Modulation Of Potassium Channels Research Articles

Negative inotropic and hypotensive effects of the superoxide dismutase mimetic tempol in pigs

Through interference with free radicals, the nitroxide tempol potentially increases bioavailability of nitric oxide (NO) and along with modulation of potassium channels reduces blood pressure (BP). We studied whether tempol in pigs lowers BP by mechanisms sensitive to inhibition of NO synthase or large conductance calcium-activated potassium channels (BKCa). The cardiovascular effects of intravenous tempol (25–50mg/kg) were examined in anesthetized pigs with myocardial function being evaluated by echocardiography. While saline-treated animals remained hemodynamically stable, tempol induced fast, dose-dependent and transient reductions in BP lasting 5–10min with a simultaneous impairment of left ventricular contraction. Pretreatment with the NO synthase (NOS) inhibitor NG-nitro-l-arginine methyl ester (l-NAME, 4mg/kg) or a blocker of BKCa (tetraethylammonium (TEA), 100mg/h) increased baseline BP but also enhanced BP reductions to tempol. Isolated myocardial trabeculae subjected to an identical protocol also demonstrated dose-related reductions in contractility to tempol. This effect was not affected by l-NAME, but attenuated by TEA. In isolated mesenteric resistance arteries contracted with noradrenaline, tempol caused small postjunctional l-NAME sensitive relaxations, while neurogenic contractions were inhibited by tempol by TEA-sensitive mechanisms and mechanisms insensitive to TEA and l-NAME. In conclusion intravenous tempol induces fast transient reductions in BP associated with simultaneous reductions in myocardial contraction. Tempol exerts direct negative inotropic effects which are partly sensitive to BKCa-blockade but independent of NOS inhibition. In addition tempol has direct vasodilatory effects despite NOS and potassium channel blockade. The negative inotropic and hypotensive effects raise concerns using tempol, or structurally similar drugs, for intravenous use.

Exome Sequencing Implicates an Increased Burden of Rare Potassium Channel Variants in the Risk of Drug-Induced Long QT Interval Syndrome

The aim of this study was to test the hypothesis that rare variants are associated with drug-induced long QT interval syndrome (diLQTS) and torsades de pointes. diLQTS is associated with the potentially fatal arrhythmia torsades de pointes. The contribution of rare genetic variants to the underlying genetic framework predisposing to diLQTS has not been systematically examined. We performed whole-exome sequencing on 65 diLQTS patients and 148 drug-exposed control subjects of European descent. We used rare variant analyses (variable threshold and sequence kernel association test) and gene-set analyses to identify genes enriched with rare amino acid coding (AAC) variants associated with diLQTS. Significant associations were reanalyzed by comparing diLQTS patients with 515 ethnically matched control subjects from the National Heart, Lung, and Blood Grand Opportunity Exome Sequencing Project. Rare variants in 7 genes were enriched in the diLQTS patients according to the sequence kernel association test or variable threshold compared with drug-exposed controls (p < 0.001). Of these, we replicated the diLQTS associations for KCNE1 and ACN9 using 515 Exome Sequencing Project control subjects (p < 0.05). A total of 37% of the diLQTS patients also had 1 or more rare AAC variants compared with 21% of control subjects (p = 0.009), in a pre-defined set of 7 congenital long QT interval syndrome (cLQTS) genes encoding potassium channels or channel modulators (KCNE1, KCNE2, KCNH2, KCNJ2, KCNJ5, KCNQ1, AKAP9). By combining whole-exome sequencing with aggregated rare variant analyses, we implicate rare variants in KCNE1 and ACN9 as risk factors for diLQTS. Moreover, diLQTS patients were more burdened by rare AAC variants in cLQTS genes encoding potassium channel modulators, supporting the idea that multiple rare variants, notably across cLQTS genes, predispose to diLQTS.

Vascular Dysfunction in Diabetes Mellitus

Vascular smooth muscle membrane potential is a key determinant of vessel tone primarily through regulation of voltage-dependent calcium channels (VDCCs). The opening probability of VDCCs is increased by membrane depolarization, facilitating influx of calcium and producing contraction of vascular smooth muscle cells (VSMCs). Control of membrane potential and VDCC activity is orchestrated by a complex series of signals that occur just below the sarcolemmal membrane. Article, see p 607 Large-conductance calcium-activated potassium channels (also known as maxi-K or BK channels) play a pivotal role in modulating vasomotor tone in both health and disease. BK channels, expressed on VSMCs, act as breaks for the increase in vascular tone that occurs after membrane depolarization and elevation of cytosolic calcium. An intracellular rise in cytosolic calcium through VDCC leads to activation of ryanodine receptors that release quanta of calcium from the sarcoplasmic reticulum (calcium sparks). It is these intracellularly generated sparks that elevate local submembrane concentrations of calcium and activate BK channels which increase potassium conductance and lower membrane potential.1 This reduces opening of VDCCs, thus modulating contraction (Figure2). Figure. Pathways for activation and modulation of vascular large-conductance calcium-activated potassium (BK) channels . Diabetes mellitus reduces BK channel activity by ≥3 mechanisms. First, as demonstrated by Nystoriak et al,2 through activation of calcineurin, dephosphorylated nuclear factor of activated T cells, c3 isoform (NFATc3) enters the nucleus where it inhibits transcription of the β1 subunit of BK, thereby reducing BK sensitivity to calcium. Second, diabetes mellitus–induced elevations in reactive oxygen species (ROS), particularly hydrogen peroxide, directly oxidize cysteine residues in the bowl region of the α subunit of BK to reduce opening. Finally, reduced insulin activation of phosphoinositol-3-kinase (PI3) kinase in diabetes mellitus reduces phosphorylation of forkhead box O family transcription factor (FOXO)-3a, allowing nuclear localization and transcription of f-box only protein …

Use of Label-free Optical Biosensors to Detect Modulation of Potassium Channels by G-protein Coupled Receptors

Ion channels control the electrical properties of neurons and other excitable cell types by selectively allowing ions to flow through the plasma membrane1. To regulate neuronal excitability, the biophysical properties of ion channels are modified by signaling proteins and molecules, which often bind to the channels themselves to form a heteromeric channel complex2,3. Traditional assays examining the interaction between channels and regulatory proteins require exogenous labels that can potentially alter the protein's behavior and decrease the physiological relevance of the target, while providing little information on the time course of interactions in living cells. Optical biosensors, such as the X-BODY Biosciences BIND Scanner system, use a novel label-free technology, resonance wavelength grating (RWG) optical biosensors, to detect changes in resonant reflected light near the biosensor. This assay allows the detection of the relative change in mass within the bottom portion of living cells adherent to the biosensor surface resulting from ligand induced changes in cell adhesion and spreading, toxicity, proliferation, and changes in protein-protein interactions near the plasma membrane. RWG optical biosensors have been used to detect changes in mass near the plasma membrane of cells following activation of G protein-coupled receptors (GPCRs), receptor tyrosine kinases, and other cell surface receptors. Ligand-induced changes in ion channel-protein interactions can also be studied using this assay. In this paper, we will describe the experimental procedure used to detect the modulation of Slack-B sodium-activated potassium (KNa) channels by GPCRs.

Use of Label-free Optical Biosensors to Detect Modulation of Potassium Channels by G-protein Coupled Receptors

Ion channels control the electrical properties of neurons and other excitable cell types by selectively allowing ions to flow through the plasma membrane(1). To regulate neuronal excitability, the biophysical properties of ion channels are modified by signaling proteins and molecules, which often bind to the channels themselves to form a heteromeric channel complex(2,3). Traditional assays examining the interaction between channels and regulatory proteins require exogenous labels that can potentially alter the protein's behavior and decrease the physiological relevance of the target, while providing little information on the time course of interactions in living cells. Optical biosensors, such as the X-BODY Biosciences BIND Scanner system, use a novel label-free technology, resonance wavelength grating (RWG) optical biosensors, to detect changes in resonant reflected light near the biosensor. This assay allows the detection of the relative change in mass within the bottom portion of living cells adherent to the biosensor surface resulting from ligand induced changes in cell adhesion and spreading, toxicity, proliferation, and changes in protein-protein interactions near the plasma membrane. RWG optical biosensors have been used to detect changes in mass near the plasma membrane of cells following activation of G protein-coupled receptors (GPCRs), receptor tyrosine kinases, and other cell surface receptors. Ligand-induced changes in ion channel-protein interactions can also be studied using this assay. In this paper, we will describe the experimental procedure used to detect the modulation of Slack-B sodium-activated potassium (KNa) channels by GPCRs.

Membrane Lipids Tune Synaptic Transmission by Direct Modulation of Presynaptic Potassium Channels

(Neuron 81, 787–799; February 19, 2014) In the version of this article published early online, the citation for Alle et al. (2011) was incorrectly deleted during the production stage, and the corresponding reference was omitted. The corrected article, including the citation and matching reference, now appears online and in print. The journal apologizes for this error. Membrane Lipids Tune Synaptic Transmission by Direct Modulation of Presynaptic Potassium ChannelsCarta et al.NeuronJanuary 30, 2014In BriefCarta et al. show that membrane-derived arachidonic acid acts as a retrograde messenger, directly modulating presynaptic Kv channels at excitatory synapses. This leads to broadening of the presynaptic action potentials and robust potentiation of synaptic transmission. Full-Text PDF Open Archive

Membrane Lipids Tune Synaptic Transmission by Direct Modulation of Presynaptic Potassium Channels

Voltage-gated potassium (Kv) channels are involved in action potential (AP) repolarization in excitable cells. Exogenous application of membrane-derived lipids, such as arachidonic acid (AA), regulates the gating of Kv channels. Whether membrane-derived lipids released under physiological conditions have an impact on neuronal coding through this mechanism is unknown. We show that AA released in an activity-dependent manner from postsynaptic hippocampal CA3 pyramidal cells acts as retrograde messenger, inducing a robust facilitation of mossy fiber (Mf) synaptic transmission over several minutes. AA acts by broadening presynaptic APs through the direct modulation of Kv channels. This form of short-term plasticity can be triggered when postsynaptic cell fires with physiologically relevant patterns and sets the threshold for the induction of the presynaptic form of long-term potentiation (LTP) at hippocampal Mf synapses. Hence, direct modulation of presynaptic Kv channels by activity-dependent release of lipids serves as a physiological mechanism for tuning synaptic transmission.

Inflammation enhanced brain-derived neurotrophic factor-induced suppression of the voltage-gated potassium currents in small-diameter trigeminal ganglion neurons projecting to the trigeminal nucleus interpolaris/caudalis transition zone

We recently indicated that brain-derived neurotrophic factor (BDNF) enhances the excitability of small-diameter trigeminal ganglion (TRG) neurons projecting onto the trigeminal nucleus interpolaris/caudalis (Vi/Vc) transition zone via a paracrine mechanism following masetter muscle (MM) inflammation. The present study investigated whether modulation of voltage-gated potassium (K) channels by BDNF contributes to this hyperexcitability effect. To induce inflammation we injected complete Freund’s adjuvant (CFA) into the MM. The escape threshold from mechanical stimulation applied to skin above the inflamed MM was significantly lower than in naïve rats. TRG neurons innervating the site of inflammation were subsequently identified by fluorogold (FG) labeling, and microbeads (MB) were used to label neurons projecting specifically to the Vi/Vc region. BDNF significantly decreased the total, transient (IA), and sustained (IK) currents in FG-/MB-labeled small-diameter TRG neurons under voltage-clamp conditions in naïve and inflamed rats. The magnitude of inhibition of IA and IK currents by BDNF in FG-/MB-labeled TRG neurons was significantly greater in inflamed rats than in naïve rats, and BDNF inhibited IA to a significantly greater extent than IK. Furthermore, co-administration of K252a, a tyrosine kinase inhibitor, abolished the suppression of IA and IK currents by BDNF. These results suggested that the inhibitory effects of BDNF on IA and IK currents in small-diameter TRG neurons projecting onto the Vi/Vc potentiate neuronal excitability, and in turn, contribute to MM inflammatory hyperalgesia. These findings support the development of voltage-gated K+ channel openers and tyrosine kinase inhibitors as potential therapeutic agents for the treatment of trigeminal inflammatory hyperalgesia.

Detection of KIR6 family protein in rat heart and liver mitochondria by immunoelectron microscopy

The electron microscopic study of thin sections of rat liver and heart using commercial specific antibodies against KIR.6.2 and secondary antibodies conjugated with colloidal gold was performed. It was found that the gold-labeled protein is localized in mitochondria of cardiomyocytes and hepatocytes but not in rough and smooth endoplasmic reticulum of hepatic cells and myofibrils of myocardium. In rat heart and liver mitochondria, the gold label was mainly located in mitochondrial cristae and was not found in mitochondrial matrix and intermembrane space. The data indicate that in heart and liver mitochondria there exists a protein similar in structure to the channel-forming subunit of a cytoplasmic potassium channel, KIR6.2. This is also supported by the presence of common modulators of cytoplasmic and mitochondrial ATP-dependent potassium channels. A possible role of the protein as a subunit of the mitochondrial ATP-dependent potassium channel is discussed.

Modulation of Sarcolemmal ATP-Sensitive Potassium Channels by Atrial Natriuretic Peptide in Ventricular Cardiomyocytes

ATP-sensitive potassium (KATP) channels couple cell metabolic status to membrane excitability and are crucial for stress adaptation in the heart. Natriuretic peptides (NPs) produced by the heart, the vasculature and the kidney exert physiologically important actions such as natriuresis and inhibition of sympathetic tone and cardiomyocyte hypertrophy. Our recent study reveals that the gaseous messenger nitric oxide evokes cardiac KATP channel stimulation via activation of soluble guanylyl cyclase and subsequently a cGMP-dependent signaling pathway. Binding of NPs like atrial natriuretic peptide (ANP) to the type 1 NP receptor (NPR-A) also increases intracellular cGMP level, through activating guanylyl cyclase intrinsic to the receptor; however, how ANP modulates the function of cardiac KATP channels remains to be established. By performing cell-attached patch recordings in ventricular cardiomyocytes acutely isolated from adult rabbits, we found that the single-channel activity of sarcolemmal KATP (sarcKATP) channels induced by sodium azide through metabolic inhibition was potentiated by addition of ANP (100 nM) (n=16), whereas the potentiation by ANP was abolished when the NPR-A antagonist anantin (1 μM) was coapplied (n=5); these findings suggest that the stimulatory action of ANP is mediated by NPR-A activation, which may depend on generation of diffusible cGMP. Indeed, sarcKATP channel stimulation by ANP in intact ventricular myocytes was significantly abated by inhibition of cGMP-dependent protein kinase (PKG) with KT5823 (1 μM) (n=5). Moreover, the ANP effect was prevented by selective suppression of extracellular signal-regulated protein kinase (ERK)1/2 (n=6) and Ca2+/calmodulin-dependent protein kinase II (CaMKII) (n=6). Collectively, here we demonstrate that the polypeptide hormone ANP positively modulates ventricular KATP channel function via activation of NPR-A, PKG, ERK1/2 and CaMKII. By opening myocardial KATP channels, this signaling pathway may regulate cardiac excitability and contribute to cytoprotection against ischemia-reperfusion injury.

New Mitochondrial Potassium Channels

Mitochondrial potassium channels play an important role in cytoprotection. The following potassium channels have been described in the inner mitochondrial membrane: the ATP-regulated potassium channel, the large conductance calcium activated potassium channel, the voltage-gated potassium channel and the twin-pore domain TASK-3 potassium channel. Potassium channels in the inner mitochondrial membrane are modulated by inhibitors and activators (potassium channel openers) previously described for plasma membrane potassium channels. The majority of mitochondrial potassium channel modulators exhibit a broad spectrum of off-target effects. These include uncoupling properties, inhibition of the respiratory chain and effects on cellular calcium homeostasis. Therefore, the rational application of channel inhibitors or activators is crucial to understanding the cellular consequences of mitochondrial channel inhibition or activation. In this paper, new observations on mitochondrial potassium channel will be discussed: 1). their molecular identity, 2). their interaction with potassium channel openers and inhibitors and 3). their functional role.

Potassium Channels: Structures, Diseases, and Modulators

Potassium channels participate in many critical biological functions and play important roles in a variety of diseases. In recent years, many significant discoveries have been made which motivate us to review these achievements. The focus of our review is mainly on three aspects. Firstly, we try to summarize the latest developments in structure determinants and regulation mechanism of all types of potassium channels. Secondly, we review some diseases induced by or related to these channels. Thirdly, both qualitative and quantitative approaches are utilized to analyze structural features of modulators of potassium channels. Our analyses further prove that modulators possess some certain natural-product scaffolds. And pharmacokinetic parameters are important properties for organic molecules. Besides, with in silico methods, some features that can be used to differentiate modulators are derived. There is no doubt that all these studies on potassium channels as possible pharmaceutical targets will facilitate future translational research. All the strategies developed in this review could be extended to studies on other ion channels and proteins as well.

Oxidative Modulation of Voltage-Gated Potassium Channels

Voltage-gated K+ channels are a large family of K+-selective ion channel protein complexes that open on membrane depolarization. These K+ channels are expressed in diverse tissues and their function is vital for numerous physiological processes, in particular of neurons and muscle cells. Potentially reversible oxidative regulation of voltage-gated K+ channels by reactive species such as reactive oxygen species (ROS) represents a contributing mechanism of normal cellular plasticity and may play important roles in diverse pathologies including neurodegenerative diseases. Studies using various protocols of oxidative modification, site-directed mutagenesis, and structural and kinetic modeling provide a broader phenomenology and emerging mechanistic insights. Physicochemical mechanisms of the functional consequences of oxidative modifications of voltage-gated K+ channels are only beginning to be revealed. In vivo documentation of oxidative modifications of specific amino-acid residues of various voltage-gated K+ channel proteins, including the target specificity issue, is largely absent. High-resolution chemical and proteomic analysis of ion channel proteins with respect to oxidative modification combined with ongoing studies on channel structure and function will provide a better understanding of how the function of voltage-gated K+ channels is tuned by ROS and the corresponding reducing enzymes to meet cellular needs.

KATP Channel Opener Diazoxide Prevents Neurodegeneration: A New Mechanism of Action via Antioxidative Pathway Activation

Pharmacological modulation of ATP-sensitive potassium channels has become a promising new therapeutic approach for the treatment of neurodegenerative diseases due to their role in mitochondrial and cellular protection. For instance, diazoxide, a well-known ATP-sensitive potassium channel activator with high affinity for mitochondrial component of the channel has been proved to be effective in animal models for different diseases such as Alzheimer’s disease, stroke or multiple sclerosis. Here, we analyzed the ability of diazoxide for protecting neurons front different neurotoxic insults in vitro and ex vivo. Results showed that diazoxide effectively protects NSC-34 motoneurons from glutamatergic, oxidative and inflammatory damage. Moreover, diazoxide decreased neuronal death in organotypic hippocampal slice cultures after exicitotoxicity and preserved myelin sheath in organotypic cerebellar cultures exposed to pro-inflammatory demyelinating damage. In addition, we demonstrated that one of the mechanisms of actions implied in the neuroprotective role of diazoxide is mediated by the activation of Nrf2 expression and nuclear translocation. Nrf2 expression was increased in NSC-34 neurons in vitro as well as in the spinal cord of experimental autoimmune encephalomyelitis animals orally administered with diazoxide. Thus, diazoxide is a neuroprotective agent against oxidative stress-induced damage and cellular dysfunction that can be beneficial for diseases such as multiple sclerosis.

Effect of ATP-dependent channel modulators on ischemia-induced arrhythmia change depending on age and gender

The number of ATP-dependent potassium channels in myocardial cells has been previously shown to change depending on gender and age. Different effects of the ATP-dependent potassium channel blocker, glybenclamide and ATP-dependent potassium channel opener, pinacidil on ischemia or reperfusion-induced arrhythmia observed in various research might depend on different ages and genders of the animals used. The aim of this study is to research the effect of ATP-dependent potassium channel modulators on ischemia-induced arrhythmia in animals of different ages and genders. Sprague-Dawley rats of different ages and genders were used in this study. Ischemia was produced by the ligation of the left coronary artery for 30 min. Electrocardiogram (ECG), blood pressure, infarct area and blood glucose were determined during the 30 min of ischemia. An arrhythmia score from an ECG recorded during 30 min of ischemia was determined by examining the duration and type of arrhythmia. Different effects of glybenclamide and pinacidil on the arrhythmias were observed in male and female young and middle-age rats. Pinacidil decreased the infarct zone in younger female rats, but differences in the type and length of ischemia-induced arrhythmias between females and males disappeared in older age. The results of this study showed that the effect of ATP-dependent potassium channel modulators on ischemia-induced arrhythmia changed due to the age and gender of rats.

Hydrogen sulphide suppresses human atrial fibroblast proliferation and transformation to myofibroblasts

Cardiac fibroblasts are crucial in pathophysiology of the myocardium whereby their aberrant proliferation has significant impact on cardiac function. Hydrogen sulphide (H2S) is a gaseous modulator of potassium channels on cardiomyocytes and has been reported to attenuate cardiac fibrosis. Yet, the mechanism of H2S in modulating proliferation of cardiac fibroblasts remains poorly understood. We hypothesized that H2S inhibits proliferative response of atrial fibroblasts through modulation of potassium channels. Biophysical property of potassium channels in human atrial fibroblasts was examined by whole-cell patch clamp technique and their cellular proliferation in response to H2S was assessed by BrdU assay. Large conductance Ca2+-activated K+ current (BKCa), transient outward K+ current (Ito) and inwardly rectifying K+ current (IKir) were found in human atrial fibroblasts. Current density of BKCa (IC50 = 69.4 μM; n = 6), Ito (IC50 = 55.1 μM; n = 6) and IKir (IC50 = 78.9 μM; n = 6) was significantly decreased (P < 0.05) by acute exposure to NaHS (a H2S donor) in atrial fibroblasts. Furthermore, NaHS (100–500 μM) inhibited fibroblast proliferation induced by transforming growth factor-β1 (TGF-β1; 1 ng/ml), Ang II (100 nM) or 20% FBS. Pre-conditioning of fibroblasts with NaHS decreased basal expression of Kv4.3 (encode Ito), but not KCa1.1 (encode BKCa) and Kir2.1 (encode IKir). Furthermore, H2S significantly attenuated TGF-β1–stimulated Kv4.3 and α-smooth muscle actin expression, which coincided with its inhibition of TGF-β–induced myofibroblast transformation. Our results show that H2S attenuates atrial fibroblast proliferation via suppression of K+ channel activity and moderates their differentiation towards myofibroblasts.

SDF-1α and LPA modulate microglia potassium channels through rho gtpases to regulate cell morphology

Microglia are the resident immune cells of the brain, which are important therapeutic targets for regulating the inflammatory responses particularly neurodegeneration in the aging human brain. The activation, chemotaxis and migration of microglia are regulated through G-protein coupled receptors by chemokines such as stromal cell-derived factor (SDF)-1α and bioactive lysophospholipids such as lysophosphatidic acid (LPA). Potassium channels play important roles in microglial function and cell fate decisions; however, the regulation of microglial potassium channels has not been fully elucidated. Here we show reciprocal action of SDF-1α and LPA, on potassium currents through Kir2.1 channels in primary murine microglia. The potassium channel modulation is mediated by the same small GTPases, Rac and Rho that regulate the actin cytoskeleton. SDF-1α rapidly increased the Kir2.1 current amplitude and cell spreading. These effects were mimicked by dialysing the cells with constitutively active Rac1 protein, and they were blocked by inhibiting the phosphatidylinositol 3-kinase (PI3K) with wortmannin. In contrast, LPA and constitutively active RhoA decreased the Kir2.1 currents and stimulated cell contraction. Thus, SDF-1α and LPA regulate both the actin cytoskeleton and the Kir2.1 potassium channels through the same Rho GTPase signaling pathways. The inhibition of Kir2.1 with chloroethylclonidine produced cell contraction independently of chemokine action. This suggests that potassium channels are essential for the morphological phenotype and functioning of microglia. In conclusion, the small GTPases, Rac and Rho, modulate Kir2.1 channels and block of Kir2.1 channels causes changes in microglia morphology.

4-Aminopyridine ameliorates mobility but not disease course in an animal model of multiple sclerosis

Neuropathological changes following demyelination in multiple sclerosis (MS) lead to a reorganization of axolemmal channels that causes conduction changes including conduction failure. Pharmacological modulation of voltage-sensitive potassium channels (KV) has been found to improve conduction in experimentally induced demyelination and produces symptomatic improvement in MS patients. Here we used an animal model of autoimmune inflammatory neurodegeneration, namely experimental autoimmune encephalomyelitis (EAE), to test the influence of the KV-inhibitor 4-aminopyridine (4-AP) on various disease and immune parameters as well as mobility in MOG35–55 immunized C57Bl/6 mice. We challenged the hypothesis that 4-AP exerts relevant immunomodulatory or neuroprotective properties.Neither prophylactic nor therapeutic treatment with 4-AP altered disease incidence or disease course of EAE. Histopathological signs of demyelination and neuronal damage as well as MRI imaging of brain volume changes were unaltered. While application of 4-AP significantly reduced the standing outward current of stimulated CD4+ T cells compared to controls, it failed to impact intracellular calcium concentrations in these cells. Compatibly, KV channel inhibition neither influenced CD4+ T cell effector functions (proliferation, IL17 or IFNγ production). Importantly however, despite equal disease severity scores 4-AP treated animals showed improved mobility as assessed by 2 independent methods, 1) foot print and 2) rotarod analysis (0.332±0.03, n=7 versus 0.399±0.08, n=14, p<0.001, respectively).Our data suggest that 4-AP while having no apparent immunomodulatory or direct neuroprotective effects, significantly ameliorates conduction abnormalities thereby improving gait and coordination. Improvement of mobility in this experimental model supports trial data and clinical experience with 4-AP in the symptomatic treatment of MS.

Dopaminergic modulation of axonal potassium channels and action potential waveform in pyramidal neurons of prefrontal cortex

Voltage-gated K(+) (KV) channels play critical roles in shaping neuronal signals. KV channels distributed in the perisomatic regions and thick dendrites of cortical pyramidal neurons have been extensively studied. However, the properties and regulation of KV channels distributed in the thin axons remain unknown. In this study, by performing somatic and axonal patch-clamp recordings from layer 5 pyramidal neurons of prefrontal cortical slices, we showed that the rapidly inactivating A-currents mediated the transient K(+) currents evoked by action potential (AP) waveform command (KAP) at the soma, whereas the rapidly activating but slowly inactivating KV1-mediated D-currents dominated the KAP at the axon. In addition, activation of D1-like receptors for dopamine decreased the axonal K(+) currents, as a result of an increase in the activity of cAMP-PKA pathway. In contrast, activation of D2-like receptors showed an opposite effect on the axonal K(+) currents. Further experiments demonstrated that functional D1-like receptors were expressed at the main axon trunk and their activation could broaden the waveforms of axonal APs. Together, these results show that axonal KV channels were subjected to dopamine modulation, and this modulation could regulate the waveforms of propagating APs at the axon, suggesting an important role of dopaminergic modulation of axonal KV channels in regulating neuronal signalling.

The Auxiliary Subunit KChIP2 Is an Essential Regulator of Homeostatic Excitability

The necessity for, or redundancy of, distinctive KChIP proteins is not known. Deletion of KChIP2 leads to increased susceptibility to epilepsy and to a reduction in IA and increased excitability in pyramidal hippocampal neurons. KChIP2 is essential for homeostasis in hippocampal neurons. Mutations in K(A) channel auxiliary subunits may be loci for epilepsy. The somatodendritic IA (A-type) K(+) current underlies neuronal excitability, and loss of IA has been associated with the development of epilepsy. Whether any one of the four auxiliary potassium channel interacting proteins (KChIPs), KChIP1-KChIP4, in specific neuronal populations is critical for IA is not known. Here we show that KChIP2, which is abundantly expressed in hippocampal pyramidal cells, is essential for IA regulation in hippocampal neurons and that deletion of Kchip2 affects susceptibility to limbic seizures. The specific effects of Kchip2 deletion on IA recorded from isolated hippocampal pyramidal neurons were a reduction in amplitude and shift in the V½ for steady-state inactivation to hyperpolarized potentials when compared with WT neurons. Consistent with the relative loss of IA, hippocampal neurons from Kchip2(-/-) mice showed increased excitability. WT cultured neurons fired only occasional single action potentials, but the average spontaneous firing rate (spikes/s) was almost 10-fold greater in Kchip2(-/-) neurons. In slice preparations, spontaneous firing was detected in CA1 pyramidal neurons from Kchip2(-/-) mice but not from WT. Additionally, when seizures were induced by kindling, the number of stimulations required to evoke an initial class 4 or 5 seizure was decreased, and the average duration of electrographic seizures was longer in Kchip2(-/-) mice compared with WT controls. Together, these data demonstrate that the KChIP2 is essential for physiologic IA modulation and homeostatic stability and that there is a lack of functional redundancy among the different KChIPs in hippocampal neurons.