Kv Channel Subunits Research Articles

Subunit-Dependent Axonal Trafficking of Distinct α Heteromeric Potassium Channel Complexes

Voltage-gated potassium (Kv) channels are critical for neuronal excitability and are targeted to specific subcellular compartments to carry out their unique functions. While it is widely believed that Kv channels exist as heteromeric complexes in neurons, direct tests of the hypothesis that specific heteromeric channel populations display divergent spatial and temporal dynamics are limited. Using a bimolecular fluorescence complementation approach, we monitored the assembly and localization of cell surface channel complexes in living cells. While PSD95-mediated clustering was subunit independent, selective visualization of heteromeric Kv complexes in rat hippocampal neurons revealed subunit-dependent localization that was not predicted by analyzing individual subunits. Assembly of Kv1.1 with Kv1.4 prevented axonal localization but not surface expression, while inclusion of Kv1.2 imparted clustering at presynaptic sites and decreased channel mobility within the axon. This mechanism by which specific Kv channel subunits can act in a dominant manner to impose unique trafficking properties to heteromeric complexes extended to Shab-related family of Kv channels. When coexpressed, Kv2.1 and Kv2.2 heteromultimers did not aggregate in somatodendritic clusters observed with expression of Kv2.1 alone. These studies demonstrate selective axonal trafficking and surface localization of distinct Kv channels based on their subunit composition.

Expression of glutamate receptors and potassium channels in vestibular nuclei neurons during development of signal processing

The principal cells of the chick tangential vestibular nucleus offer a simple neuron model to study signal processing in second-order, vestibular reflex projection neurons. The principal cells represent a relatively uniform population of vestibular nuclei neurons which receive a major input from the primary vestibular fibers and send axons to targets mainly involved in the vestibuloocular reflexes. Here, studies performed on ion channels involved in the emergence and establishment of signal processing in this morphologically-identified subset of vestibular nuclei neurons are reviewed, including the AMPA glutamate receptor subunits GluR1, GluR2, GluR3, and GluR4 and the potassium channel subunits Kv1.1 and Kv1.2.

Dynamic Kv4.3–CaMKII unit in heart: an intrinsic negative regulator for CaMKII activation

Reduction of transient outward current (I(to)) and excessive activation of Ca(2+)/Calmodulin-dependent kinase II (CaMKII) are general features of ventricular myocytes in heart failure. We hypothesize that alterations of I(to) directly regulate CaMKII activation in cardiomyocytes. A dynamic coupling of I(to) channel subunit Kv4.3 and inactive CaMKII was discovered in cardiomyocytes with the membrane predominant distribution by co-immunoprecipitation and fluorescence resonance energy transfer techniques. CaMKII dissociation from Kv4.3-CaMKII units caused a significant increase in CaMKII autophosphorylation and L-type calcium current (I(Ca)) facilitation. I(Ca) facilitation was blunted by the compartmental Ca²(+) chelator BAPTA but unaffected by bulk Ca²(+) chelator EGTA, implicating membrane-localized CaMKII. Kv4.3 overexpression reduced basal CaMKII autophosphorylation in myocytes and eliminated Ca²(+)-induced CaMKII activation. Kv4.3 blocks CaMKII activation by binding to the calmodulin binding sites, whereas Kv4.3 uncoupling releases these sites and leads to a substantial CaMKII activation. Our results uncovered an important mechanism that regulates CaMKII activation in the heart and implicate I(to) channel alteration in pathological CaMKII activation.

Chronic deficit in the expression of voltage-gated potassium channel Kv3.4 subunit in the hippocampus of pilocarpine-treated epileptic rats

Voltage gated K + channels (Kv) are a highly diverse group of channels critical in determining neuronal excitability. Deficits of Kv channel subunit expression and function have been implicated in the pathogenesis of epilepsy. In this study, we investigate whether the expression of the specific subunit Kv3.4 is affected during epileptogenesis following pilocarpine-induced status epilepticus. For this purpose, we used immunohistochemistry, Western blotting assays and comparative analysis of gene expression using TaqMan-based probes and delta-delta cycle threshold (ΔΔCT) method of quantitative real-time polymerase chain reaction (qPCR) technique in samples obtained from age-matched control and epileptic rats. A marked down-regulation of Kv3.4 immunoreactivity was detected in the stratum lucidum and hilus of dentate gyrus in areas corresponding to the mossy fiber system of chronically epileptic rats. Correspondingly, a 20% reduction of Kv3.4 protein levels was detected in the hippocampus of chronic epileptic rats. Real-time quantitative PCR analysis of gene expression revealed that a significant 33% reduction of transcripts for Kv3.4 (gene Kcnc4) occurred after 1 month of pilocarpine-induced status epilepticus and persisted during the chronic phase of the model. These data indicate a reduced expression of Kv3.4 channels at protein and transcript levels in the epileptic hippocampus. Down-regulation of Kv3.4 in mossy fibers may contribute to enhanced presynaptic excitability leading to recurrent seizures in the pilocarpine model of temporal lobe epilepsy.

Kv4.2 block of long-term potentiation is partially dependent on synaptic NMDA receptor remodeling

Proper expression of synaptic NMDA receptors (NMDARs) is necessary to regulate synaptic Ca 2+ influx and the induction the long-term potentiation (LTP) in the mammalian hippocampus. Previously we reported that expressing the A-type K + channel subunit Kv4.2 in CA1 neurons of organotypic slice cultures reduced synaptic NR2B-containing NMDAR expression and completely blocked LTP induced by a pairing protocol. As pretreatment with an NMDAR antagonist (APV) overnight blocked the reduction of NR2B-containing receptors in neurons expressing EGFP-labeled Kv4.2 (Kv4.2g), we hypothesized that LTP would be rescued in Kv4.2g neurons by overnight treatment with APV. We report here that the overnight APV pretreatment in Kv4.2g-expressing neurons only partially restored potentiation. This partial potentiation was completely blocked by inhibition of the CAMKII kinase. These results indicate that A-type K + channels must regulate synaptic integration and plasticity through another mechanism in addition to their regulation of synaptic NR2 subunit composition. We suggest that dendritic excitability, which is regulated by Kv4.2 expression, also contributes to synaptic plasticity.

Ancillary Subunits Associated With Voltage-Dependent K+Channels

Since the first discovery of Kvbeta-subunits more than 15 years ago, many more ancillary Kv channel subunits were characterized, for example, KChIPs, KCNEs, and BKbeta-subunits. The ancillary subunits are often integral parts of native Kv channels, which, therefore, are mostly multiprotein complexes composed of voltage-sensing and pore-forming Kvalpha-subunits and of ancillary or beta-subunits. Apparently, Kv channels need the ancillary subunits to fulfill their many different cell physiological roles. This is reflected by the large structural diversity observed with ancillary subunit structures. They range from proteins with transmembrane segments and extracellular domains to purely cytoplasmic proteins. Ancillary subunits modulate Kv channel gating but can also have a great impact on channel assembly, on channel trafficking to and from the cellular surface, and on targeting Kv channels to different cellular compartments. The importance of the role of accessory subunits is further emphasized by the number of mutations that are associated in both humans and animals with diseases like hypertension, epilepsy, arrhythmogenesis, periodic paralysis, and hypothyroidism. Interestingly, several ancillary subunits have in vitro enzymatic activity; for example, Kvbeta-subunits are oxidoreductases, or modulate enzymatic activity, i.e., KChIP3 modulates presenilin activity. Thus different modes of beta-subunit association and of functional impact on Kv channels can be delineated, making it difficult to extract common principles underlying Kvalpha- and beta-subunit interactions. We critically review present knowledge on the physiological role of ancillary Kv channel subunits and their effects on Kv channel properties.

Dendritic Kv3.3 Potassium Channels in Cerebellar Purkinje Cells Regulate Generation and Spatial Dynamics of Dendritic Ca2+Spikes

Purkinje cell dendrites are excitable structures with intrinsic and synaptic conductances contributing to the generation and propagation of electrical activity. Voltage-gated potassium channel subunit Kv3.3 is expressed in the distal dendrites of Purkinje cells. However, the functional relevance of this dendritic distribution is not understood. Moreover, mutations in Kv3.3 cause movement disorders in mice and cerebellar atrophy and ataxia in humans, emphasizing the importance of understanding the role of these channels. In this study, we explore functional implications of this dendritic channel expression and compare Purkinje cell dendritic excitability in wild-type and Kv3.3 knockout mice. We demonstrate enhanced excitability of Purkinje cell dendrites in Kv3.3 knockout mice, despite normal resting membrane properties. Combined data from local application pharmacology, voltage clamp analysis of ionic currents, and assessment of dendritic Ca(2+) spike threshold in Purkinje cells suggest a role for Kv3.3 channels in opposing Ca(2+) spike initiation. To study the physiological relevance of altered dendritic excitability, we measured [Ca(2+)](i) changes throughout the dendritic tree in response to climbing fiber activation. Ca(2+) signals were specifically enhanced in distal dendrites of Kv3.3 knockout Purkinje cells, suggesting a role for dendritic Kv3.3 channels in regulating propagation of electrical activity and Ca(2+) influx in distal dendrites. These findings characterize unique roles of Kv3.3 channels in dendrites, with implications for synaptic integration, plasticity, and human disease.

Specific and rapid effects of acoustic stimulation on the tonotopic distribution of Kv3.1b potassium channels in the adult rat

Recent studies have demonstrated that total cellular levels of voltage-gated potassium channel subunits can change on a time scale of minutes in acute slices and cultured neurons, raising the possibility that rapid changes in the abundance of channel proteins contribute to experience-dependent plasticity in vivo. In order to investigate this possibility, we took advantage of the medial nucleus of the trapezoid body (MNTB) sound localization circuit, which contains neurons that precisely phase-lock their action potentials to rapid temporal fluctuations in the acoustic waveform. Previous work has demonstrated that the ability of these neurons to follow high-frequency stimuli depends critically upon whether they express adequate amounts of the potassium channel subunit Kv3.1. To test the hypothesis that net amounts of Kv3.1 protein would be rapidly upregulated when animals are exposed to sounds that require high frequency firing for accurate encoding, we briefly exposed adult rats to acoustic environments that varied according to carrier frequency and amplitude modulation (AM) rate. Using an antibody directed at the cytoplasmic C-terminus of Kv3.1b (the adult splice isoform of Kv3.1), we found that total cellular levels of Kv3.1b protein—as well as the tonotopic distribution of Kv3.1b-labeled cells—was significantly altered following 30 min of exposure to rapidly modulated (400 Hz) sounds relative to slowly modulated (0–40 Hz, 60 Hz) sounds. These results provide direct evidence that net amounts of Kv3.1b protein can change on a time scale of minutes in response to stimulus-driven synaptic activity, permitting auditory neurons to actively adapt their complement of ion channels to changes in the acoustic environment.

A PATIENT WITH EPISODIC ATAXIA AND PARAMYOTONIA CONGENITA DUE TO MUTATIONS IN KCNA1 AND SCN4A

Channelopathies are mendelian disorders arising from mutations in genes that encode ion channel subunits.1 Mutations in SCN4A, which encodes the α-subunit of the muscle sodium channel Nav1.4, underlie paramyotonia congenita (PMC),2 while mutations in KCNA1, which encodes the potassium channel subunit Kv1.1, cause episodic ataxia type 1 (EA1) and isolated myokymia.3 We describe the clinical, genetic, and functional expression studies in an unusual patient who harbors mutations in both SCN4A and KCNA1 and report that sodium channel dysfunction dominates the phenotype. ### Case history. A 34-year-old woman had a lifelong history of muscle cramps and weakness affecting upper and lower limbs. Symptoms were exacerbated by exercise and cold weather, which induced both muscle stiffness and difficulty releasing her grip. Her father, paternal uncle, and second son are similarly affected. Examination demonstrated weakness of intrinsic hand muscles (Medical Research Council 4/5) with paradoxical myotonia of hand grip and eye closure. Neurophysiology confirmed a diagnosis of PMC, with a cooling test showing a rapid decrement in amplitude and area, which did not recover with rewarming. A McManis test was positive, showing a >40% decrement in compound muscle action potential (CMAP) during exercise with recovery during the prolonged rest period. Unexpectedly, needle EMG demonstrated sustained doublets, triplets, and multiplets interspersed with the myotonic discharges in the right abductor digiti minimi, biceps, tibialis anterior, and thoracic paraspinals (figure A). Because generalized myokymic discharges are not a recognized feature of PMC, this prompted further direct questioning about her symptoms. She reported that since the age of 16, she had experienced brief attacks of slurred speech and unsteadiness lasting seconds to minutes. …

Contribution of Kv channel subunits to glutamate‐induced apoptosis in cultured rat hippocampal neurons

Potassium channel dysfunction has been implicated in apoptosis in many pathological conditions. However, which Kv channel subunit is involved in glutamate-induced apoptosis remains unknown. In this study, the contributions of nine Kv alpha and three Kv beta subunits to glutamate-induced hippocampal neuronal apoptosis were investigated. Results showed that neuronal apoptosis was not obvious with 12 hr incubation of glutamate but increased markedly after 18 hr, which was attenuated by the Kv channel blocker TEA. Among all the Kv subunits investigated, gene and protein expression of Kv2.1 increased significantly before the appearance of neuronal apoptosis, whereas the Kv1.1 mRNA level decreased quickly, and protein expression was reduced gradually after the insult. Seven other Kv alpha subunits and three Kv beta subunits were not obviously affected over time. In addition, Kv1.1 overexpression could reduce glutamate-induced hippocampal neuronal apoptosis. Therefore, the alterations of Kv1.1 and Kv2.1 might contribute to glutamate-induced toxicity in hippocampal neurons. These findings suggest that these two Kv channel subunits may represent potential therapeutic targets for neuropathological conditions in which glutamate-induced toxicity is thought to contribute to neuronal dysfunction.

The voltage-gated K+ channel subunit Kv1.1 links kidney and brain

Analysis of Mendelian Mg2+ wasting disorders helps us to unravel the mechanisms of Mg2+ homeostasis. In this issue of the JCI, Glaudemans andcolleagues show that mutations in voltage-gated K+ channel subtype 1.1(Kv1.1) cause autosomal dominant hypomagnesemia in humans (see the related article beginning on page 936). Interestingly, other mutations in the same protein cause the neurological disease episodic ataxia type 1. The authors show, using cells with heterologous expression of the wild-type and mutant channels, that the mutant channel is dysfunctional and speculate that Mg2+ wasting results from changes in apical membrane voltage along the nephron. Mechanisms by which the apical voltage is generated and howKv1.1 fits within this context are discussed herein.

Stromatoxin‐1‐sensitive voltage‐gated K+ channels regulate rat urinary bladder smooth muscle contractility

Recent studies report that stromatoxin‐1 (ScTx1), a 34‐amino acid peptide, selectively inhibits the voltage‐gated K+ (Kv) channel members, Kv2.1, Kv2.2, Kv4.2, and Kv2.1/9.3 heteromultimers with high affinity. The objective of this study was to identify if any voltage‐gated Kv channels sensitive to ScTx1 participate in the regulation of rat urinary bladder smooth muscle (UBSM) contractility. Freshly isolated rat UBSM strips and single UBSM cells were studied using molecular, immunological, and physiological techniques. RT‐PCR experiments on isolated UBSM cells show mRNA expression of Kv2.1, Kv2.2, and Kv9.3 but not Kv4.2 subunits (n=3). Protein expression of Kv2.1 and Kv2.2 was further confirmed in single UBSM cells by applying immunological methods with specific antibodies (n=3). Isometric UBSM tension recordings of isolated UBSM strips showed that ScTx1 (100 nM) increased spontaneous phasic contraction amplitude and muscle force by about 20% without any effect on contraction frequency or muscle tone (n=5). The ScTx1 (100 nM) effect on UBSM contraction amplitude was observed even after the UBSM strips were depolarized with 20 mM K+ (n=3). Our studies indicate that Kv2.1 and Kv2.2 members of the Kv family play a key role in shaping rat UBSM contractility. New drugs targeting specific Kv channel subunits in the UBSM may prove useful for overactive bladder.Supported by NIH DK‐070909 to G.V. Petkov.

Chondroitin sulfate proteoglycan-based extracellular matrix in chicken ( Gallus domesticus) brain

A specialised form of extracellular matrix consisting of large aggregating chondroitin sulphate proteoglycans connected to hyaluronan and tenascins, as main components, is termed perineuronal nets. These perineuronal nets surround subpopulations of neurons in many vertebrates including man. In this study we investigated the distribution and the postnatal development of perineuronal nets in the brain of the domestic chicken using immunohistochemical, lectin-histochemical and biochemical methods. Perineuronal nets could be identified very early, already on the first postnatal day throughout various regions and nuclei in chicken fore- and midbrains, most expressively in nidopallium, hyperpallium, lateral striatum, globus pallidus and mesopallium. These mostly delicate, scanty structures around the cell bodies of neurons thicken and complete during the first 2 weeks, however, differ in shape and clearness of contours from the mature form of perineuronal nets found in the adult, 3 year old animals. Perineuronal nets frequently co-localized with the potassium channel subunit Kv3.1b characteristic for fast spiking neurons but remained unrevealed around cholinergic or monoaminergic neurons. The early appearance of perineuronal nets in the precocial birds' brain is probably due to the rapid establishment of neuronal morphology and function which is required for the immediate functional and behavioural performance of chicken.

MinK-dependent internalization of the IKs potassium channel

KCNQ1-MinK potassium channel complexes (4alpha:2beta stoichiometry) generate IKs, the slowly activating human cardiac ventricular repolarization current. The MinK ancillary subunit slows KCNQ1 activation, eliminates its inactivation, and increases its unitary conductance. However, KCNQ1 transcripts outnumber MinK transcripts five to one in human ventricles, suggesting KCNQ1 also forms other heteromeric or even homomeric channels there. Mechanisms governing which channel types prevail have not previously been reported, despite their significance: normal cardiac rhythm requires tight control of IKs density and kinetics, and inherited mutations in KCNQ1 and MinK can cause ventricular fibrillation and sudden death. Here, we describe a novel mechanism for this control. Whole-cell patch-clamping, confocal immunofluorescence microscopy, antibody feeding, biotin feeding, fluorescent transferrin feeding, and protein biochemistry techniques were applied to COS-7 cells heterologously expressing KCNQ1 with wild-type or mutant MinK and dynamin 2 and to native IKs channels in guinea-pig myocytes. KCNQ1-MinK complexes, but not homomeric KCNQ1 channels, were found to undergo clathrin- and dynamin 2-dependent internalization (DDI). Three sites on the MinK intracellular C-terminus were, in concert, necessary and sufficient for DDI. Gating kinetics and sensitivity to XE991 indicated that DDI decreased cell-surface KCNQ1-MinK channels relative to homomeric KCNQ1, decreasing whole-cell current but increasing net activation rate; inhibiting DDI did the reverse. The data redefine MinK as an endocytic chaperone for KCNQ1 and present a dynamic mechanism for controlling net surface Kv channel subunit composition-and thus current density and gating kinetics-that may also apply to other alpha-beta type Kv channel complexes.

De novo expression of Kv6.3 contributes to changes in vascular smooth muscle cell excitability in a hypertensive mice strain

Essential hypertension involves a gradual and sustained increase in total peripheral resistance, reflecting an increased vascular tone. This change associates with a depolarization of vascular myocytes, and relies on a change in the expression profile of voltage-dependent ion channels (mainly Ca(2+) and K(+) channels) that promotes arterial contraction. However, changes in expression and/or modulation of voltage-dependent K(+) channels (Kv channels) are poorly defined, due to their large molecular diversity and their vascular bed-specific expression. Here we endeavor to characterize the molecular and functional expression of Kv channels in vascular smooth muscle cells (VSMCs) and their regulation in essential hypertension, by using VSMCs from resistance (mesenteric) or conduit (aortic) arteries obtained from a hypertensive inbred mice strain, BPH, and the corresponding normotensive strain, BPN. Real-time PCR reveals a differential distribution of Kv channel subunits in the different vascular beds as well as arterial bed-specific changes under hypertension. In mesenteric arteries, the most conspicuous change was the de novo expression of Kv6.3 (Kcng3) mRNA in hypertensive animals. The functional relevance of this change was studied by using patch-clamp techniques. VSMCs from BPH arteries were more depolarized than BPN ones, and showed significantly larger capacitance values. Moreover, Kv current density in BPH VSMCs is decreased mainly due to the diminished contribution of the Kv2 component. The kinetic and pharmacological profile of Kv2 currents suggests that the expression of Kv6.3 could contribute to the natural development of hypertension.

Pentameric Assembly of Potassium Channel Tetramerization Domain-Containing Protein 5

We report the X-ray crystal structure of human potassium channel tetramerization domain-containing protein 5 (KCTD5), the first member of the family to be so characterized. Four findings were unexpected. First, the structure reveals assemblies of five subunits while tetramers were anticipated; pentameric stoichiometry is observed also in solution by scanning transmission electron microscopy mass analysis and analytical ultracentrifugation. Second, the same BTB ( bric-a-brac, tramtrack, broad complex) domain surface mediates the assembly of five KCTD5 and four voltage-gated K + (Kv) channel subunits; four amino acid differences appear crucial. Third, KCTD5 complexes have well-defined N- and C-terminal modules separated by a flexible linker that swivels by ∼ 30 o; the C-module shows a new fold and is required to bind Golgi reassembly stacking protein 55 with ∼ 1 μM affinity, as judged by surface plasmon resonance and ultracentrifugation. Fourth, despite the homology reflected in its name, KCTD5 does not impact the operation of Kv4.2, Kv3.4, Kv2.1, or Kv1.2 channels.

Kv4.2 is a locus for PKC and ERK/MAPK cross-talk

Transient outward K+ currents are particularly important for the regulation of membrane excitability of neurons and repolarization of action potentials in cardiac myocytes. These currents are modulated by PKC (protein kinase C) activation, and the K+- channel subunit Kv4.2 is a major contributor to these currents. Furthermore, the current recorded from Kv4.2 channels expressed in oocytes is reduced by PKC activation. The mechanism underlying PKC regulation of Kv4.2 currents is unknown. In the present study, we determined that PKC directly phosphorylates the Kv4.2 channel protein. In vitro phosphorylation of the intracellular N- and C-termini of Kv4.2 GST (glutathione transferase) tagged fusion protein revealed that the C-terminal of Kv4.2 was phosphorylated by PKC, whereas the N-terminal was not. Amino acid mapping and site-directed mutagenesis revealed that the phosphorylated residues on the Kv4.2 C-terminal were Ser447 and Ser537. A phospho-site-specific antibody showed that phosphorylation at the Ser537 site was increased in the hippocampus in response to PKC activation. Surface biotinylation experiments revealed that mutation to alanine of both Ser447 and Ser537 in order to block phosphorylation at both of the PKC sites increased surface expression compared with wild-type Kv4.2. Electrophysiological recordings of the wild-type and both the alanine and aspartate mutant Kv4.2 channels expressed with KChIP3 (Kv4 channel-interacting protein 3) revealed no significant difference in the half-activation or half-inactivation voltage of the channel. Interestingly, Ser537 lies within a possible ERK (extracellular-signal-regulated kinase)/MAPK (mitogen-activated protein kinase) recognition (docking) domain in the Kv4.2 C-terminal sequence. We found that phosphorylation of Kv4.2 by PKC enhanced ERK phosphorylation of the channel in vitro. These findings suggest the possibility that Kv4.2 is a locus for PKC and ERK cross-talk.

Rapid, Bidirectional Remodeling of Synaptic NMDA Receptor Subunit Composition by A-type K+ Channel Activity in Hippocampal CA1 Pyramidal Neurons

The transient, A-type K+ current (IA) controls the excitability of CA1 pyramidal neuron dendrites by regulating the back-propagation of action potentials and by shaping synaptic input. Dendritic A-type K+ channels are targeted for modulation during long-term potentiation (LTP) and we have recently shown that activity-dependent internalization of the A-type channel subunit Kv4.2 enhances synaptic currents. However, the effect of changes in IA on the ability to induce subsequent synaptic plasticity (metaplasticity) has not been investigated. Here, we show that altering functional Kv4.2 expression level leads to a rapid, bidirectional remodeling of CA1 synapses. Neurons exhibiting enhanced IA showed a decrease in relative synaptic NR2B/NR2A subunit composition and did not exhibit LTP. Conversely, reducing IA by expression of a Kv4.2 dominant-negative or through genomic knockout of Kv4.2 led to an increased fraction of synaptic NR2B/NR2A and enhanced LTP. Bidirectional synaptic remodeling was mimicked in experiments manipulating intracellular Ca2+ and dependent on spontaneous activation of NMDA receptors and CaMKII activity. Our data suggest that A-type K+ channels are an integral part of a synaptic complex that regulates Ca2+ signaling through spontaneous NMDAR activation to control synaptic NMDAR expression and plasticity.

Chemical sympathetic denervation, suppression of myocardial transient outward potassium current, and ventricular fibrillation in the rat

Sympathetic denervation is frequently observed in heart disease. To investigate the linkage of sympathetic denervation and cardiac arrhythmia, we developed a rat model of chemical sympathectomy by subcutaneous injections of 6-hydroxydopamine (6-OHDA). Cardiac sympathetic innervation was visualized by means of a glyoxylic catecholaminergic histofluorescence method. Transient outward current (Ito) of ventricular myocytes was recorded with the whole-cell configuration of the patch clamp technique. We observed that sympathectomy (i) decreased cardiac sympathetic nerve density and norepinephrine level, (ii) reduced the protein expression of Kv4.2, Kv1.4, and Kv channel-interacting protein 2 (KChIP2), (iii) decreased current densities and delayed activation of Ito channels, (iv) reduced the phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) and cAMP response element-binding protein (CREB), and (v) increased the severity of ventricular fibrillation induced by rapid pacing. Three weeks after 6-OHDA injections, which allowed time for sympathetic regeneration, we found cardiac sympathetic nerve density, norepinephrine levels, expression levels of Kv4.2 and KChIP2 proteins, and I(to) densities were partially normalized and ventricular fibrillation severity was decreased. We conclude that chemical sympathectomy downregulates the expression of selective Kv channel subunits and decreases myocardial I(to) channel activities, contributing to the elevated susceptibility to ventricular fibrillation.

Protein Kinase A Mediates Activity-Dependent Kv4.2 Channel Trafficking

The A-type potassium channel subunit Kv4.2 influences hippocampal function through regulation of dendritic excitability, and changes in Kv4.2 surface expression alter synaptic plasticity. Recent data from our laboratory demonstrate that EGFP (enhanced green fluorescent protein)-tagged Kv4.2 channels located in dendritic spines are internalized in an activity-dependent manner after synaptic stimulation and during chemically induced long-term potentiation. However, the molecular trigger for Kv4.2 internalization remains unknown. Here we examined the role of protein kinase A (PKA) in Kv4.2 activity-dependent trafficking. In hippocampal neurons, PKA activation with forskolin or 8-Br-cAMP induced Kv4.2 internalization from dendritic spines, whereas PKA inhibition with H89 prevented AMPA-induced internalization. Furthermore, introduction of a point mutation at the C-terminal PKA phosphorylation site of Kv4.2 (S552A) prevented the AMPA-induced internalization of Kv4.2. Together, these data demonstrate that Kv4.2 activity-dependent internalization requires PKA phosphorylation of Kv4.2 at serine 522.