no major blockade of the basal r-ERG current, but impaired the TRH inhibitory effect on r-ERG. Indeed, the TRH effect on r-ERG was also reduced when GH3 cells were transfected with siRNAs against either Erk1 or Erk2. Using antibodies, we found that TRH treatment also causes activating phosphorylation of Rsk. The TRH effect on r-ERG current was also impaired when cells were transfected with any of two different siRNAs mixtures against Rsk1. However, treatment of GH3 cells with 20nM EGF for 5min, which causes ERK and RSK activation, had no effect on the r-ERG currents. Therefore, we conclude that in the native GH3 cell system, ERK and RSK are involved in the pathway linking TRH receptor to r-ERG channel inhibition, but additional components must participate to cause such inhibition.

Voltage-dependent gating of KCNH potassium channels lacking a covalent link between voltage-sensing and pore domains.

Voltage-gated channels open paths for ion permeation upon changes in membrane potential, but how voltage changes are coupled to gating is not entirely understood. Two modules can be recognized in voltage-gated potassium channels, one responsible for voltage sensing (transmembrane segments S1 to S4), the other for permeation (S5 and S6). It is generally assumed that the conversion of a conformational change in the voltage sensor into channel gating occurs through the intracellular S4-S5 linker that provides physical continuity between the two regions. Using the pathophysiologically relevant KCNH family, we show that truncated proteins interrupted at, or lacking the S4-S5 linker produce voltage-gated channels in a heterologous model that recapitulate both the voltage-sensing and permeation properties of the complete protein. These observations indicate that voltage sensing by the S4 segment is transduced to the channel gate in the absence of physical continuity between the modules.

Interactions between the N-terminal tail and the gating machinery of hERG K⁺ channels both in closed and open/inactive states.

The N-terminal-most N-tail of the human ether-à-go-go-related gene (hERG) potassium channel is a crucial modulator of deactivation through its interactions with the S4-S5 loop and/or the C-linker/cNBD, leading to a stabilization of the channel's open state. Not only the N-terminal, but also the initial C-terminal region of the channel can modulate the transitions between the open and closed states either by direct or by indirect/allosteric interactions with the gating machinery. However, while a physical proximity of the N-tail to the gating machinery has been demonstrated in the closed state, data about their possible interaction in other channel conformations have been lacking. Using a site-directed cysteine mutagenesis and disulfide chemistry approach, we present here evidence that a physical proximity between the N-tail and the gating-related structures can also exist in channels held between pulses in the open/inactive state, highlighting the physiological and functional relevance of the direct interactions between the N-terminal tail and the S4-S5 loop and/or C-linker structures for modulation of channel.

Control of seizures in different stages of partial epilepsy: LACO-EXP, a Spanish retrospective study of lacosamide.

Lacosamide is approved as adjunctive therapy for focal epilepsies. The number of antiepileptic drugs (AEDs) tried is associated with prognosis. This multicenter, retrospective, observational study (LACO-EXP) in Spain in 500 adult patients with focal epilepsies examined the efficacy and tolerability of add-on lacosamide. Factors associated with better efficacy/tolerability were analyzed. After 12months, the responder rate (≥50% reduction in seizure frequency) was 57.1%, and the seizure-free rate was 14.9%. Efficacy was better when lacosamide was the first or second add-on AED, although there was a small chance to be seizure-free even for patients who had received ≤10 prior AEDs. The mechanism of action of concomitant AEDs is important in all the stages, but differences are smaller in the early stages. Lacosamide was generally well tolerated. A slower dosage-titration schedule was associated with a lower adverse event rate. Further investigation of the timing of initiation of lacosamide add-on therapy and ideal combinations of AEDs is required.

Nonglial tumors of the brainstem.

Nonglial tumors of the brainstem constitute a histologically heterogeneous group of lesions with quite a different behavior and aggressiveness. Therefore, the diverse therapeutic options depend on a correct and prompt diagnosis. We can limit their differential diagnosis by using clinical and demographic data and imaging findings, which in most cases will be a translation of their histologic characteristics. The main clinical, neuroimaging, and pathologic features of these lesions are described according to the last updated classification of the World Health Organization for central nervous system tumors. We provide some useful clues, based on the direct correlation of the imaging appearance with its gross pathologic and histologic appearance, for a comprehensive diagnostic approach. Embryonic tumors (medulloblastoma and primitive neuroectodermal tumor), cavernoma, lymphoma, hemangioblastoma, and ganglionic and mixed tumors as long as lesions affecting the central nervous system by external compression (arising from the skull, cerebrospinal fluid spaces, or extraaxial nervous and vascular elements) are included. All cases presented belong to the archive data of our hospital.

Mapping of interactions between the N- and C-termini and the channel core in HERG K+ channels.

The characteristic gating properties of the HERG [human eag (ether-a-go-go)-related gene] potassium channel determine its contribution to cardiac repolarization and in setting the electrical behaviour of a variety of cells. In the present study we analysed, using a site-directed cysteine and disulfide chemistry approach, whether the eag/PAS (Per/Arnt/Sim) and proximal domains at the HERG N-terminus exert a role in controlling the access of the N-terminal flexible tail to its binding site in the channel core for interaction with the gating machinery. Whereas the eag/PAS domain is necessary for disulfide bridging, plus the cysteine residues introduced at positions 3 and 542 of the HERG sequence, the presence of the proximal domain seems to be dispensable. The state-dependent formation of a disulfide bridge between Cys3 and an endogenous cysteine residue at position 723 in the C-terminal C-linker suggests that the N-terminal tail of HERG can also get into close proximity with the C-linker structures located at the bottom of helix S6. Therefore the intrinsic flexibility of the N-tail and its proximity to both the S4-S5 loop and the C-linker may dynamically contribute to the modulation of HERG channel gating.

Relief of intractable posthypoxic myoclonus after administration of agomelatine.

Chronic posthypoxic myoclonus is characterized by myoclonic jerks that are specifically triggered by action. It is a rare but devastating sequela of hypoxic encephalopathy. We report a 42-year-old female patient with treatment-resistant chronic posthypoxic myoclonus, which improved with administration of the drug agomelatine. We suggest that agomelatine could represent a useful therapeutic option in the treatment of chronic posthypoxic myoclonus.

Cytoplasmic domains and voltage-dependent potassium channel gating.

The basic architecture of the voltage-dependent K(+) channels (Kv channels) corresponds to a transmembrane protein core in which the permeation pore, the voltage-sensing components and the gating machinery (cytoplasmic facing gate and sensor-gate coupler) reside. Usually, large protein tails are attached to this core, hanging toward the inside of the cell. These cytoplasmic regions are essential for normal channel function and, due to their accessibility to the cytoplasmic environment, constitute obvious targets for cell-physiological control of channel behavior. Here we review the present knowledge about the molecular organization of these intracellular channel regions and their role in both setting and controlling Kv voltage-dependent gating properties. This includes the influence that they exert on Kv rapid/N-type inactivation and on activation/deactivation gating of Shaker-like and eag-type Kv channels. Some illustrative examples about the relevance of these cytoplasmic domains determining the possibilities for modulation of Kv channel gating by cellular components are also considered.

Cell type influences the molecular mechanisms involved in hormonal regulation of ERG K+ channels.

While the thyrotropin-releasing hormone (TRH) effect of raising intracellular Ca(2+) levels has been shown to rely on G(q/11) and PLC activation, the molecular mechanisms involved in the regulation of ERG K(+) channels by TRH are still partially unknown. We have analysed the effects of ßγ scavengers, Akt/PKB inactivation, and TRH receptor (TRH-R) overexpression on such regulation in native and heterologous expression cell systems. In native rat pituitary GH(3) cells ß-ARK/CT, Gα(t), and phosducin significantly reduced TRH inhibition of rERG currents, whereas in HEK-H36/T1 cells permanently expressing TRH-R and hERG, neither of the ßγ scavengers affected the TRH-induced shift in V (1/2). Use of specific siRNAs to knock Akt/PKB expression down abolished the TRH effect on HEK-H36/T1 cell hERG, but not on rERG from GH(3) cells. Indeed, wortmannin or long insulin pretreatment also blocked TRH regulation of ERG currents in HEK-H36/T1 but not in GH(3) cells. To determine whether these differences could be related to the amount of TRH-Rs in the cell, we studied the TRH concentration dependence of the Ca(2+) and ERG responses in GH(3) cells overexpressing the receptors. The data indicated that independent of the receptor number additional cellular factor(s) contribute differently to couple the TRH-R to hERG channel modulation in HEK-H36/T1 cells. We conclude that regulation of ERG currents by TRH and its receptor is transduced in GH(3) and HEK-H36/T1 cell systems through common and different elements, and hence that the cell type influences the signalling pathways involved in the TRH-evoked responses.

An inhibitor of neuronal exocytosis (DD04107) displays long-lasting in vivo activity against chronic inflammatory and neuropathic pain.

Small peptides patterned after the N terminus of the synaptosomal protein of 25 kDa, a member of the protein complex implicated in Ca(2+)-dependent neuronal exocytosis, inhibit in vitro the release of neuromodulators involved in pain signaling, suggesting an in vivo analgesic activity. Here, we report that compound DD04107 (palmitoyl-EEMQRR-NH(2)), a 6-mer palmitoylated peptide that blocks the inflammatory recruitment of ion channels to the plasma membrane of nociceptors and the release of calcitonin gene-related peptide from primary sensory neurons, displays potent and long-lasting in vivo antihyperalgesia and antiallodynia in chronic models of inflammatory and neuropathic pain, such as the complete Freund's adjuvant, osteosarcoma, chemotherapy, and diabetic neuropathic models. Subcutaneous administration of the peptide produced a dose-dependent antihyperalgesic and antiallodynic activity that lasted ≥24 h. The compound showed a systemic distribution, characterized by a bicompartmental pharmacokinetic profile. Safety pharmacology studies indicated that the peptide is largely devoid of side effects and substantiated that the in vivo activity is not caused by locomotor impairment. Therefore, DD04107 is a potent and long-lasting antinociceptive compound that displays a safe pharmacological profile. These findings support the notion that neuronal exocytosis of receptors and neuronal algogens pivotally contribute to chronic inflammatory and neuropathic pain and imply a central role of peptidergic nociceptor sensitization to the pathogenesis of pain.

Screening for mutations in Spanish families with myotonia. Functional analysis of novel mutations in CLCN1 gene.

Myotonia congenita is an inherited muscle disorder caused by mutations in the CLCN1 gene, a voltage-gated chloride channel of skeletal muscle. We have studied 48 families with myotonia, 32 out of them carrying mutations in CLCN1 gene and eight carry mutations in SCN4A gene. We have found 26 different mutations in CLCN1 gene, including 13 not reported previously. Among those 26 mutations, c.180+3A>T in intron 1 is present in nearly one half of the Spanish families in this series, the largest one analyzed in Spain so far. Although scarce data have been published on the frequency of mutation c.180+3A>T in other populations, our data suggest that this mutation is more frequent in Spain than in other European populations. In addition, expression in HEK293 cells of the new missense mutants Tyr137Asp, Gly230Val, Gly233Val, Tyr302His, Gly416Glu, Arg421Cys, Asn567Lys and Gln788Pro, demonstrated that these DNA variants are disease-causing mutations that abrogate chloride currents.

Molecular determinants of interactions between the N-terminal domain and the transmembrane core that modulate hERG K+ channel gating.

A conserved eag domain in the cytoplasmic amino terminus of the human ether-a-go-go-related gene (hERG) potassium channel is critical for its slow deactivation gating. Introduction of gene fragments encoding the eag domain are able to restore normal deactivation properties of channels from which most of the amino terminus has been deleted, and also those lacking exclusively the eag domain or carrying a single point mutation in the initial residues of the N-terminus. Deactivation slowing in the presence of the recombinant domain is not observed with channels carrying a specific Y542C point mutation in the S4-S5 linker. On the other hand, mutations in some initial positions of the recombinant fragment also impair its ability to restore normal deactivation. Fluorescence resonance energy transfer (FRET) analysis of fluorophore-tagged proteins under total internal reflection fluorescence (TIRF) conditions revealed a substantial level of FRET between the introduced N-terminal eag fragments and the eag domain-deleted channels expressed at the membrane, but not between the recombinant eag domain and full-length channels with an intact amino terminus. The FRET signals were also minimized when the recombinant eag fragments carried single point mutations in the initial portion of their amino end, and when Y542C mutated channels were used. These data suggest that the restoration of normal deactivation gating by the N-terminal recombinant eag fragment is an intrinsic effect of this domain directed by the interaction of its N-terminal segment with the gating machinery, likely at the level of the S4-S5 linker.