Amyloid-ß Protein Precursor Regulates Electrophysiological Properties in the Hippocampus via Altered Kv1.4 Expression and Function in Mice.

BACKGROUND: Amyloid-ß protein precursor (AßPP) is enriched in neurons. However, the mechanism underlying AßPP regulation of neuronal activity is poorly understood. Potassium channels are critically involved in neuronal excitability. In hippocampus, A-type potassium channels are highly expressed and involved in determining neuronal spiking. OBJECTIVE: We explored hippocampal local field potential (LFP) and spiking in the presence and absence of AßPP, and the potential involvement of an A-type potassium channel. METHODS: We used in vivo extracellular recording and whole-cell patch-clamp recording to determine neuronal activity, current density of A-type potassium currents, and western blot to detect changes in related protein levels. RESULTS: Abnormal LFP was observed in AßPP-/- mice, including reduced beta and gamma power, and increased epsilon and ripple power. The firing rate of glutamatergic neurons reduced significantly, in line with an increased action potential rheobase. Given that A-type potassium channels regulate neuronal firing, we measured the protein levels and function of two major A-type potassium channels and found that the post-transcriptional level of Kv1.4, but not Kv4.2, was significantly increased in the AßPP-/- mice. This resulted in a marked increase in the peak time of A-type transient outward potassium currents in both glutamatergic and gamma-aminobutyric acid-ergic (GABAergic) neurons. Furthermore, a mechanistic experiment using human embryonic kidney 293 (HEK293) cells revealed that the AßPP deficiency-induced increase in Kv1.4 may not involve protein-protein interaction between AßPP and Kv1.4. CONCLUSION: This study suggests that AßPP modulates neuronal firing and oscillatory activity in the hippocampus, and Kv1.4 may be involved in mediating the modulation.

miR-448 regulates potassium voltage-gated channel subfamily A member 4 (KCNA4) in ischemia and heart failure.

BACKGROUND: MicroRNA miR-448 mediates some of the effects of ischemia on arrhythmic risk. Potassium voltage-gated channel subfamily A member 4 (KCNA4) encodes a Kv1.4 current that opens in response to membrane depolarization and is essential for regulating the action potential duration in heart. KCNA4 has a miR-448 binding site. OBJECTIVE: We investigated whether miR-448 was involved in the regulation of KCNA4 messenger RNA expression in ischemia. METHODS: Quantitative real-time reverse-transcriptase polymerase chain reaction was used to investigate the expression of KCNA4 and miR-448. Pull-down assays were used to examine the interaction between miR-448 and KCNA4. miR-448 decoy and binding site mutation were used to examine the specificity of the effect for KCNA4. RESULTS: The expression of KCNA4 is diminished in ischemia and human heart failure tissues with ventricular tachycardia. Previously, we have shown that miR-448 is upregulated in ischemia and inhibition can prevent arrhythmic risk after myocardial infarction. The 3'-untranslated region of KCNA4 has a conserved miR-448 binding site. miR-448 bound to this site directly and reduced KCNA4 expression and the transient outward potassium current. Inhibition of miR-448 restored KCNA4. CONCLUSION: These findings showed a link between Kv1.4 downregulation and miR-448-mediated upregulation in ischemia, suggesting a new mechanism for the antiarrhythmic effect of miR-448 inhibition.

Voltage-gated potassium channel dysfunction in dorsal root ganglia contributes to the exaggerated exercise pressor reflex in rats with chronic heart failure.

An exaggerated exercise pressor reflex (EPR) causes excessive sympathoexcitation and exercise intolerance during physical activity in the chronic heart failure (CHF) state. Muscle afferent sensitization contributes to the genesis of the exaggerated EPR in CHF. However, the cellular mechanisms underlying muscle afferent sensitization in CHF remain unclear. Considering that voltage-gated potassium (Kv) channels critically regulate afferent neuronal excitability, we examined the potential role of Kv channels in mediating the sensitized EPR in male rats with CHF. Real-time reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting experiments demonstrate that both mRNA and protein expressions of multiple Kv channel isoforms (Kv1.4, Kv3.4, Kv4.2, and Kv4.3) were downregulated in lumbar dorsal root ganglions (DRGs) of CHF rats compared with sham rats. Immunofluorescence data demonstrate significant decreased Kv channel staining in both NF200-positive and IB4-positive lumbar DRG neurons in CHF rats compared with sham rats. Data from patch-clamp experiments demonstrate that the total Kv current, especially IA, was dramatically decreased in medium-sized IB4-negative muscle afferent neurons (a subpopulation containing mostly Aδ neurons) from CHF rats compared with sham rats, indicating a potential functional loss of Kv channels in muscle afferent Aδ neurons. In in vivo experiments, adenoviral overexpression of Kv4.3 in lumbar DRGs for 1 wk attenuated the exaggerated EPR induced by muscle static contraction and the mechanoreflex by passive stretch without affecting the blunted cardiovascular response to hindlimb arterial injection of capsaicin in CHF rats. These data suggest that Kv channel dysfunction in DRGs plays a critical role in mediating the exaggerated EPR and muscle afferent sensitization in CHF.NEW & NOTEWORTHY The primary finding of this manuscript is that voltage-gated potassium (Kv) channel dysfunction in DRGs plays a critical role in mediating the exaggerated EPR and muscle afferent sensitization in chronic heart failure (CHF). We propose that manipulation of Kv channels in DRG neurons could be considered as a potential new approach to reduce the exaggerated sympathoexcitation and to improve exercise intolerance in CHF, which can ultimately facilitate an improved quality of life and reduce mortality.

Human adrenal glomerulosa cells express K2P and GIRK potassium channels that are inhibited by ANG II and ACTH.

In whole cell patch clamp recordings, it was discovered that normal human adrenal zona glomerulosa (AZG) cells express members of the three major families of K+ channels. Among these are a two-pore (K2P) leak-type and a G protein-coupled, inwardly rectifying (GIRK) channel, both inhibited by peptide hormones that stimulate aldosterone secretion. The K2P current displayed properties identifying it as TREK-1 (KCNK2). This outwardly rectifying current was activated by arachidonic acid and inhibited by angiotensin II (ANG II), adrenocorticotrophic hormone (ACTH), and forskolin. The activation and inhibition of TREK-1 was coupled to AZG cell hyperpolarization and depolarization, respectively. A second K2P channel, TASK-1 (KCNK3), was expressed at a lower density in AZG cells. Human AZG cells also express inwardly rectifying K+ current(s) (KIR) that include quasi-instantaneous and time-dependent components. This is the first report demonstrating the presence of KIR in whole cell recordings from AZG cells of any species. The time-dependent current was selectively inhibited by ANG II, and ACTH, identifying it as a G protein-coupled (GIRK) channel, most likely KIR3.4 (KCNJ5). The quasi-instantaneous KIR current was not inhibited by ANG II or ACTH and may be a separate non-GIRK current. Finally, AZG cells express a voltage-gated, rapidly inactivating K+ current whose properties identified as KV1.4 (KCNA4), a conclusion confirmed by Northern blot. These findings demonstrate that human AZG cells express K2P and GIRK channels whose inhibition by ANG II and ACTH is likely coupled to depolarization-dependent secretion. They further demonstrate that human AZG K+ channels differ fundamentally from the widely adopted rodent models for human aldosterone secretion.

Studies of Conorfamide-Sr3 on Human Voltage-Gated Kv1 Potassium Channel Subtypes.

Recently, Conorfamide-Sr3 (CNF-Sr3) was isolated from the venom of Conus spurius and was demonstrated to have an inhibitory concentration-dependent effect on the Shaker K+ channel. The voltage-gated potassium channels play critical functions on cellular signaling, from the regeneration of action potentials in neurons to the regulation of insulin secretion in pancreatic cells, among others. In mammals, there are at least 40 genes encoding voltage-gated K+ channels and the process of expression of some of them may include alternative splicing. Given the enormous variety of these channels and the proven use of conotoxins as tools to distinguish different ligand- and voltage-gated ion channels, in this work, we explored the possible effect of CNF-Sr3 on four human voltage-gated K+ channel subtypes homologous to the Shaker channel. CNF-Sr3 showed a 10 times higher affinity for the Kv1.6 subtype with respect to Kv1.3 (IC50 = 2.7 and 24 µM, respectively) and no significant effect on Kv1.4 and Kv1.5 at 10 µM. Thus, CNF-Sr3 might become a novel molecular probe to study diverse aspects of human Kv1.3 and Kv1.6 channels.

Sphingosine kinase 2 inhibitor ABC294640 suppresses neuronal excitability and inhibits multiple endogenously and exogenously expressed voltage-gated ion channels in cultured cells.

Sphingolipids regulate multiple cellular processes, including proliferation, autophagy, and apoptosis. Sphingosine kinases, the key enzymes in the metabolism of sphingolipids, are overexpressed in many cancers, making them important targets for the development of antitumor drugs. ABC294640 is a selective sphingosine kinase 2 (SK2) inhibitor that shows good antitumor activity in vitro. One phase I clinical study of ABC294640 reported that ABC294640 caused a variety of neurological disorders. The mechanism of these phenomena, however, remains unclear. In the present study, we used in vitro cell experiments to test the effects of ABC294640 on the nervous system. We found that ABC294640 suppressed the firing of action potentials in cultured hippocampal neurons from neonatal mice and inhibited endogenous sodium, potassium, and calcium currents in both cultured neurons and SH-SY5Y cells. In addition, we tested four types of human voltage-gated potassium channels transiently expressed in HEK293T cells. All were inhibited by ABC294640, of which KV4.2 and KV1.4 were more sensitive than BK and K2P2.1. The effect of ABC294640 on ion channels was different from another SK2 inhibitor K145 and was not affected by S1P. The fast onset and recovery of the inhibition indicated that ABC294640 was likely to inhibit ion channels by acting directly on channel proteins, rather than by inhibiting SK2. These results revealed the mechanism by which ABC294640 interferes with the nervous system. To develop future antitumor drugs, researchers should modify the structure of ABC294640 to avoid its effects on ion channels or should develop compounds that target SK2 or downstream molecules.

Functional role of endogenous Kv1.4 in experimental demyelination.

During neuroinflammation, the shaker type potassium channel Kv1.4 is re-expressed in oligodendrocytes (Ol), but not immune cells. Here, we analyze the role of endogenous Kv1.4 in two demyelinating animal models of multiple sclerosis. While Kv1.4 deficiency in primary murine Ol led to a decreased proliferation rate in vitro, it did not exert an effect on Ol proliferation or on the extent of de- or remyelination in the cuprizone model in vivo. However, in experimental autoimmune encephalomyelitis, Kv1.4-/- mice exhibited a milder disease course and reduced Th1 responses. These data argue for an indirect effect of Kv1.4 on immune cells, possibly via glial cells.

Altered gating of Kv1.4 in the nucleus accumbens suppresses motivation for reward.

Deficient motivation contributes to numerous psychiatric disorders, including withdrawal from drug use, depression, schizophrenia, and others. Nucleus accumbens (NAc) has been implicated in motivated behavior, but it remains unclear whether motivational drive is linked to discrete neurobiological mechanisms within the NAc. To examine this, we profiled cohorts of Sprague-Dawley rats in a test of motivation to consume sucrose. We found that substantial variability in willingness to exert effort for reward was not associated with operant responding under low-effort conditions or stress levels. Instead, effort-based motivation was mirrored by a divergent NAc shell transcriptome with differential regulation at potassium and dopamine signaling genes. Functionally, motivation was inversely related to excitability of NAc principal neurons. Furthermore, neuronal and behavioral outputs associated with low motivation were linked to faster inactivation of a voltage-gated potassium channel, Kv1.4. These results raise the prospect of targeting Kv1.4 gating in psychiatric conditions associated with motivational dysfunction.

Interleukin 6 decreases nociceptor expression of the potassium channel KV1.4 in a rat model of hand-arm vibration syndrome.

Chronic muscle pain is a prominent symptom of the hand-arm vibration syndrome (HAVS), an occupational disease induced by exposure to vibrating power tools, but the underlying mechanism remains unknown. We evaluated the hypothesis that vibration induces an interleukin 6 (IL-6)-mediated downregulation of the potassium voltage-gated channel subfamily A member 4 (KV1.4) in nociceptors leading to muscle pain. Adult male rats were submitted to a protocol of mechanical vibration of the right hind limb. Twenty-four hours after vibration, muscle hyperalgesia was observed, concomitant to increased levels of IL-6 in the gastrocnemius muscle and decreased expression of KV1.4 in the dorsal root ganglia. Local injection of neutralizing antibodies against IL-6 attenuated the muscle hyperalgesia induced by vibration, whereas antisense knockdown of this channel in the dorsal root ganglia mimicked the muscle hyperalgesia observed in the model of HAVS. Finally, knockdown of the IL-6 receptor signaling subunit glycoprotein 130 (gp130) attenuated both vibration-induced muscle hyperalgesia and downregulation of KV1.4. These results support the hypothesis that IL-6 plays a central role in the induction of muscle pain in HAVS. This likely occurs through intracellular signaling downstream to the IL-6 receptor subunit gp130, which decreases the expression of KV1.4 in nociceptors.

ADAM23 is a negative regulator of Kv1.1/Kv1.4 potassium currents.

Background ADAM22 and ADAM23 are transmembrane proteins that bind the secreted synaptic protein LGI1 and associate with Kv1.1/Kv1.4 potassium channels. However, the roles of these proteins in regulated voltage-gated potassium currents are poorly understood. Methods Cultured cells were transfected to express ADAM22, ADAM23, and Kv1.1/Kv1.4. Voltage-gated potassium currents were measured by whole-cell patch-clamp. Immunostaining Kv1.1 with fluorescent antibodies and fluorescently tagged Kv1.1 subunits was used to measure the effects of ADAM proteins on cell-surface and total expression of Kv1.1 channels. LGI1-conditioned media was added to assess the effect on LGI1 on Kv1.1 currents. Results Cells transfected with Kv1.1/Kv1.4 showed voltage-gated potassium currents (Kv1.1 currents). ADAM23 was a powerful negative regulator of Kv1.1 currents and caused decreased surface expression of Kv1.1 subunits. This decrease in current was not mediated by clathrin-dependent endocytosis. LGI1-conditioned media did not affect the negative regulation of Kv1.1 currents by ADAM23. ADAM22 had no significant effect on Kv1.1 currents by itself, but in the presence of LGI1-conditioned media markedly potentiated Kv1.1 currents without changing channel activation kinetics. Conclusions ADAM22 and ADAM23 have opposite effects on Kv1.1 currents. The relative expression of these proteins, and the availability of LGI1 may shape the expression of Kv1.1 currents in different neuronal membrane domains.

Cytometric cell-based assays for anti-striational antibodies in myasthenia gravis with myositis and/or myocarditis.

The purposes of the present study were to identify anti-striational antibodies in myasthenia gravis (MG) patients with myositis and/or myocarditis using a combination of cell-based assays and flow cytometry (cytometric cell-based assays) and to describe the main clinical implications. Among 2,609 stored samples collected from all over Japan between 2003 and 2016, we had serum samples from 30 MG patients with myositis and/or myocarditis. Cytometric cell-based assays with titin, ryanodine receptor, and voltage-gated Kv1.4 were performed. Autoantibodies were determined by differences in phycoerythin fluorescence between the 293F cells and titin-transfected cells. MG patients with myositis and/or myocarditis as well as late-onset and thymoma-associated MG had anti-titin, anti-ryanodine receptor, and anti-Kv1.4 antibodies. In contrast, patients with early-onset MG, those with other myopathies and healthy controls did not have anti-titin or anti-Kv1.4 antibodies with some exceptions, but they possessed anti-ryanodine receptor antibodies. Thirty MG patients with myositis and/or myocarditis showed a severe generalized form, and 21 of them had thymoma. Anti-titin and anti-Kv1.4 antibodies were found in 28 (93%) and 15 (50%) patients, respectively, and all patients had at least one of these antibodies. Cytometric cell-based assays thus demonstrated that anti-striational antibodies are biomarkers of MG with myositis and/or myocarditis.

Antidepressive and anxiolytic effects of ostruthin, a TREK-1 channel activator.

We screened a library of botanical compounds purified from plants of Vietnam for modulators of the activity of a two-pore domain K+ channel, TREK-1, and we identified a hydroxycoumarin-related compound, ostruthin, as an activator of this channel. Ostruthin increased whole-cell TREK-1 channel currents in 293T cells at a low concentration (EC50 = 5.3 µM), and also activity of the TREK-2 channel (EC50 = 3.7 mM). In contrast, ostruthin inhibited other K+ channels, e.g. human ether-à-go-go-related gene (HERG1), inward-rectifier (Kir2.1), voltage-gated (Kv1.4), and two-pore domain (TASK-1) at higher concentrations, without affecting voltage-gated potassium channel (KCNQ1 and 3). We tested the effect of this compound on mouse anxiety- and depression-like behaviors and found anxiolytic activity in the open-field, elevated plus maze, and light/dark box tests. Of note, ostruthin also showed antidepressive effects in the forced swim and tail suspension tests, although previous studies reported that inhibition of TREK-1 channels resulted in an antidepressive effect. The anxiolytic and antidepressive effect was diminished by co-administration of a TREK-1 blocker, amlodipine, indicating the involvement of TREK-1 channels. Administration of ostruthin suppressed the stress-induced increase in anti-c-Fos immunoreactivity in the lateral septum, without affecting immunoreactivity in other mood disorder-related nuclei, e.g. the amygdala, paraventricular nuclei, and dorsal raphe nucleus. Ostruthin may exert its anxiolytic and antidepressive effects through a different mechanism from current drugs.

Regulation of Kv1.4 potassium channels by PKC and AMPK kinases.

Over the last years extensive kinase-mediated regulation of a number of voltage-gated potassium (Kv) channels important in cardiac electrophysiology has been reported. This includes regulation of Kv1.5, Kv7.1 and Kv11.1 cell surface expression, where the kinase-mediated regulation appears to center around the ubiquitin ligase Nedd4-2. In the present study we examined whether Kv1.4, constituting the cardiac Ito,s current, is subject to similar regulation. In the epithelial Madin-Darby Canine Kidney (MDCK) cell line, which constitutes a highly reproducible model system for addressing membrane targeting, we find, by confocal microscopy, that Kv1.4 cell surface expression is downregulated by activation of protein kinase C (PKC) and AMP-activated protein kinase (AMPK). In contrast, manipulating the activities of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) and serum and glucocorticoid-regulated kinase 1 (SGK1) were without effect on channel localization. The PKC and AMPK-mediated downregulation of Kv1.4 membrane surface localization was confirmed by two-electrode voltage clamp in Xenopus laevis oocytes, where pharmacological activation of PKC and AMPK reduced Kv1.4 current levels. We further demonstrate that unlike related Kv channels, Kv1.4 current levels in Xenopus laevis oocytes are not reduced by co-expression of Nedd4-2, or the related Nedd4-1 ubiquitin ligase. In conclusion, we demonstrate that the surface expression of Kv1.4 is downregulated by the two kinases AMPK and PKC, but is unaffected by PI3K-SGK1 signaling, as well as Nedd4-1/Nedd4-2 activity. In the light of previous reports, our results demonstrate an impressive heterogeneity in the molecular pathways controlling the surface expression of highly related potassium channel subunits.

Ergodicity breaking on the neuronal surface emerges from random switching between diffusive states.

Stochastic motion on the surface of living cells is critical to promote molecular encounters that are necessary for multiple cellular processes. Often the complexity of the cell membranes leads to anomalous diffusion, which under certain conditions it is accompanied by non-ergodic dynamics. Here, we unravel two manifestations of ergodicity breaking in the dynamics of membrane proteins in the somatic surface of hippocampal neurons. Three different tagged molecules are studied on the surface of the soma: the voltage-gated potassium and sodium channels Kv1.4 and Nav1.6 and the glycoprotein CD4. In these three molecules ergodicity breaking is unveiled by the confidence interval of the mean square displacement and by the dynamical functional estimator. Ergodicity breaking is found to take place due to transient confinement effects since the molecules alternate between free diffusion and confined motion.

Amino Acid Properties of Trafficking Determinants in the Outer Pore-Forming Region of Kv1 Potassium Channels in Cell Lines.

Different classes of Kv1 potassium channels have different trafficking patterns despite having very similar amino acid sequences. Two amino acids responsible for these differences have been identified in the outer pore turret region of Kv1.1 and Kv1.4. Here we tested a series of substitutions at these two determinants on Kv1.4. All P506 substitutions tested resulted in a significant decrease in surface protein, total protein, and protein half-life, indicating that proline is required at 506 to stabilize protein conformation and increase trafficking to the cell surface. All K533 substitutions tested had no effect on total protein, suggesting that the lysine at 533 is not important for maintaining Kv1.4 protein conformation. However, a basic or long polar amino acid, such as K, R, or Q, at this position favored high surface protein and efficient trafficking of Kv1.4, whereas an acidic or short amino acid, such as D, E, S, L, N, or H, at this position induced partial high endoplasmic reticulum-retention. This intracellular retention was not due to protein misfolding. We propose that these four prolines and four lysines in a Kv1.4 homotetramer might provide a binding site for a putative endoplasmic reticulum-export molecule to ensure high cell surface protein expression of the channel.

Elucidating distinct ion channel populations on the surface of hippocampal neurons via single-particle tracking recurrence analysis.

Protein and lipid nanodomains are prevalent on the surface of mammalian cells. In particular, it has been recently recognized that ion channels assemble into surface nanoclusters in the soma of cultured neurons. However, the interactions of these molecules with surface nanodomains display a considerable degree of heterogeneity. Here, we investigate this heterogeneity and develop statistical tools based on the recurrence of individual trajectories to identify subpopulations within ion channels in the neuronal surface. We specifically study the dynamics of the K^{+} channel Kv1.4 and the Na^{+} channel Nav1.6 on the surface of cultured hippocampal neurons at the single-molecule level. We find that both these molecules are expressed in two different forms with distinct kinetics with regards to surface interactions, emphasizing the complex proteomic landscape of the neuronal surface. Further, the tools presented in this work provide new methods for the analysis of membrane nanodomains, transient confinement, and identification of populations within single-particle trajectories.

Effects of ginseng-spikenard heart-nourishing capsule on inactivation of C-type Kv1.4 potassium channel.

This research is to explore the effects of traditional Chinese medicine Ginseng-spikenard heart-nourishing capsule on the inactivation of c-type Kv1.4 channels (Kv1.4∆N) in Xenopus laevis oocytes with two-electrode voltageclamp technique. Defolliculated oocytes (stage V-VI) were injected with transcribed cRNAs of ferret Kv1.4δN channels. During recording, oocytes were continuously perfused with ND96 solution (control group) and solution prepared from Ginseng-spikenard heart-nourishing capsule (experimental group). Results found that, at the command potential of +50 mV, the current of experimental group was reduced to 48.33±4.0% of that in control group. The inactivation time constants in control and experimental groups were 2962.56±175.35 ms and 304.13±36.22ms, respectively (P<0.05, n=7). The recovery time of fKv1.4∆N channel after inactivation in control group and experimental groups was 987±68.39 ms and 1734.15±98.45 ms, respectively (P<0.05, n=5). Ginseng-spikenard heart-nourishing capsule can inhibit the Kv1.4δN channel, which may be one of the mechanisms of underlying antiarrhythmia.

Polyunsaturated fatty acids inhibit Kv1.4 by interacting with positively charged extracellular pore residues.

Polyunsaturated fatty acids (PUFAs) modulate voltage-gated K(+) channel inactivation by an unknown site and mechanism. The effects of ω-6 and ω-3 PUFAs were investigated on the heterologously expressed Kv1.4 channel. PUFAs inhibited wild-type Kv1.4 during repetitive pulsing as a result of slowing of recovery from inactivation. In a mutant Kv1.4 channel lacking N-type inactivation, PUFAs reversibly enhanced C-type inactivation (Kd, 15-43 µM). C-type inactivation was affected by extracellular H(+) and K(+) as well as PUFAs and there was an interaction among the three: the effect of PUFAs was reversed during acidosis and abolished on raising K(+) Replacement of two positively charged residues in the extracellular pore (H508 and K532) abolished the effects of the PUFAs (and extracellular H(+) and K(+)) on C-type inactivation but had no effect on the lipoelectric modulation of voltage sensor activation, suggesting two separable interaction sites/mechanisms of action of PUFAs. Charge calculations suggest that the acidic head group of the PUFAs raises the pKa of H508 and this reduces the K(+) occupancy of the selectivity filter, stabilizing the C-type inactivated state.

The episodic ataxia type 1 mutation I262T alters voltage-dependent gating and disrupts protein biosynthesis of human Kv1.1 potassium channels.

Voltage-gated potassium (Kv) channels are essential for setting neuronal membrane excitability. Mutations in human Kv1.1 channels are linked to episodic ataxia type 1 (EA1). The EA1-associated mutation I262T was identified from a patient with atypical phenotypes. Although a previous report has characterized its suppression effect, several key questions regarding the impact of the I262T mutation on Kv1.1 as well as other members of the Kv1 subfamily remain unanswered. Herein we show that the dominant-negative effect of I262T on Kv1.1 current expression is not reversed by co-expression with Kvß1.1 or Kvß2 subunits. Biochemical examinations indicate that I262T displays enhanced protein degradation and impedes membrane trafficking of Kv1.1 wild-type subunits. I262T appears to be the first EA1 mutation directly associated with impaired protein stability. Further functional analyses demonstrate that I262T changes the voltage-dependent activation and Kvß1.1-mediated inactivation, uncouples inactivation from activation gating, and decelerates the kinetics of cumulative inactivation of Kv1.1 channels. I262T also exerts similar dominant effects on the gating of Kv1.2 and Kv1.4 channels. Together our data suggest that I262T confers altered channel gating and reduced functional expression of Kv1 channels, which may account for some of the phenotypes of the EA1 patient.

Inhibition of Kv channel expression by NSAIDs depolarizes membrane potential and inhibits cell migration by disrupting calpain signaling.

Clinical use of non-steroidal anti-inflammatory drugs (NSAIDs) is well known to cause gastrointestinal ulcer formation via several mechanisms that include inhibiting epithelial cell migration and mucosal restitution. The drug-affected signaling pathways that contribute to inhibition of migration by NSAIDs are poorly understood, though previous studies have shown that NSAIDs depolarize membrane potential and suppress expression of calpain proteases and voltage-gated potassium (Kv) channel subunits. Kv channels play significant roles in cell migration and are targets of NSAID activity in white blood cells, but the specific functional effects of NSAID-induced changes in Kv channel expression, particularly on cell migration, are unknown in intestinal epithelial cells. Accordingly, we investigated the effects of NSAIDs on expression of Kv1.3, 1.4, and 1.6 in vitro and/or in vivo and evaluated the functional significance of loss of Kv subunit expression. Indomethacin or NS-398 reduced total and plasma membrane protein expression of Kv1.3 in cultured intestinal epithelial cells (IEC-6). Additionally, depolarization of membrane potential with margatoxin (MgTx), 40mM K(+), or silencing of Kv channel expression with siRNA significantly reduced IEC-6 cell migration and disrupted calpain activity. Furthermore, in rat small intestinal epithelia, indomethacin and NS-398 had significant, yet distinct, effects on gene and protein expression of Kv1.3, 1.4, or 1.6, suggesting that these may be clinically relevant targets. Our results show that inhibition of epithelial cell migration by NSAIDs is associated with decreased expression of Kv channel subunits, and provide a mechanism through which NSAIDs inhibit cell migration and may contribute to NSAID-induced gastrointestinal (GI) toxicity.