Testosterone Enhances KV Currents and Airway Smooth Muscle Relaxation Induced by ATP and UTP through P2Y4 Receptors and Adenylyl Cyclase Pathway.

Numerous studies suggest the involvement of adenosine-5'-triphosphate (ATP) and similar nucleotides in the pathophysiology of asthma. Androgens, such as testosterone (TES), are proposed to alleviate asthma symptoms in young men. ATP and uridine-5'-triphosphate (UTP) relax the airway smooth muscle (ASM) via purinergic P2Y2 and P2Y4 receptors and K+ channel opening. We previously demonstrated that TES increased the expression of voltage-dependent K+ (KV) channels in ASM. This study investigates how TES may potentiate ASM relaxation induced by ATP and UTP. Tracheal tissues treated with or without TES (control group) from young male guinea pigs were used. In organ baths, tracheas exposed to TES (40 nM for 48 h) showed enhanced ATP- and UTP-evoked relaxation. Tetraethylammonium, a K+ channel blocker, annulled this effect. Patch-clamp experiments in tracheal myocytes showed that TES also increased ATP- and UTP-induced K+ currents, and this effect was abolished with flutamide (an androgen receptor antagonist). KV channels were involved in this phenomenon, which was demonstrated by inhibition with 4-aminopyridine. RB2 (an antagonist of almost all P2Y receptors except for P2Y2), as well as N-ethylmaleimide and SQ 22,536 (inhibitors of G proteins and adenylyl cyclase, respectively), attenuated the enhancement of the K+ currents induced by TES. Immunofluorescence and immunohistochemistry studies revealed that TES did not modify the expression of P2Y4 receptors or COX-1 and COX-2, while we have demonstrated that this androgen augmented the expression of KV1.2 and KV1.5 channels in ASM. Thus, TES leads to the upregulation of P2Y4 signaling and KV channels in guinea pig ASM, enhancing ATP and UTP relaxation responses, which likely limits the severity of bronchospasm in young males.

STINGing away the pain: the role of interferon-stimulated genes.

Pain and inflammation are biologically intertwined responses that warn the body of potential danger. In this issue of the JCI, Defaye, Bradaia, and colleagues identified a functional link between inflammation and pain, demonstrating that inflammation-induced activation of stimulator of IFN genes (STING) in dorsal root ganglia nociceptors reduced pain-like behaviors in a rodent model of inflammatory pain. Utilizing mice with a gain-of-function STING mutation, Defaye, Bradaia, and colleagues identified type I IFN regulation of voltage-gated potassium channels as the mechanism of this pain relief. Further investigation into mechanisms by which proinflammatory pathways can reduce pain may reveal druggable targets and insights into new approaches for treating persistent pain.

A novel isoquinoline alkaloid HJ-69 isolated from Zanthoxylum bungeanum attenuates inflammatory pain by inhibiting voltage-gated sodium and potassium channels.

ETHNOPHARMACOLOGY RELEVANCE: Zanthoxylum bungeanum Maxim. (Z. bungeanum), a member of the Rutaceae family, has a rich history of traditional use in Asia for treating arthritis and toothache conditions. As characteristic chemical components, numerous kinds of alkaloids have been extracted from plants and their diverse biological activities have been reported. However, research on the isoquinoline alkaloid, a specific type of alkaloids, in Z. bungeanum was scarce. AIM OF THE STUDY: The study aimed to isolate a novel isoquinoline alkaloid from Z. bungeanum and explore its pharmacological activity in vitro and analgesic activity in vivo. MATERIALS AND METHODS: Isoquinoline alkaloid isolation and identification from Z. bungeanum were conducted using chromatographic and spectroscopic methods. The whole-cell patch-clamp technique was applied to assess its impact on neuronal excitability, and endogenous voltage-gated potassium (Kv) and sodium (Nav) currents in acutely isolated mouse small-diameter dorsal root ganglion (DRG) neurons. Its inhibitory impacts on channels were further validated with HEK293 cells stably expressing Nav1.7 and Nav1.8, and Chinese hamster ovary (CHO) cells transiently expressing Kv2.1. The formalin inflammatory pain model was utilized to evaluate the potential analgesic activity in vivo. RESULTS: A novel isoquinoline alkaloid named HJ-69 (N-13-(3-methoxyprop-1-yl)rutaecarpine) was isolated and identified from Z. bungeanum for the first time. HJ-69 significantly suppressed the firing frequency and amplitudes of action potentials in DRG neurons. Consistently, it state-dependently inhibited endogenous Nav currents of DRG neurons, with half maximal inhibitory concentration (IC50) values of 13.06 ± 2.06 µM and 30.19 ± 2.07 µM for the inactivated and resting states, respectively. HJ-69 significantly suppressed potassium currents in DRG neurons, which notably inhibited the delayed rectifier potassium (IK) currents (IC50 = 6.95 ± 1.29 µM) and slightly affected the transient outward potassium (IA) currents (IC50 = 523.50 ± 39.16 µM). Furtherly, HJ-69 exhibited similar potencies on heterologously expressed Nav1.7, Nav1.8, and Kv2.1 channels, which correspondingly represent the main components in neurons. Notably, intraperitoneal administration of 30 mg/kg and 100 mg/kg HJ-69 significantly alleviated pain behaviors in the mouse inflammatory pain model induced by formalin. CONCLUSION: The study concluded that HJ-69 is a novel and active isoquinoline alkaloid, and the inhibition of Nav and Kv channels contributes to its analgesic activity. HJ-69 may be a promising prototype for future analgesic drug discovery based on the isoquinoline alkaloid.

The antidiabetic drug ipragliflozin induces vasorelaxation of rabbit femoral artery by activating a Kv channel, the SERCA pump, and the PKA signaling pathway.

We explored the vasorelaxant effects of ipragliflozin, a sodium-glucose cotransporter-2 inhibitor, on rabbit femoral arterial rings. Ipragliflozin relaxed phenylephrine-induced pre-contracted rings in a dose-dependent manner. Pre-treatment with the ATP-sensitive K+ channel inhibitor glibenclamide (10 µM), the inwardly rectifying K+ channel inhibitor Ba2+ (50 µM), or the Ca2+-sensitive K+ channel inhibitor paxilline (10 µM) did not influence the vasorelaxant effect. However, the voltage-dependent K+ (Kv) channel inhibitor 4-aminopyridine (3 mM) reduced the vasorelaxant effect. Specifically, the vasorelaxant response to ipragliflozin was significantly attenuated by pretreatment with the Kv7.X channel inhibitors linopirdine (10 µM) and XE991 (10 µM), the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) pump inhibitors thapsigargin (1 µM) and cyclopiazonic acid (10 µM), and the cAMP/protein kinase A (PKA)-associated signaling pathway inhibitors SQ22536 (50 µM) and KT5720 (1 µM). Neither the cGMP/protein kinase G (PKG)-associated signaling pathway nor the endothelium was involved in ipragliflozin-induced vasorelaxation. We conclude that ipragliflozin induced vasorelaxation of rabbit femoral arteries by activating Kv channels (principally the Kv7.X channel), the SERCA pump, and the cAMP/PKA-associated signaling pathway independent of other K+ (ATP-sensitive K+, inwardly rectifying K+, and Ca2+-sensitive K+) channels, cGMP/PKG-associated signaling, and the endothelium.

Inhibition of voltage-gated potassium channel by aripiprazole in rabbit coronary arterial smooth muscle cells.

Aripiprazole, a third-generation antipsychotic, has been widely used to treat schizophrenia. In this study, we evaluated the effect of aripiprazole on voltage-gated potassium (Kv) channels in rabbit coronary arterial smooth muscle cells using the patch clamp technique. Aripiprazole reduced the Kv current in a concentration-dependent manner with a half-maximal inhibitory concentration of 0.89 ± 0.20 µM and a Hill coefficient of 1.30 ± 0.25. The inhibitory effect of aripiprazole on Kv channels was voltage-dependent, and an additional aripiprazole-induced decrease in the Kv current was observed in the voltage range of full channel activation. The decay rate of Kv channel inactivation was accelerated by aripiprazole. Aripiprazole shifted the steady-state activation curve to the right and the inactivation curve to the left. Application of a repetitive train of pulses (1 and 2 Hz) promoted inhibition of the Kv current by aripiprazole. Furthermore, the recovery time constant from inactivation increased in the presence of aripiprazole. Pretreatment of Kv1.5 subtype inhibitor reduced the inhibitory effect of aripiprazole. However, pretreatment with Kv 7 and Kv2.1 subtype inhibitors did not change the degree of aripiprazole-induced inhibition of the Kv current. We conclude that aripiprazole inhibits Kv channels in a concentration-, voltage-, time-, and use (state)-dependent manner by affecting the gating properties of the channels.

KCTD10 regulates brain development by destabilizing brain disorder-associated protein KCTD13.

KCTD10 belongs to the KCTD (potassiumchannel tetramerization domain) family, many members of which are associated with neuropsychiatric disorders. However, the biological function underlying the association with brain disorders remains to be explored. Here, we reveal that Kctd10 is highly expressed in neuronal progenitors and layer V neurons throughout brain development. Kctd10 deficiency triggers abnormal proliferation and differentiation of neuronal progenitors, reduced deep-layer (especially layer V) neurons, increased upper-layer neurons, and lowered brain size. Mechanistically, we screened and identified a unique KCTD10-interacting protein, KCTD13, associated with neurodevelopmental disorders. KCTD10 mediated the ubiquitination-dependent degradation of KCTD13 and KCTD10 ablation resulted in a considerable increase of KCTD13 expression in the developing cortex. KCTD13 overexpression in neuronal progenitors led to reduced proliferation and abnormal cell distribution, mirroring KCTD10 deficiency. Notably, mice with brain-specific Kctd10 knockout exhibited obvious motor deficits. This study uncovers the physiological function of KCTD10 and provides unique insights into the pathogenesis of neurodevelopmental disorders.

Role of oncogenic long noncoding RNA KCNQ1OT1 in colon cancer.

The role of lncRNA KCNQ1 opposite strand/antisense transcript 1 (KCNQ1OT1) in colon cancer involves various tumorigenic processes and has been studied widely. However, the mechanism by which it promotes colon cancer remains unclear. Retroviral vector pSEB61 was retrofitted in established HCT116-siKCN and SW480-siKCN cells to silence KCNQ1OT1. Cellular proliferation was measured using CCK8 assay, and flow cytometry (FCM) detected cell cycle changes. RNA sequencing (RNA-Seq) analysis showed differentially expressed genes (DEGs). Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were carried out to analyze enriched functions and signaling pathways. RT-qPCR, immunofluorescence, and western blotting were carried out to validate downstream gene expressions. The effects of tumorigenesis were evaluated in BALB/c nude mice by tumor xenografts. Our data revealed that the silencing of KCNQ1OT1 in HCT116 and SW480 cells slowed cell growth and decreased the number of cells in the G2/M phase. RNA-Seq analysis showed the data of DEGs enriched in various GO and KEGG pathways such as DNA replication and cell cycle. RT-qPCR, immunofluorescence, and western blotting confirmed downstream CCNE2 and PCNA gene expressions. HCT116-siKCN cells significantly suppressed tumorigenesis in BALB/c nude mice. Our study suggests that lncRNA KCNQ1OT1 may provide a promising therapeutic strategy for colon cancer.

A computational model predicts sex-specific responses to calcium channel blockers in mammalian mesenteric vascular smooth muscle.

The function of the smooth muscle cells lining the walls of mammalian systemic arteries and arterioles is to regulate the diameter of the vessels to control blood flow and blood pressure. Here, we describe an in silico model, which we call the 'Hernandez-Hernandez model', of electrical and Ca2+ signaling in arterial myocytes based on new experimental data indicating sex-specific differences in male and female arterial myocytes from murine resistance arteries. The model suggests the fundamental ionic mechanisms underlying membrane potential and intracellular Ca2+ signaling during the development of myogenic tone in arterial blood vessels. Although experimental data suggest that KV1.5 channel currents have similar amplitudes, kinetics, and voltage dependencies in male and female myocytes, simulations suggest that the KV1.5 current is the dominant current regulating membrane potential in male myocytes. In female cells, which have larger KV2.1 channel expression and longer time constants for activation than male myocytes, predictions from simulated female myocytes suggest that KV2.1 plays a primary role in the control of membrane potential. Over the physiological range of membrane potentials, the gating of a small number of voltage-gated K+ channels and L-type Ca2+ channels are predicted to drive sex-specific differences in intracellular Ca2+ and excitability. We also show that in an idealized computational model of a vessel, female arterial smooth muscle exhibits heightened sensitivity to commonly used Ca2+ channel blockers compared to male. In summary, we present a new model framework to investigate the potential sex-specific impact of antihypertensive drugs.

High blood pressure is a major risk factor for heart disease, which is one of the leading causes of death worldwide. While drugs are available to control blood pressure, male and female patients can respond differently to treatment. However, the biological mechanisms behind this sex difference are not fully understood. Blood pressure is controlled by cells lining the artery walls called smooth muscle cells which alter the width of blood vessels. On the surface of smooth muscle cells are potassium and calcium channels which control the cell's electrical activity. When calcium ions enter the cell via calcium channels, this generates an electrical signal that causes the smooth muscle to contract and narrow the blood vessel. Potassium ions then flood out of the cell via potassium channels to dampen the rise in electrical activity, causing the muscle to relax and widen the artery. There are various sub-types of potassium and calcium channels in smooth muscle cells. Here, Hernandez-Hernandez et al. set out to find how these channels differ between male and female mice, and whether these sex differences could alter the response to blood pressure medication. The team developed a computational model of a smooth muscle cell, incorporating data from laboratory experiments measuring differences in cells isolated from the arteries of male and female mice. The model predicted that the sub-types of potassium and calcium channels in smooth muscle cells varied between males and females, and how the channels impacted electrical activity also differed. For instance, the potassium channel Kv2.1 was found to have a greater role in controlling electrical activity in female mice, and this sex difference impacted blood vessel contraction. The model also predicted that female mice were more sensitive than males to calcium channel blockers, a drug commonly prescribed to treat high blood pressure. The findings by Hernandez-Hernandez et al. provide new insights into the biological mechanisms underlying sex differences in response to blood pressure medication. They also demonstrate how computational models can be used to predict the effects of drugs on different individuals. In the future, these predictions may help researchers to identify better, more personalized treatments for blood pressure.

Kv3.1 Voltage-gated Potassium Channels Modulate Anxiety-like Behaviors in Female Mice.

Inhibitory parvalbumin (PV) interneurons regulate the activity of neural circuits within brain regions involved in emotional processing, including the prefrontal cortex (PFC). Recently, rodent studies have implicated a stress-induced increase in prefrontal PV neuron activity in the development of anxiety behaviors, particularly in females. However, the mechanisms through which stress increases activity of prefrontal PV neurons remain unknown. The fast-spiking properties of PV neurons in part come from their expression of voltage-gated potassium (K+) ion channels, particularly Kv3.1 channels. We therefore suggest that stress-induced changes in Kv3.1 channels contribute to the appearance of an anxious phenotype following chronic stress in female mice. Here, we first showed that unpredictable chronic mild stress (UCMS) increased expression of Kv3.1 channels on prefrontal PV neurons in female mice, a potential mechanism underlying the previously observed hyperactivity of these neurons after stress. We then showed that female mice deficient in Kv3.1 channels displayed resilience to UCMS-induced anxiety-like behaviors. Altogether, our findings implicate Kv3.1 channels in the development of anxiety-like behaviors following UCMS, particularly in females, providing a novel mechanism to understand sex-specific vulnerabilities to stress-induced psychopathologies.

Identification, structural, and biophysical characterization of a positive modulator of human Kv3.1 channels.

Voltage-gated potassium channels (Kv) are tetrameric membrane proteins that provide a highly selective pathway for potassium ions (K+) to diffuse across a hydrophobic cell membrane. These unique voltage-gated cation channels detect changes in membrane potential and, upon activation, help to return the depolarized cell to a resting state during the repolarization stage of each action potential. The Kv3 family of potassium channels is characterized by a high activation potential and rapid kinetics, which play a crucial role for the fast-spiking neuronal phenotype. Mutations in the Kv3.1 channel have been shown to have implications in various neurological diseases like epilepsy and Alzheimer's disease. Moreover, disruptions in neuronal circuitry involving Kv3.1 have been correlated with negative symptoms of schizophrenia. Here, we report the discovery of a novel positive modulator of Kv3.1, investigate its biophysical properties, and determine the cryo-EM structure of the compound in complex with Kv3.1. Structural analysis reveals the molecular determinants of positive modulation in Kv3.1 channels by this class of compounds and provides additional opportunities for rational drug design for the treatment of associated neurological disorders.

Mechanistic understanding of KCNQ1 activating polyunsaturated fatty acid analogs.

The KCNQ1 channel is important for the repolarization phase of the cardiac action potential. Loss of function mutations in KCNQ1 can cause long QT syndrome (LQTS), which can lead to cardiac arrythmia and even sudden cardiac death. We have previously shown that polyunsaturated fatty acids (PUFAs) and PUFA analogs can activate the cardiac KCNQ1 channel, making them potential therapeutics for the treatment of LQTS. PUFAs bind to KCNQ1 at two different binding sites: one at the voltage sensor (Site I) and one at the pore (Site II). PUFA interaction at Site I shifts the voltage dependence of the channel to the left, while interaction at Site II increases maximal conductance. The PUFA analogs, linoleic-glycine and linoleic-tyrosine, are more effective than linoleic acid at Site I, but less effective at Site II. Using both simulations and experiments, we find that the larger head groups of linoleic-glycine and linoleic-tyrosine interact with more residues than the smaller linoleic acid at Site I. We propose that this will stabilize the negatively charged PUFA head group in a position to better interact electrostatically with the positively charges in the voltage sensor. In contrast, the larger head groups of linoleic-glycine and linoleic-tyrosine compared with linoleic acid prevent a close fit of these PUFA analogs in Site II, which is more confined. In addition, we identify several KCNQ1 residues as critical PUFA-analog binding residues, thereby providing molecular models of specific interactions between PUFA analogs and KCNQ1. These interactions will aid in future drug development based on PUFA-KCNQ1 channel interactions.

Vitamin D Receptor Deficiency Upregulates Pulmonary Artery Kv7 Channel Activity.

Recent evidence suggests that vitamin D is involved in the development of pulmonary arterial hypertension (PAH). The aim of this study was to analyze the electrophysiological and contractile properties of pulmonary arteries (PAs) in vitamin D receptor knockout mice (Vdr-/-). PAs were dissected and mounted in a wire myograph. Potassium membrane currents were recorded in freshly isolated PA smooth muscle cells (PASMCs) using the conventional whole-cell configuration of the patch-clamp technique. Potential vitamin D response elements (VDREs) in Kv7 channels coding genes were studied, and their protein expression was analyzed. Vdr-/- mice did not show a pulmonary hypertensive phenotype, as neither right ventricular hypertrophy nor endothelial dysfunction was apparent. However, resistance PA from these mice exhibited increased response to retigabine, a Kv7 activator, compared to controls and heterozygous mice. Furthermore, the current sensitive to XE991, a Kv7 inhibitor, was also higher in PASMCs from knockout mice. A possible VDRE was found in the gene coding for KCNE4, the regulatory subunit of Kv7.4. Accordingly, Vdr-/- mice showed an increased expression of KCNE4 in the lungs, with no changes in Kv7.1 and Kv7.4. These results indicate that the absence of Vdr in mice, as occurred with vitamin D deficient rats, is not sufficient to induce PAH. However, the contribution of Kv7 channel currents to the regulation of PA tone is increased in Vdr-/- mice, resembling animals and humans suffering from PAH.

Pharmacological modulation of Kv3 voltage-gated potassium channels regulates fear discrimination and expression in a response-dependent manner.

Various psychiatric diseases are characterized by aberrant cognition and emotional regulation. This includes inappropriately attributing affective salience to innocuous cues, which can be investigated using translationally relevant preclinical models of fear discrimination. Activity in the underpinning corticolimbic circuitry is governed by parvalbumin-expressing GABAergic interneurons, which also regulate fear discrimination. Kv3 voltage-gated potassium channels are highly expressed in these neurons and are important for controlling their activity, suggesting that pharmacological Kv3 modulation may regulate fear discrimination. We determined the effect of the positive Kv3 modulator AUT00206 given systemically to female rats undergoing limited or extended auditory fear discrimination training, which we have previously shown results in more discrimination or generalization, respectively, based on freezing at retrieval. We also characterized darting and other active fear-related responses. We found that limited training resulted in more discrimination based on freezing, which was unaffected by AUT00206. In contrast, extended training resulted in more generalization based on freezing and the emergence of discrimination based on darting during training and, to a lesser extent, at retrieval. Importantly, AUT00206 given before extended training had dissociable effects on fear discrimination and expression at retrieval depending on the response examined. While AUT00206 mitigated generalization without affecting expression based on freezing, it reduced expression without affecting discrimination based on darting, although darting levels were low overall. These results indicate that pharmacological Kv3 modulation regulates fear discrimination and expression in a response-dependent manner. They also raise the possibility that targeting Kv3 channels may ameliorate perturbed cognition and emotional regulation in psychiatric disease.

Methionine-isoleucine dichotomy at a key position in scorpion toxins inhibiting voltage-gated potassium channels.

Previous studies have identified some key amino acid residues in scorpion toxins blocking potassium channels. In particular, the most numerous toxins belonging to the α-KTx family and affecting voltage-gated potassium channels (KV) present a conserved K-C-X-N motif in the C-terminal half of their sequence. Here, we show that the X position of this motif is almost always occupied by either methionine or isoleucine. We compare the activity of three pairs of peptides that differ just by this residue on a panel of KV1 channels and find that toxins bearing methionine affect preferentially KV1.1 and 1.6 isoforms. The refined K-C-M/I-N motif stands out as the principal structural element of α-KTx conferring high affinity and selectivity to KV channels.

Mechanistic insights into robust cardiac IKs potassium channel activation by aromatic polyunsaturated fatty acid analogues.

Voltage-gated potassium (KV) channels are important regulators of cellular excitability and control action potential repolarization in the heart and brain. KV channel mutations lead to disordered cellular excitability. Loss-of-function mutations, for example, result in membrane hyperexcitability, a characteristic of epilepsy and cardiac arrhythmias. Interventions intended to restore KV channel function have strong therapeutic potential in such disorders. Polyunsaturated fatty acids (PUFAs) and PUFA analogues comprise a class of KV channel activators with potential applications in the treatment of arrhythmogenic disorders such as long QT syndrome (LQTS). LQTS is caused by a loss-of-function of the cardiac IKs channel - a tetrameric potassium channel complex formed by KV7.1 and associated KCNE1 protein subunits. We have discovered a set of aromatic PUFA analogues that produce robust activation of the cardiac IKs channel, and a unique feature of these PUFA analogues is an aromatic, tyrosine head group. We determine the mechanisms through which tyrosine PUFA analogues exert strong activating effects on the IKs channel by generating modified aromatic head groups designed to probe cation-pi interactions, hydrogen bonding, and ionic interactions. We found that tyrosine PUFA analogues do not activate the IKs channel through cation-pi interactions, but instead do so through a combination of hydrogen bonding and ionic interactions.

Effect of Terahertz Electromagnetic Field on the Permeability of Potassium Channel Kv1.2.

In this paper, the influence of external terahertz electromagnetic fields with different frequencies of 4 THz, 10 THz, 15 THz, and 20 THz on the permeability of the Kv1.2 voltage-gated potassium ion channel on the nerve cell membrane was studied using the combined model of the "Constant Electric Field-Ion Imbalance" method by molecular dynamics. We found that although the applied terahertz electric field does not produce strong resonance with the -C=O groups of the conservative sequence T-V-G-Y-G amino acid residue of the selective filter (SF) of the channel, it would affect the stability of the electrostatic bond between potassium ions and the carbonyl group of T-V-G-Y-G of SF, and it would affect the stability of the hydrogen bond between water molecules and oxygen atoms of the hydroxyl group of the 374THR side chain at the SF entrance, changing the potential and occupied states of ions in the SF and the occurrence probability of the permeation mode of ions and resulting in the change in the permeability of the channel. Compared with no external electric field, when the external electric field with 15 THz frequency is applied, the lifetime of the hydrogen bond is reduced by 29%, the probability of the "soft knock on" mode is decreased by 46.9%, and the ion flux of the channel is activated by 67.7%. Our research results support the view that compared to "direct knock-on", "soft knock-on" is a slower permeation mode.

The role of native cysteine residues in the oligomerization of KCNQ1 channels.

KCNQ1, the major component of the slow-delayed rectifier potassium channel, is responsible for repolarization of cardiac action potential. Mutations in this channel can lead to a variety of diseases, most notably long QT syndrome. It is currently unknown how many of these mutations change channel function and structure on a molecular level. Since tetramerization is key to proper function and structure of the channel, it is likely that mutations modify the stability of KCNQ1 oligomers. Presently, the C-terminal domain of KCNQ1 has been noted as the driving force for oligomer formation. However, truncated versions of this protein lacking the C-terminal domain still tetramerize. Therefore, we explored the role of native cysteine residues in a truncated construct of human KCNQ1, amino acids 100-370, by blocking potential interactions of cysteines with a nitroxide based spin label. Mobility of the spin labels was investigated with continuous wave electron paramagnetic resonance (CW-EPR) spectroscopy. The oligomerization state was examined by gel electrophoresis. The data provide information on tetramerization of human KCNQ1 without the C-terminal domain. Specifically, how blocking the side chains of native cysteines residues reduces oligomerization. A better understanding of tetramer formation could provide improved understanding of the molecular etiology of long QT syndrome and other diseases related to KCNQ1.

Mechanism of external K+ sensitivity of KCNQ1 channels.

KCNQ1 voltage-gated K+ channels are involved in a wide variety of fundamental physiological processes and exhibit the unique feature of being markedly inhibited by external K+. Despite the potential role of this regulatory mechanism in distinct physiological and pathological processes, its exact underpinnings are not well understood. In this study, using extensive mutagenesis, molecular dynamics simulations, and single-channel recordings, we delineate the molecular mechanism of KCNQ1 modulation by external K+. First, we demonstrate the involvement of the selectivity filter in the external K+ sensitivity of the channel. Then, we show that external K+ binds to the vacant outermost ion coordination site of the selectivity filter inducing a diminution in the unitary conductance of the channel. The larger reduction in the unitary conductance compared to whole-cell currents suggests an additional modulatory effect of external K+ on the channel. Further, we show that the external K+ sensitivity of the heteromeric KCNQ1/KCNE complexes depends on the type of associated KCNE subunits.

LGI3/2-ADAM23 interactions cluster Kv1 channels in myelinated axons to regulate refractory period.

Along myelinated axons, Shaker-type potassium channels (Kv1) accumulate at high density in the juxtaparanodal region, directly adjacent to the paranodal axon-glia junctions that flank the nodes of Ranvier. However, the mechanisms that control the clustering of Kv1 channels, as well as their function at this site, are still poorly understood. Here we demonstrate that axonal ADAM23 is essential for both the accumulation and stability of juxtaparanodal Kv1 complexes. The function of ADAM23 is critically dependent on its interaction with its extracellular ligands LGI2 and LGI3. Furthermore, we demonstrate that juxtaparanodal Kv1 complexes affect the refractory period, thus enabling high-frequency burst firing of action potentials. Our findings not only reveal a previously unknown molecular pathway that regulates Kv1 channel clustering, but they also demonstrate that the juxtaparanodal Kv1 channels that are concealed below the myelin sheath, play a significant role in modifying axonal physiology.

The in vitro anticancer effects of FS48 from salivary glands of Xenopsylla cheopis on NCI-H460 cells via its blockage of voltage-gated K+ channels.

Voltage-gated K+ (Kv) channels play a role in the cellular processes of various cancer cells, including lung cancer cells. We previously identified and reported a salivary protein from the Xenopsylla cheopis, FS48, which exhibited inhibitory activity against Kv1.1-1.3 channels when assayed in HEK 293T cells. However, whether FS48 has an inhibitory effect on cancer cells expressing Kv channels is unclear. The present study aims to reveal the effects of FS48 on the Kv channels and the NCI-H460 human lung cancer cells through patch clamp, MTT, wound healing, transwell, gelatinase zymography, qRT-PCR and WB assays. The results demonstrated that FS48 can be effective in suppressing the Kv currents, migration, and invasion of NCI-H460 cells in a dose-dependent manner, despite the failure to inhibit the proliferation. Moreover, the expression of Kv1.1 and Kv1.3 mRNA and protein were found to be significantly reduced. Finally, FS48 decreases the mRNA level of MMP-9 while increasing TIMP-1 mRNA level. The present study highlights for the first time that blood-sucking arthropod saliva-derived protein can inhibit the physiological activities of tumour cells via the Kv channels. Furthermore, FS48 can be taken as a hit compound against the tumour cells expressing Kv channels.