Potassium Channel Kv1 Research Articles

The anti-tumor drug 2-hydroxyoleic acid regulates the oncogenic potassium channel Kv10.1

Background2-hydroxyoleic acid (2OHOA) is a synthetic fatty acid with antitumor properties that alters membrane composition and structure, which in turn influences the functioning of membrane proteins and cell signaling. In this study, we propose a novel antitumoral mechanism of 2OHOA accomplished through the regulation of Kv10.1 channels. We evaluated the effects of 2OHOA on Kv10.1 channels expressed in HEK-293 cells by using electrophysiological techniques and a cell proliferation assay.Results2OHOA increased Kv10.1 channel currents in a voltage-dependent manner, shifted its conductance-voltage relationship towards negative potentials, and accelerated its activation kinetics. Moreover, 2OHOA reduced proliferation of cells that exogenously (HEK-293) and endogenously (MCF-7) expressed Kv10.1 channels. It is worth noting that the antiproliferative effect of 2OHOA was maintained in HEK-293 cells expressing a non-conducting mutant of Kv10.1 channel (Kv10.1-F456A), while it did not affect HEK-293 cells not expressing Kv10.1 channels, suggesting that 2OHOA interferes with a non-conducting function of Kv10.1 channels involved in cell proliferation. Finally, we found that 2OHOA can act synergistically with astemizole, a Kv10.1 channel blocker, to decrease cell proliferation more efficiently.ConclusionOur data suggest that 2OHOA decreases cell proliferation, at least in part, by regulating Kv10.1 channels.

ER-PM Junctions on GABAergic Interneurons Are Organized by Neuregulin 2/VAP Interactions and Regulated by NMDA Receptors.

Neuregulins (NRGs) signal via ErbB receptors to regulate neural development, excitability, synaptic and network activity, and behaviors relevant to psychiatric disorders. Bidirectional signaling between NRG2/ErbB4 and NMDA receptors is thought to homeostatically regulate GABAergic interneurons in response to increased excitatory neurotransmission or elevated extracellular glutamate levels. Unprocessed proNRG2 forms discrete clusters on cell bodies and proximal dendrites that colocalize with the potassium channel Kv2.1 at specialized endoplasmic reticulum-plasma membrane (ER-PM) junctions, and NMDA receptor activation triggers rapid dissociation from ER-PM junctions and ectodomain shedding by ADAM10. Here, we elucidate the mechanistic basis of proNRG2 clustering at ER-PM junctions and its regulation by NMDA receptors. Importantly, we demonstrate that proNRG2 promotes the formation of ER-PM junctions by directly binding the ER-resident membrane tether VAP, like Kv2.1. The proNRG2 intracellular domain harbors two non-canonical, low-affinity sites that cooperatively mediate VAP binding. One of these is a cryptic and phosphorylation-dependent VAP binding motif that is dephosphorylated following NMDA receptor activation, thus revealing how excitatory neurotransmission promotes the dissociation of proNRG2 from ER-PM junctions. Therefore, proNRG2 and Kv2.1 can independently function as VAP-dependent organizers of neuronal ER-PM junctions. Based on these and prior studies, we propose that proNRG2 and Kv2.1 serve as co-regulated downstream effectors of NMDA receptors to homeostatically regulate GABAergic interneurons.

Kv1.3 as a therapeutic target for immunocytoprotection after reperfusion in ischemic stroke.

The voltage-gated potassium channel Kv1.3, which is expressed on activated, disease-associated microglia and memory T cells, constitutes an attractive target for immunocytoprotection after endovascular thrombectomy (EVT). Using young male mice and rats we previously demonstrated that the Kv1.3 blocker PAP-1 when started 12 hours after reperfusion dose-dependently reduces infarction and improves neurological deficit on day-8 after ischemic stroke. However, these proof-of-concept findings are of limited translational value because the majority of strokes occur in patients over 65 and, when considering overall lifetime risk, in females. We therefore tested whether Kv1.3 deletion or delayed pharmacological therapy would be beneficial in females and aged animals. Transient middle cerebral artery occlusion (tMCAO, 60-min) was induced in 16-week and 80-week-old male and female wild-type C57BL/6J and Kv1.3-/- mice. Stroke outcomes were assessed daily with the 14-score tactile and proprioceptive limp-placing test and on day-8 by T2-weighted MRI. Young and old female mice were treated twice daily with 40 mg/kg of the Kv1.3 blocker PAP-1 starting 12 hours after reperfusion. Kv1.3 deletion provided no significant benefit in young females but improved outcomes in young males, old males and old females compared with wild-type controls of the same sex. PAP-1 treatment improved outcomes in both young and old females. In old females, Kv1.3 deletion and PAP-1 treatment significantly reduced microglia activation and T cell infiltration in the infarcted hemisphere. Our preclinical studies using aged and female mice further validate Kv1.3 inhibitors as potential adjunctive treatments for reperfusion therapy in stroke by providing both genetic and pharmacological verification. Using young male rats, we further explored the treatment window for Kv1.3 inhibitors and found that PAP-1 treatment still provided benefit and reduced inflammatory marker expression in the brain when started 72 hours after reperfusion.

Competition between Slc7a5 and LMAN2 for modulation of Kv1.2 gating.

The voltage-gated potassium channel Kv1.2 is a prominent ion channel in the CNS, where it regulates electrical activity. Kv1.2 structure and function are well understood, but its regulation by auxiliary proteins has not been widely studied. We identified novel regulators of Kv1.2 by a mass spectrometry approach, including the neutral amino acid transporter Slc7a5, which causes a dramatic hyperpolarizing shift of channel activation. In contrast, LMAN2 is a recently identified candidate regulator that has the opposite effect on gating: large depolarizing voltages are required to activate Kv1.2 channels co-expressed with LMAN2. In this study, we characterized the functional interaction between LMAN2 and Slc7a5 on Kv1.2 gating properties, and identified key structural elements that underlie sensitivity to either regulator. When LMAN2 and Slc7a5 are expressed together, Kv1.2 activation exhibits a bimodal voltage-dependence, suggesting two distinct populations of channels regulated either by LMAN2 or Slc7a5, but not both. Kv1.5 is a close homolog of Kv1.2, but insensitive to both LMAN2 and Slc7a5. Using a Kv1.2:1.5 chimeric approach, we identified the S1 segment of the voltage sensing domain (VSD) as essential for Slc7a5 sensitivity, and the S2-S3 linker of the VSD for LMAN2 sensitivity. By replacing either segment with sequence from Kv1.5, modulation by the corresponding regulator was selectively abolished. Replacement of the S1-S3 segment of Kv1.2 with sequence from Kv1.5 rendered the channel insensitive to both forms of regulation. These results suggest that Slc7a5 and LMAN2 compete for interaction with the Kv1.2 voltage sensor, leading to complex voltage-dependence of channel activity when both regulators are present in the cell. Although the structural basis for this modulation remains uncertain, Kv1.2 clearly exhibits a propensity for modulation over a wide range of voltages.

Mitochondrial dynamics control of mitoKv1.3 during cell cycle progression.

The voltage-gated potassium channel Kv1.3 plays a crucial role in adipose tissue. Kv1.3 is involved in the development of obesity and diabetes, thus becoming a potential therapeutical target. Kv1.3 locates at the plasma membrane and at mitochondria, controlling cell proliferation and differentiation or apoptosis, respectively. While the plasma membrane targeting is by a COPII-dependent mechanism and driven by several forward-traffic motifs, little is known about the mitochondrial route. In this work, the mitochondrial Kv1.3 interactome was obtained in order to decipher the mitochondrial components that participate in mitoKv1.3 import pathway. We report for the first time that, although Kv1.3 is a multispanning protein with no defined N-terminal presequence, the channel follows an unconventional routing through the TIM23 complex. The targeting is mediated by the information encoded along the channel sequence and regulated by the cytosolic Hsp70/90 chaperon complex. Once in mitochondria, mitoKv1.3 shows a higher affinity for perinuclear populations. In addition, mitoKv1.3 expression is cell cycle-dependent, increasing at the G1/S phase. The development of a knocked-down preadipocyte cell line let us describe that Kv1.3 promotes the preadipocyte proliferation by regulating mitochondrial dynamics. Throughout the cell cycle, there are several fusion-fission events that are key for a correct proliferation. We found that Kv1.3 expression mediates the G1/S phase hyperfusion and, consequently, the hyperpolarization of mitochondria. Our results unravel the unconventional import pathway of Kv1.3 into the inner mitochondrial membrane. Moreover, it sheds light into Kv1.3 control of mitochondrial morphology and dynamics at the G1 phase fine-tuning proliferation. Supported by the Ministerio de Ciencia e Innovación (MICINN/AEI), Spain (PID2020-112647RB-I00 and 10.13039/501100011033) and European Regional Development Fund (FEDER).

Modulation of Kv1.2 redox-sensitive gating by the transmembrane lectin LMAN2.

The voltage-gated potassium channel Kv1.2 plays a pivotal role in the nervous system, where it regulates membrane excitability and action potential propagation. Compared to other Kv1 channels, Kv1.2 exhibits dynamic voltage-dependent gating with dramatic cell-to-cell variability that is attributed to regulation by unidentified external factors. Recent progress from our lab has identified extrinsic factors that shift the voltage-dependence of Kv1.2 gating. For example, the neutral amino acid transporter Slc7a5 causes a dramatic hyperpolarizing shift of Kv1.2 channel activation. A counteracting effect is that extracellular reducing conditions promote a depolarizing shift in channel activation. However, the underlying mechanism of redox sensitivity has been challenging to identify. Using a mass-spectrometry approach to identify novel Kv1.2 accessory proteins, followed by screening candidates with whole-cell patch clamp electrophysiology, we observed that co-expression with the transmembrane lectin LMAN2 caused a pronounced depolarizing shift of Kv1.2 voltage-dependence. Overexpression of LMAN2 enhanced Kv1.2 sensitivity to extracellular reducing conditions, while shRNA knockdown of endogenous LMAN2 abrogated the redox effects. Using a chimeric approach between Kv1.2 and the LMAN2 insensitive Kv1.5 subunit, we found the S2-S3 linker of the VSD to be essential for sensitivity to both LMAN2 and reducing conditions. Biotinylation experiments revealed that LMAN2 maturation to the plasma membrane is required for its effects on Kv1.2 gating. Furthermore, extracellular application of the membrane impermeable reducing agent TCEP to dorsal root ganglion (DRG) neurons, decelerated the activation kinetics of isolated Kv1.2 currents and increased excitability. In conclusion, we identified a novel accessory protein (the transmembrane Lectin, LMAN2) that alters Kv1.2 gating properties and contributes to redox sensitivity in heterologous cells and DRG neurons.

Inactivation mechanism of an immunoglobulin modulator of the T-cell potassium channel Kv1.3.

Kv1.3 belongs to the Shaker family of voltage-activated potassium channels that are abundantly expressed in the T lymphocytes, where it plays an important role in T-cell activation. This role has made Kv1.3 a target for therapeutic immunomodulation for autoimmune diseases like multiple sclerosis, type 1 diabetes, and rheumatoid arthritis. Here, through cryo-electron microscopy (cryo-EM) and electrophysiology, we have investigated the modulatory mechanism of an inhibitory nanobody A0194009G09 (Ablynx/Sanofi). Functional studies reveal that the nanobody inhibits Kv1.3 by promoting slow C-type inactivation and that multiple nanobodies can bind to individual Kv1.3 channels. The structure of the human Kv1.3 in apo state (without modulators) showed a dynamic selectivity filter (SF) with two conformations (D1, D2) that are dilated when compared to reference structures where the SF is conducting. In the presence of the nanobody, the structure displays a SF in the D1 conformation, similar to the apo protein, but also a distinct conformation where the SF was further dilated (D3). Further, the nanobody-bound structure reveals how the nanobody bridges the turret loop within the pore domain and the S1-S2 loop within the voltage sensing domains to promote C-type inactivation. To explore how the nanobody promotes inactivation, we mutated residues at the interface between the nanobody and channel in both the S1-S2 loop and the turret loop, as well as a residue in the SF (H451) known to control inactivation in Kv1.3. Interestingly, the mutations that prevent inactivation in Kv1.3 largely attenuated the inhibitory action of the nanobody, similar to the mutations at the Kv1.3-nanobody binding interface. Taken together, our results demonstrate that the nanobody inhibits Kv1.3 by promoting inactivation, and we are currently solving mutant structures to elucidate the molecular mechanisms of how nanobody binding promotes inactivation.

Molecular simulations of state-specific drug interactions with multiple cardiac ion channels to reveal mechanisms of arrhythmogenesis.

Unintended drug block of cardiac ion channels remains a major problem in drug development. The voltage-gated potassium channel KV11.1 also known as hERG is a major drug anti-target binding a diverse set of small molecule drugs that potently reduce the critical repolarizing current IKr. Many drugs that bind the hERG channel promote deadly arrythmias while some hERG blockers present significantly lower proarrhythmic risk. Two hypotheses were proposed to elucidate this discrepancy: (1) preferential drug binding to the inactivated state of the hERG channel confers greater proarrhythmic risk and (2) simultaneous drug binding to other cardiac ion channels can ameliorate the risk associated with hERG channel block. Here, we present a state-specific molecular modeling assessment of drug binding to different conformations of the hERG and voltage-gated sodium NaV1.5 and calcium CaV1.2 channels. We have developed structural models of these cardiac ion channels in open and inactivated conformations and performed all-atom molecular dynamics (MD) simulations to validate structural stabilities and assess ion conduction. Ligand docking was then performed using Site Identification by Ligand Competitive Saturation (SILCS), a pre-compute ensemble molecular docking technique. SILCS allows us to perform a high-throughput assessment of ligand binding affinities using molecular fragment energy maps derived from MD simulations of state-specific ion channel models. Bayesian machine learning was used to provide improved correlation of SILCS-computed affinities with experimental data. Using SILCS multi-ligand docking we also estimated interactions of drugs with sex hormones in the hERG channel pore to assess a potential molecular mechanism for an increased proarrhythmia risk in females. We aim to use SILCS computed state-specific drug affinity data to inform multi-scale functional kinetic models of cardiac electrophysiology to estimate emergent drug effects on the cardiac action potential and heart rhythm.

Point mutations in the voltage-gated potassium channel Kv7.2/7.3, and their possible involvement in epilepsy and channel gating.

Benign familial neonatal epilepsy (BFNE) is a genetic disorder that causes seizures, starting during the 3rd - 7th day after birth. Most of the causes of this condition are associated with a malfunction of the M-channel, a heteromeric potassium channel consisting of Kv7.2 and Kv7.3 subunits. The demand for better antiepileptic drugs has made this channel an important target to be explored, equally to treat BFNE as well as some other forms of epilepsy. Understanding in detail the function of this channel in health and disease is fundamental to develop new and more effective drug treatments. Using a combination of mutagenesis, concatemers, molecular modeling, and two-electrode voltage clamp electrophysiology we have characterized some functional aspects of mutations, identified on the ClinVar database as pathogenic or likely pathogenic. The identity and location of these mutations, and how they affect channel function, may guide us to a deeper understanding of the severity of this disease. These mutations also suggest which biophysical properties are important for normal gating and stability in the membrane. Here we describe a significant reduction in the magnitude of the current as a direct consequence of the mutations and discuss some of the possible mechanistic reasons for this reduction.

Effects of potassium channel knockdown on peripheral blood T lymphocytes and NFAT signaling pathway in Xinjiang Kazak patients with hypertension

ABSTRACT Backgroud and aim T lymphocytes are involved in the occurrence and development of essential hypertension, and potassium channels are thought to be critical for lymphocyte activation. This study is to examine the roles of the voltage-gated potassium channels (Kv1.3) and calcium-activated potassium channels (KCa3.1) in peripheral blood T lymphocytes in Kazakh hypertensive patients of Xinjiang, China, mainly focusing on the effects of these channels on nuclear factor of activated T cells (NFAT) and inflammatory cytokines of T lymphocytes. Method Kv1.3 and KCa3.1 gene silencing were performed in cultured T lymphocytes from Kazakh patients with severe hypertension. T cell proliferation after gene silencing was measured using CCK-8. The mRNA and protein expression levels were measured using RT-qPCR and Western blot analysis, respectively. Nuclear translocation of NFAT was observed using laser confocal fluorescence microscopy. Inflammatory cytokine levels were detected with ELISA. Results Compared with control group, gene silencing of Kv1.3 and KCa3.1 respectively inhibited the proliferation of T cells. Moreover, compared with the control group, the mRNA expression levels of NFAT, IL-6 and IFN-γ were significantly decreased after gene silencing. Furthermore, the NFAT protein expression level was significantly down-regulated. In addition, the levels of IFN-γ and IL-6 in the cell culture supernatant were significantly decreased. Conclusion Both Kv1.3 and KCa3.1 potassium channels activated T lymphocytes and enhanced the cytokine secretion possibly through CaN/NFAT signaling pathway, which may in turn induce micro-inflammatory responses and trigger the occurrence and progression of hypertension.

The association between gene polymorphisms in voltage-gated potassium channels Kv2.1 and Kv4.2 and susceptibility to autism spectrum disorder.

Autism spectrum disorder (ASD) is a heritable form of neurodevelopmental disorder that arises through synaptic dysfunction. Given the involvement of voltage-gated potassium (Kv) channels in the regulation of synaptic plasticity, we aimed to explore the relationship between the genetic variants in the KCNB1 and KCND2 genes (encoding Kv2.1 and Kv4.2, respectively) and the risk of developing ASD. A total of 243 patients with ASD and 243 healthy controls were included in the present study. Sixty single nucleotide polymorphisms (SNPs) (35 in KCNB1 and 25 in KCND2) were genotyped using the Sequenom Mass Array. There were no significant differences in the distribution of allele frequencies and genotype frequencies in KCNB1 between cases and controls. However, the differences were significant in the allelic distribution of KCND2 rs1990429 (p Bonferroni < 0.005) and rs7793864 (p Bonferroni < 0.005) between the two groups. KCND2 rs7800545 (p FDR = 0.045) in the dominant model and rs1990429 (p FDR < 0.001) and rs7793864 (p FDR < 0.001) in the over-dominant model were associated with ASD risk. The G/A genotype of rs1990429 in the over-dominant model and the G/A-G/G genotype of rs7800545 in the dominant model were correlated with lower severity in the Autism Diagnostic Interview-Revised (ADI-R) restricted repetitive behavior (RRB) domain. Our results provide evidence that KCND2 gene polymorphism is strongly associated with ASD susceptibility and the severity of RRB.

Spinal voltage-gated potassium channel Kv1.3 contributes to neuropathic pain via the promotion of microglial M1 polarization and activation of the NLRP3 inflammasome.

Studies have shown that the activation of microglia is the main mechanism of neuropathic pain. Kv1.3 channel is a novel therapeutic target for treating neuroinflammatory disorders due to its crucial role in subsets of microglial cells. As such, it may be involved in the processes of neuropathic pain, however, whether Kv1.3 plays a role in neuroinflammation following peripheral nerve injury is unclear. The spared nerve injury model (SNI) was used to establish neuropathic pain. Western blot and immunofluorescence were used to examine the effect of Kv1.3 in the SNI rats. PAP-1, a Kv1.3 specific blocker was administered to alleviate neuropathic pain in the SNI rats. Neuropathic pain and allodynia occurred after SNI, the levels of M1 (CD68, iNos) and M2 (CD206, Arg-1) phenotypes were up-regulated in the spinal cord, and the protein levels of NLRP3, caspase-1 and IL-1β were also increased. Pharmacological blocking of Kv1.3 with PAP-1 alleviated hyperpathia induced by SNI. Meanwhile, intrathecal injection of PAP-1 reduced M1 polarization and decreased NLRP3, caspase-1 and IL-1β expressions of protein levels. Our research indicates that the Kv1.3 channel in the spinal cord contributes to neuropathic pain by promoting microglial M1 polarization and activating the NLRP3 inflammasome.

Identification and pharmacological characterization of the voltage-gated potassium channel Shab in diamondback moth, Plutella xylostella.

Voltage-gated potassium channel Kv2 is the primarily delayed rectifier in insect nerves and muscles involved in several crucial biological processes, including action potential regulation, photoreceptor performance, and larval locomotor. It is a potential molecular target for developing a novel pesticide for mosquitos. However, there are few studies on the Kv2 channel in agricultural pests. The only α-subunit gene of the Kv2 channel in Plutella xylostella (L.), PxShab, was cloned, and its expression profile was analyzed. The relative expression level of PxShab was highest in the pupal stage of both sexes and male adults but lowest in female adults. Meanwhile, PxShab had the highest expression in the head in both larvae and adults. Then, PxShab was stably expressed in the HEK-293 T cell line. Whole cell patch clamp recordings showed an outward current whose current-voltage relationship conformed to a typical delayed-rectifier potassium channel. 20 μM quinidine could effectively inhibit the potassium current, while the channel was insensitive to 4-AP even at 10 mM. Several potential compounds and botanical pesticides were assessed, and carvedilol (IC50 = 0.53 μM) and veratrine (IC50 = 2.22 μM) had a good inhibitory effect on the channel. This study revealed the pharmacological properties of PxShab and screened out several high potency inhibitors, which laid the foundation for further functional research of PxShab and provides new insight into designing novel insecticides. © 2022 Society of Chemical Industry.

Depletion of voltage‐gated potassium channel Kv1.1 contributes to behavioral deficits in transgenic mouse model of Alzheimer’s disease

AbstractBackgroundPatients with Alzheimer's disease (AD) are at risk for seizures and accelerated cognitive decline. Mechanisms of seizures and related synaptic dysfunction in AD are active areas of investigation. Alterations in ion channels have been identified in transgenic mouse models of AD, but levels of voltage‐gated potassium channels (VGKCs) have not been fully explored. In particular, little is known about Kv1.1 channels in AD. Mutations in Kv1.1 or autoantibodies against Kv1.1 cause neuronal overexcitation in several human diseases, including episodic ataxia type 1, epilepsy, myokymia, and limbic encephalitis, indicating that Kv1.1 is critical for regulating neuronal excitability.MethodTo explore VGKCs in AD, we used hAPP‐J20 mice ages 4‐6 months and determined mRNA and protein levels of four highly expressed VGKCs ‐ Kv1.1, Kv1.2, Kv1.4, and Kv4.2 ‐ in dentate gyrus, entorhinal cortex, motor cortex, and somatosensory cortex (SSC).ResultUsing RT‐qPCR, we identified reductions in Kv1.1 mRNA in the SSC of hAPP‐J20 mice. Western blotting revealed reductions in the Kv1.1 channel in the SSC of hAPP‐J20 mice and human parietal cortex in mild and moderate stages of AD. Immunolabeling revealed that the reductions in Kv1.1 channels in the SSC occurred in pyramidal cells and GABAergic interneurons. Kv1.1 levels in the SSC were lower in hAPP‐J20 mice, which overexpress mutant hAPP and have high Aβ(1‐42) levels, than in the wild‐type hAPP line I5, suggesting some dependence on Aβ(1‐42) levels. Levetiracetam treatment (75 mg/kg/day) administered for 28 days did not affect Kv1.1 levels in hAPP‐J20 mice. To assess Kv1.1 depletion in an AD model, we crossed Kv1.1 heterozygous mice (Kv1.1 +/‐) with hAPP‐J9 mice, which have lower expression of mutant hAPP than J20 mice. We performed behavioral tests on mice ages 4‐9 months. While Kv1.1 +/‐ mice and hAPP‐J9 mice had normal survival, the double mutant (Kv1.1 +/‐ hAPP‐J9) mice had premature mortality and impairments in elevated plus maze and light‐dark box tests (n=16‐25 mice/genotype) of anxiety.ConclusionThese data indicate that elevated Aβ(1‐42) levels may act synergistically with Kv1.1 depletion to exacerbate cognitive deterioration in AD. Ongoing investigation will further characterize the relationships between Kv1.1 loss and AD‐related hyperexcitability.

New Treatment for the Cognitive and Emotional Deficits Linked with Paclitaxel-Induced Peripheral Neuropathy in Mice.

Chemotherapy-provoked peripheral neuropathy and its linked comorbidities severely reduce the quality of a patient's life. Its therapy is not completely resolved and has become an important clinical challenge. The protective actions of molecular hydrogen (H2) in many neurological disorders have been described, but its effects on memory and the emotional deficits accompanying neuropathic pain induced by chemotherapy remain unknown. In this study, using male mice injected with paclitaxel (PTX), we examined the effects of systemic treatment with hydrogen-rich water (HRW) in: (i) the mechanical and thermal allodynia provoked by PTX and the pathways involved; (ii) the memory deficits, anxiety- and depressive-like behaviors associated with PTX-induced peripheral neuropathy (PIPN); and (iii) the plasticity (p-extracellular signal-regulated protein kinase; p-ERK ½), nociceptive (p-protein kinase B, p-Akt), inflammatory (p-nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha; p-IKBα), and oxidative (4-hydroxynonenal: 4-HNE) alterations provoked by PIPN in the prefrontal cortex (PFC). The results revealed: (1) the antiallodynic actions of HRW administered at one or two times per day during 7 and 3 consecutive days; (2) the participation of Kv7 potassium channels and the Nrf2-heme oxygenase 1-NAD(P)H: quinone oxidoreductase 1 pathway in the painkiller effects of HRW; (3) the inhibition of memory deficits and the anxiodepressive-like behaviors related with PIPN induced by HRW; and (4) the normalization of p-ERK ½, p-Akt and 4-HNE up-regulation and the activation of antioxidant enzymes produced by this treatment in PFC. This study proposes HRW as a possible effective and safe therapy for PIPN and its associated cognitive and emotional deficits.

Blockade of Kv1.3 Potassium Channel Inhibits Microglia-Mediated Neuroinflammation in Epilepsy

Epilepsy is a chronic neurological disorder whose pathophysiology relates to inflammation. The potassium channel Kv1.3 in microglia has been reported as a promising therapeutic target in neurological diseases in which neuroinflammation is involved, such as multiple sclerosis (MS), Alzheimer's disease (AD), Parkinson's disease (PD), and middle cerebral artery occlusion/reperfusion (MCAO/R). Currently, little is known about the relationship between Kv1.3 and epilepsy. In this study, we found that Kv1.3 was upregulated in microglia in the KA-induced mouse epilepsy model. Importantly, blocking Kv1.3 with its specific small-molecule blocker 5-(4-phenoxybutoxy)psoralen (PAP-1) reduced seizure severity, prolonged seizure latency, and decreased neuronal loss. Mechanistically, we further confirmed that blockade of Kv1.3 suppressed proinflammatory microglial activation and reduced proinflammatory cytokine production by inhibiting the Ca2+/NF-κB signaling pathway. These results shed light on the critical function of microglial Kv1.3 in epilepsy and provided a potential therapeutic target.

Olfactory Sensitivity Is Associated with Body Mass Index and Polymorphism in the Voltage-Gated Potassium Channels Kv1.3.

Smell strongly contributes to food choice and its hedonistic evaluation. A reduction or loss of smell has been related to malnutrition problems, resulting in excessive weight loss or gain. Voltage-gated potassium channels Kv1.3 are widely expressed in the olfactory bulb, and contribute mainly to the value of the resting membrane potential and to the frequency of action potentials. Mutations in the Kv1.3 gene are associated with alterations in glycemic homeostasis and olfactory sensitivity. We evaluated the olfactory performance in 102 healthy subjects and its association with BMI and polymorphism in the human Kv1.3 gene. Olfactory performance, based on the olfactory threshold, discrimination and identification scores and their summed score (TDI), was measured using the “Sniffin’ Sticks” test. Subjects were genotyped for the rs2821557 polymorphism of the Kv1.3 gene, whose major allele T was associated with a super-smeller phenotype, lower plasma glucose levels and resistance to diet-induced obesity as compared with the minor allele C. Based on the Kv1.3 genotype, the TDI and I olfactory scores obtained by the subjects were the following: TT > TC > CC. Subjects who were TT homozygous or heterozygous exhibited lower BMIs and reached higher olfactory scores than those with the CC genotype. The results were sex-dependent: heterozygous females performed better than heterozygous males. These findings show an inverse relationship between olfactory function and BMI, and a significant effect of the Kv1.3 genotypes on the olfactory functions and on the BMIs of the subjects. Finally, they suggest that the sex-related differences in the olfactory function can be partially ascribed to the Kv1.3 gene’s polymorphism.

Radiochemical Synthesis and Evaluation of 3-[11C]Methyl-4-aminopyridine in Rodents and Nonhuman Primates for Imaging Potassium Channels in the CNS.

Demyelination, the loss of the insulating sheath of neurons, causes failed or slowed neuronal conduction and contributes to the neurological symptoms in multiple sclerosis, traumatic brain and spinal cord injuries, stroke, and dementia. In demyelinated neurons, the axonal potassium channels Kv1.1 and Kv1.2, generally under the myelin sheath, become exposed and upregulated. Therefore, imaging these channels using positron emission tomography can provide valuable information for disease diagnosis and monitoring. Here, we describe a novel tracer for Kv1 channels, [11C]3-methyl-4-aminopyridine ([11C]3Me4AP). [11C]3Me4AP was efficiently synthesized via Pd(0)-Cu(I) comediated Stille cross-coupling of a stannyl precursor containing a free amino group. Evaluation of its imaging properties in rats and nonhuman primates showed that [11C]3Me4AP has a moderate brain permeability and slow kinetics. Additional evaluation in monkeys showed that the tracer is metabolically stable and that a one-tissue compartment model can accurately model the regional brain time-activity curves. Compared to the related tracers [18F]3-fluoro-4-aminopyridine ([18F]3F4AP) and [11C]3-methoxy-4-aminopyridine ([11C]3MeO4AP), [11C]3Me4AP shows lower initial brain uptake, which indicates reduced permeability to the blood-brain barrier and slower kinetics, suggesting higher binding affinity consistent with in vitro studies. While the slow kinetics and strong binding affinity resulted in a tracer with less favorable properties for imaging the brain than its predecessors, these properties may make 3Me4AP useful as a therapeutic.

A Fluorescent Peptide Toxin for Selective Visualization of the Voltage-Gated Potassium Channel KV1.3.

Upregulation of the voltage-gated potassium channel KV1.3 is implicated in a range of autoimmune and neuroinflammatory diseases, including rheumatoid arthritis, psoriasis, multiple sclerosis, and type I diabetes. Understanding the expression, localization, and trafficking of KV1.3 in normal and disease states is key to developing targeted immunomodulatory therapies. HsTX1[R14A], an analogue of a 34-residue peptide toxin from the scorpion Heterometrus spinifer, binds KV1.3 with high affinity (IC50 of 45 pM) and selectivity (2000-fold for KV1.3 over KV1.1). We have synthesized a fluorescent analogue of HsTX1[R14A] by N-terminal conjugation of a Cy5 tag. Electrophysiology assays show that Cy5-HsTX1[R14A] retains activity against KV1.3 (IC50 ∼ 0.9 nM) and selectivity over a range of other potassium channels (KV1.2, KV1.4, KV1.5, KV1.6, KCa1.1 and KCa3.1), as well as selectivity against heteromeric channels assembled from KV1.3/KV1.5 tandem dimers. Live imaging of CHO cells expressing green fluorescent protein-tagged KV1.3 shows co-localization of Cy5-HsTX1[R14A] and KV1.3 fluorescence signals at the cell membrane. Moreover, flow cytometry demonstrated that Cy5-HsTX1[R14A] can detect KV1.3-expressing CHO cells. Stimulation of mouse microglia by lipopolysaccharide, which enhances membrane expression of KV1.3, was associated with increased staining by Cy5-HsTX1[R14A], demonstrating that it can be used to identify KV1.3 in disease-relevant models of inflammation. Furthermore, the biodistribution of Cy5-HsTX1[R14A] could be monitored using ex vivo fluorescence imaging of organs in mice dosed subcutaneously with the peptide. These results illustrate the utility of Cy5-HsTX1[R14A] as a tool for visualizing KV1.3, with broad applicability in fundamental investigations of KV1.3 biology, and the validation of novel disease indications where KV1.3 inhibition may be of therapeutic value.

A Biodistribution Study of the Radiolabeled Kv1.3-Blocking Peptide DOTA-HsTX1[R14A] Demonstrates Brain Uptake in a Mouse Model of Neuroinflammation.

The voltage-gated potassium channel Kv1.3 regulates the pro-inflammatory function of microglia and is highly expressed in the post-mortem brains of individuals with Alzheimer's and Parkinson's diseases. HsTX1[R14A] is a selective and potent peptide inhibitor of the Kv1.3 channel (IC50 ∼ 45 pM) that has been shown to decrease cytokine levels in a lipopolysaccharide (LPS)-induced mouse model of inflammation. Central nervous system exposure to HsTX1[R14A] was previously detected in this mouse model using liquid chromatography with tandem mass spectrometry, but this technique does not report on the spatial distribution of the peptide in the different brain regions or peripheral organs. Herein, the in vivo distribution of a [64Cu]Cu-labeled DOTA conjugate of HsTX1[R14A] was observed for up to 48 h by positron emission tomography (PET) in mice. After subcutaneous administration to untreated C57BL/6J mice, considerable uptake of the radiolabeled peptide was observed in the kidney, but it was undetectable in the brain. Biodistribution of a [68Ga]Ga-DOTA conjugate of HsTX1[R14A] was then investigated in the LPS-induced mouse model of neuroinflammation to assess the effects of inflammation on uptake of the peptide in the brain. A control peptide with very weak Kv1.3 binding, [68Ga]Ga-DOTA-HsTX1[R14A,Y21A,K23A] (IC50 ∼ 6 μM), was also tested. Significantly increased uptake of [68Ga]Ga-DOTA-HsTX1[R14A] was observed in the brains of LPS-treated mice compared to mice treated with control peptide, implying that the enhanced uptake was due to increased Kv1.3 expression rather than simply increased blood-brain barrier disruption. PET imaging also showed accumulation of [68Ga]Ga-DOTA-HsTX1[R14A] in inflamed joints and decreased clearance from the kidneys in LPS-treated mice. These biodistribution data highlight the potential of HsTX1[R14A] as a therapeutic for the treatment of neuroinflammatory diseases mediated by overexpression of Kv1.3.