Activity-dependent regulation of the potassium channel subunits Kv1.1 and Kv3.1

Afferent activity, especially in young animals, can have profound influences on postsynaptic neuronal structure, function and metabolic processes. Most studies evaluating activity regulation of cellular components have examined the expression of ubiquitous cellular proteins as opposed to molecules that are specialized in the neurons of interest. Here we consider the regulation of two proteins (voltage-gated potassium channel subunits Kv1.1 and Kv3.1) that auditory brainstem neurons in birds and mammals express at uniquely high levels. Unilateral removal of the avian cochlea leads to rapid and dramatic reduction in the expression of both proteins in the nucleus magnocellularis (NM; a division of the avian cochlear nucleus) neurons as detected by immunocytochemistry. Uniform downregulation of Kv1.1 was reliable by 3 hours after cochlea removal, was sustained through 96 hours, and returned to control levels in the surviving neurons by 2 weeks. The activity-dependent changes in Kv3.1 appear to be bimodal and are more transient, being observed at 3 hours after cochlea removal and recovering to control levels within 24 hours. We also explored the functional properties of Kv1.1 in NM neurons deprived of auditory input for 24 hours by whole-cell recordings. Low-threshold potassium currents in deprived NM neurons were not significantly different from control neurons in their amplitude or sensitivity to dendrotoxin-I, a selective K+ channel antagonist. We conclude that the highly specialized abundant expression of Kv1.1 and 3.1 channel subunits is not permanently regulated by synaptic activity and that changes in overall protein levels do not predict membrane pools.     

Kaliotoxin, a Kv1.1 and Kv1.3 channel blocker, improves associative learning in rats

Olfactory associative learning was used to investigate the involvement of Kv channels containing Kv1.1 and Kv1.3 alpha-subunits in learning and memory. Kaliotoxin (KTX), a specific inhibitor of these Kv channels, was injected intracerebroventricularly in the rat brain, at a dose of 10 ng that did not disturb the rats' locomotor activity or drinking behaviour. In the first paradigm (odour-reward training), KTX improved learning but not information consolidation. Moreover, KTX increased the long-term retrieval of an odour-reward association tested by a reversal test 1 month after the odour-reward training. The second paradigm (successive odour-pair training) tested reference memory. The first session was an acquisition session where the rats learned a new odour-discrimination problem with the same procedure. The second was a retention session held 24 h later to test retrieval of the learned information. KTX injected before the acquisition or retention session improved performance, but no effect was found when KTX was injected immediately after acquisition. We showed that these effects were not due to the action of KTX on attention processes. Thus, these results suggest that the blockage of Kv1.1 or Kv1.3 channels by KTX facilitates cognitive processes as learning, in particular in a reference representation.     

Age-related changes in the distribution of Kv1.1 and Kv1.2 channel subunits in the rat cerebellum

We have revealed age-related changes in the expression patterns of Kv1.1 and Kv1.2 in the rat cerebellum for the first time. In the aged rat, immunoreactivity for Kv1.1 was increased in the cell bodies of Purkinje cells, while the staining intensity was significantly decreased in the granule cells. The cell bodies of cerebellar output neurons showed strong Kv1.1 immunoreactivity in the nucleus medialis, interpositus and lateralis of the aged rat. Kv1.2 immunoreactivity was found in some interneurons with their processes in this region of the aged rat. Image analysis demonstrated that immunoreactivities for Kv1.1 and Kv1.2 were increased specifically in the cell bodies of cerebellar output neurons of the aged rat. This study may provide useful data for future investigations on the channels that cause brain diseases and age-related disorders.     

Transplanting the N-terminus from Kv1.4 to Kv1.1 generates an inwardly rectifying K+ channel

A chimeric channel, 4N/1, was generated from two outwardly rectifying K+ channels by linking the N-terminal cytoplasmic domain of hKv1.4 (N terminus ball and chain of hKv1.4) with the transmembrane body of hKv1.1 (delta78N1 construct of hKv1.1). The recombinant channel has properties similar to the six transmembrane inward rectifiers and opens on hyperpolarization with a threshold of activation at -90 mV. Outward currents are seen on depolarization provided the channel is first exposed to a hyperpolarizing pulse of -100 mV or more. Hyperpolarization at and beyond -130 mV provides evidence of channel deactivation. Delta78N1 does not show inward currents on hyperpolarization but does open on depolarizing from -80 mV with characteristics similar to native hKv1.1. The outward currents seen in both delta78N1 and 4N/1 inactivate slowly at rates consistent with C-type inactivation. The inward rectification of the 4N/1 chimera is consistent with the inactivation gating mechanism. This implies that the addition of the N-terminus from hKv1.4 to hKv1.1 shifts channel activation to hyperpolarizing potentials. These results suggest a mechanism involving the N-terminal cytoplasmic domain for conversion of outward rectifiers to inward rectifiers.     

电压门控钾通道Kv1.1在戊四唑致痫大鼠中的表达

目的通过检测戊四唑(PTZ)致痫大鼠钾通道Kv1.1蛋白表达,探讨钾通道Kv1.1与癫痫发病的相关性。方法40只SD大鼠分成实验组30只和正常对照组10只。实验组30只大鼠通过腹腔注射PTZ建立全身强直-阵挛发作大鼠癫痫模型,取成功致痫鼠24只均分成3组,分别于致痫后3个时间段(1h、24h、48h)取脑组织。用免疫组化法和Westernblot法检测大鼠钾通道Kv1.1蛋白。结果实验组大鼠海马区钾通道Kv1.1蛋白表达水平在致痫后3个时间段(1h、24h、48h)均明显低于对照组(P0.05),但3个时间段之间钾通道Kv1.1蛋白表达水平差异无统计学意义(P0.05)。结论大鼠海马区钾通道Kv1.1表达的减少与全身强直阵挛发作大鼠癫痫发病密切相关。     

Truncation of the Shaker-like voltage-gated potassium channel, Kv1.1, causes megencephaly

The megencephaly mouse, mceph/mceph, displays dramatically increased brain volume and hypertrophic brain cells. Despite overall enlargement, the mceph/mceph brain appears structurally normal, without oedema, hydrocephaly or leukodystrophy, and with only minor astrocytosis. Furthermore, it presents striking disturbances in expression of trophic and neuromodulating factors within the hippocampus and cortex. Using a positional cloning approach we have identified the mceph mutation. We show that mceph/mceph mice carry an 11-base-pair deletion in the gene encoding the Shaker-like voltage-gated potassium channel subtype 1, Kcna1. The mutation leads to a frame shift and the predicted MCEPH protein is truncated at amino acid 230 (out of 495), terminating with six aberrant amino acids. The expression of Kcna1 mRNA is increased in the mceph/mceph brain. However, the C-terminal domains of the corresponding Kv1.1 protein are absent. The putative MCEPH protein retains only the N-terminal domains for channel assembly and may congregate nonfunctional complexes of multiple Shaker-like subunits. Indeed, whereas Kcna2 and Kcna3 mRNA expression is normal, the mceph/mceph hippocampus displays decreased amounts of Kv1.2 and Kv1.3 proteins, suggesting interactions at the protein level. We show that mceph/mceph mice have disturbed brain electrophysiology and experience recurrent behavioural seizures, in agreement with the abnormal electrical brain activity found in Shaker mutants. However, in contrast to the commonly demonstrated epilepsy-induced neurodegeneration, we find that the mceph mutation leads to seizures with a concomitant increase in brain size, without overt neural atrophy.     

Episodic ataxia and myokymia syndrome: A new mutation of potassium channel gene Kv1.1

Episodic ataxia and myokymia syndrome is an autosomal dominant disorder characterized by persistent myokymia and attacks of unsteadiness, slurred speech, and tremulousness. This disease has been associated with point mutations in the potassium channel gene Kv1.1 (KCNA1), located at chromosome 12p13. Here, we describe a novel mutation within this gene in a newly diagnosed family.     

Episodic ataxia type 1 mutations in the KCNA1 gene impair the fast inactivation properties of the human potassium channels Kv1.4-1.1/Kvbeta1.1 and Kv1.4-1.1/Kvbeta1.2

Episodic ataxia type 1 (EA1) is an autosomal dominant neurological disorder characterized by constant muscle rippling movements (myokymia) and episodic attacks of ataxia. Several heterozygous point mutations have been found in the coding sequence of the voltage-gated potassium channel gene KCNA1 (hKv1.1), which alter the delayed-rectifier function of the channel. Shaker-like channels of different cell types may be formed by unique hetero-oligomeric complexes comprising Kv1.1, Kv1.4 and Kvbeta1.x subunits. Here we show that the human Kvbeta1.1 and Kvbeta1.2 subunits modulated the functional properties of tandemly linked Kv1.4-1.1 wild-type channels expressed in Xenopus laevis oocytes by (i) increasing the rate and amount of N-type inactivation, (ii) slowing the recovery rate from inactivation, (iii) accelerating the cumulative inactivation of the channel and (iv) negatively shifting the voltage dependence of inactivation. To date, the role of the human Kv1.4-1.1, Kv1.4-1.1/Kvbeta1.1 and Kv1.4-1.1/Kvbeta1.2 channels in the aetiopathogenesis of EA1 has not been investigated. Here we also show that the EA1 mutations E325D, V404I and V408A, which line the ion-conducting pore, and I177N, which resides within the S1 segment, alter the fast inactivation and repriming properties of the channels by decreasing both the rate and degree of N-type inactivation and by accelerating the recovery from fast inactivation. Furthermore, the E325D, V404I and I177N mutations shifted the voltage dependence of the steady-state inactivation to more positive potentials. The results demonstrate that the human Kvbeta1.1 and Kvbeta1.2 subunits regulate the proportion of wild-type Kv1.4-1.1 channels that are available to open. Furthermore, EA1 mutations alter heteromeric channel availability which probably modifies the integration properties and firing patterns of neurones controlling cognitive processes and body movements.     

Developmental regulation and adult maintenance of potassium channel proteins (Kv 1.1 and Kv 1.2) in the cochlear nucleus of the rat

The development and maintenance of the adult expression and distribution of Kv 1.1 and Kv 1.2, two voltage-dependent potassium channel subunits, were investigated in the anteroventral cochlear nucleus (AVCN) of the rat. Both Kv 1.1 and Kv 1.2 were found in AVCN neuronal cell bodies at birth, as detected by in situ hybridization and immunocytochemistry. However, Kv 1.1 and Kv 1.2 were not seen in axons until the end of the third postnatal week. From postnatal day 21 through adulthood, labeling for both potassium channels was in axonal processes, whereas the number of cell bodies labeled for Kv 1.1 decreased and there were no cell bodies labeled for Kv 1.2. Therefore, these two potassium channel proteins are targeted to their final subcellular destinations in axons well after hearing onset. Once the adult distribution pattern of Kv 1.1 and Kv 1.2 is attained, its maintenance does not depend on signals from auditory nerve synapses. Eliminating auditory nerve input to the cochlear nucleus by means of bilateral cochleotomy did not change Kv 1.1 or Kv 1.2 expression or distribution, as seen by in situ hybridization, immunocytochemistry and Western blot. Thus, normal excitatory synaptic input in adult animals is not a requirement to regulate the expression and cellular and subcellular distribution of these potassium channel proteins.     

Kv1.1 and Kv1.2: similar channels, different seizure models

Voltage-gated K(+) channels (Kv) represent the largest family of genes in the K(+) channel family. The Kv1 subfamily plays an essential role in the initiation and shaping of action potentials, influencing action potential firing patterns and controlling neuronal excitability. Overlapping patterns with differential expression and precise localization of Kv1.1 and Kv1.2 channels targeted to specialized subcellular compartments contribute to distinctive patterns of neuronal excitability. Dynamic regulation of the components in these subcellular domains help to finely tune the cellular and regional networks. Disruption of the expression, distribution, and density of these channels through deletion or mutation of the genes encoding these channels, Kcna1 and Kcna2, is associated with neurologic pathologies including epilepsy and ataxia in humans and in rodent models. Kv1.1 and Kv1.2 knockout mice both have seizures beginning early in development; however, each express a different seizure type (pathway), although the channels are from the same subfamily and are abundantly coexpressed. Voltage-gated ion channels clustered in specific locations may present a novel therapeutic target for influencing excitability in neurologic disorders associated with some channelopathies.     

Hyperexcitability of CA3 pyramidal cells in mice lacking the potassium channel subunit Kv1.1

PURPOSE: To investigate further the membrane properties and postsynaptic potentials of the CA3 pyramidal cells in mice that display spontaneous seizures because of a targeted deletion of the Kcna1 potassium channel gene (encoding the Kv1.1 protein subunit). METHODS: Intracellular recordings were obtained from CA3 pyramidal cells in hippocampal slices prepared from Kcna1-null and control littermates. CA3 pyramidal cells were activated: orthodromically, by stimulating mossy fibers; antidromically, by activating Schaffer collaterals; and by injecting intracellular pulses of current. Responses evoked under these conditions were compared in both genotypes in normal extracellular medium (containing 3 mM potassium) and in medium containing 6 mM potassium. RESULTS: Recordings from CA3 pyramidal cells in Kcna1-null and littermate control slices showed similar membrane and action-potential properties. However, in 33% of cells studied in Kcna1-null slices bathed in normal extracellular medium, orthodromic stimulation evoked synaptically driven bursts of action potentials that followed a short-latency excitatory postsynaptic potential (EPSP)-inhibitory PSP (IPSP) sequence. Such bursts were not seen in cells from control slices. The short-latency gamma-aminobutyric acid (GABA)A-mediated IPSP event appeared similar in null and control slices. When extracellular potassium was elevated and excitatory synaptic transmission was blocked, antidromic activation or short pulses of intracellular depolarizing current evoked voltage-dependent bursts of action potentials in the majority of cells recorded in Kcna1 null slices, but only single spikes in control slices. CONCLUSIONS: Lack of Kv1.1 potassium channel subunits in CA3 pyramidal cells leads to synaptic hyperexcitability, as reflected in the propensity of these cells to generate multiple action potentials. The action-potential burst did not appear to result from loss of GABAA receptor-mediated inhibition. This property of CA3 neurons, seen particularly when tissue conditions become abnormal (e.g., elevated extracellular potassium), helps to explain the high seizure susceptibility of Kcna1-null mice.     

Age-related changes in the distribution of Kv1.1 and Kv3.1 in rat cochlear nuclei

Abstract

Objectives: To identify age-related changes in voltage-gated K⁺ (Kv) channels that contribute to temporal processing in neurons of the central auditory system, we investigated the distribution of Kv1.1 and Kv3.1 in the auditory brainstem of adult and aged rats.

Methods: Immunohistochemistry was performed in accordance with the free-floating method described earlier.

Results: Among the auditory nuclei, only the posterior ventral cochlear nucleus (PVCN) showed age-related changes. Kv1.1 immunoreactivity was increased in the octopus cell bodies, while the staining intensity was significantly decreased in the neuropil. Image analysis demonstrated the specific increase in Kv1.1 immunoreactivity in aged cochlear nucleus neurons although the mean density of the entire selection was significantly decreased. In contrast, the number of Kv1.1-immunoreactive neurons was not significantly different between control and aged groups. The immunoreactivity for Kv3.1 was decreased in the octopus cells and neuropil of aged PVCN, which was confirmed by image analysis. The number of Kv3.1-positive cells was also significantly decreased in aged PVCN.

Discussion: This study may provide useful data to compare age-related changes in Kv1.1 and Kv3.1 with known physiological properties of auditory neurons.     

Developmental change in expression and subcellular localization of two shaker-related potassium channel proteins (Kv1.1 and Kv1.2) in the chick tangential vestibular nucleus

The chick tangential nucleus is a major avian vestibular nucleus whose principal cells participate in two vestibular reflexes. Intracellular recordings have shown that the principal cells acquire their mature firing pattern gradually during development. At embryonic day 16 (E16), most principal cells fire a single spike, whereas shortly after hatching (H) the vast majority fire repetitively on depolarization. The transition in firing pattern was likely due in part to a downregulation of a low-threshold, sustained, dendrotoxin-sensitive (DTX) potassium current, I(DS). Since the DTX-sensitive potassium channel subunits Kv1.1 and Kv1.2 generate sustained currents, in the present study we applied fluorescence immunocytochemistry and confocal microscopy to characterize their developmental expression at E16, H1, and H9. At E16, both Kv1.1 and Kv1.2 staining were confined to the principal cell bodies. Immunolabeling decreased significantly for both proteins at H1, and more so by H9. Double-labeling with a monoclonal antibody against microtubule-associated protein 2 (MAP2) in hatchlings showed that some Kv1.1 remained as clusters within the cell body, at the base of the dendrites, and in the axon initial segment. In hatchlings, Kv1.2 staining decreased in the cell bodies and simultaneously appeared in the neuropil, colocalized with biocytin-labeled primary vestibular fibers and vestibular "spoon" terminals. Also, double-labeling with synaptotagmin showed that Kv1.2 colocalized with many nonvestibular terminals surrounding the principal cell bodies. These results identified developmental decreases in the staining of these two potassium channel protein subunits and changes in their subcellular localization corresponding to the downregulation of I(DS) defined electrophysiologically around hatching. Accordingly, both of these protein subunits could be involved in regulating excitability of the principal cells.     

Age-related changes in the distribution of Kv1.1 and Kv3.1 in rat cochlear nuclei

OBJECTIVES: To identify age-related changes in voltage-gated K(+) (Kv) channels that contribute to temporal processing in neurons of the central auditory system, we investigated the distribution of Kv1.1 and Kv3.1 in the auditory brainstem of adult and aged rats. METHODS: Immunohistochemistry was performed in accordance with the free-floating method described earlier. RESULTS: Among the auditory nuclei, only the posterior ventral cochlear nucleus (PVCN) showed age-related changes. Kv1.1 immunoreactivity was increased in the octopus cell bodies, while the staining intensity was significantly decreased in the neuropil. Image analysis demonstrated the specific increase in Kv1.1 immunoreactivity in aged cochlear nucleus neurons although the mean density of the entire selection was significantly decreased. In contrast, the number of Kv1.1-immunoreactive neurons was not significantly different between control and aged groups. The immunoreactivity for Kv3.1 was decreased in the octopus cells and neuropil of aged PVCN, which was confirmed by image analysis. The number of Kv3.1-positive cells was also significantly decreased in aged PVCN. DISCUSSION: This study may provide useful data to compare age-related changes in Kv1.1 and Kv3.1 with known physiological properties of auditory neurons.     

Expression of the Kv1.1 ion channel subunit in the auditory brainstem of the big brown bat,Eptesicus fuscus

Voltage-gated potassium channels play an important role in shaping membrane properties that underlie neurons' discharge patterns and the ways in which they transform their input. In the auditory system, low threshold potassium currents such as those created by Kv1.1 subunits contribute to precise phaselocking and to transient onset responses that provide time markers for temporal features of sounds. The purpose of the present study was to compare information about the distribution of neurons expressing the KV 1.1 in the brainstem auditory nuclei with the distribution of neurons with known functional properties in the auditory system of the big brown bat, Eptesicus fuscus. We used immunocytochemistry and light microscopy to look at the distribution of Kv1.1 subunits in the brainstem auditory nuclei. There was prominent expression in cell types known to contain high levels of Kv1.1 in other species and known to respond to auditory signals with high temporal precision. These included octopus cells and spherical bushy cells of the cochlear nucleus and principal neurons of the medial nucleus of the trapezoid body. In addition, we found high levels of Kv1.1 in neurons of the columnar subdivision of the ventral nucleus of the lateral lemniscus and in ventral periolivary cell groups. Neurons with high levels of Kv1.1 were differentially distributed in the intermediate nucleus of the lateral lemniscus and in the inferior colliculus, suggesting that these structures contain functionally distinct cell populations, some of which may be involved in high-precision temporal processing.     

Block of neural Kv1.1 potassium channels for neuroinflammatory disease therapy

OBJECTIVE: We asked whether blockade of voltage-gated K+ channel Kv1.1, whose altered axonal localization during myelin insult and remyelination may disturb nerve conduction, treats experimental autoimmune encephalomyelitis (EAE). METHODS: Electrophysiological, cell proliferation, cytokine secretion, immunohistochemical, clinical, brain magnetic resonance imaging, and spectroscopy studies assessed the effects of a selective blocker of Kv1.1, BgK-F6A, on neurons and immune cells in vitro and on EAE-induced neurological deficits and brain lesions in Lewis rats. RESULTS: BgK-F6A increased the frequency of miniature excitatory postsynaptic currents in neurons and did not affect T-cell activation. EAE was characterized by ventriculomegaly, decreased apparent diffusion coefficient, and decreased (phosphocreatine + beta-adenosine triphosphate)/inorganic phosphate ratio. Reduced apparent diffusion coefficient and impaired energy metabolism indicate astrocytic edema. Intracerebroventricularly BgK-F6A-treated rats showed attenuated clinical EAE with unexpectedly reduced ventriculomegaly and preserved apparent diffusion coefficient values and (phosphocreatine + beta-adenosine triphosphate)/inorganic phosphate ratio. Thus, under BgK-F6A treatment, brain damage was dramatically reduced and energy metabolism maintained. INTERPRETATION: Kv1.1 blockade may target neurons and astrocytes, and modulate neuronal activity and neural cell volume, which may partly account for the attenuation of the neurological deficits. We propose that Kv1.1 blockade has a broad therapeutic potential in neuroinflammatory diseases (multiple sclerosis, stroke, and trauma).     

Effects of temperature on motor unit action potentials during isometric contraction

The effect of temperature on motor unit action potential (MUAP) configuration and recruitment was studied using automatic decomposition electromyography (ADEMG) recordings from a concentric needle electrode placed in the first dorsal interosseous (FDI) muscle of 10 normal adult subjects during isometric contraction. Focally cooling the FDI resulted in prolonged MUAP duration (P < 0.001, ANOVA), a finding congruent with those of Buchthal. Focal ulnar cooling at the elbow resulted in the increased MUAP frequency. In contrast to previous studies, there were no significant differences in amplitude or turns. Greater understanding of normal motor unit electrophysiology is necessary to improve diagnostic accuracy of EMG testing © 1995 John Wiley & Sons, Inc.     

蛇床子素对癫痫大鼠电压门控钾通道Kv1.2表达的影响

目的通过检测大鼠海马区钾通道Kv1.2蛋白表达的差异,探讨蛇床子素(osthole,OST)对海人酸(KA)致痫大鼠神经元的保护作用及其机制。方法 60只SD雄性大鼠随机分成空白对照组、模型组、OST组各20只。OST组首先给予OST灌胃,对照组、模型组给予等量的生理盐水灌胃,10d后,OST组与模型组通过颈内皮下注射KA致痫,对照组经颈内皮下注射等量的生理盐水。用免疫组化法和Western blot方法检测大鼠海马CA3区瞬时外向钾离子通道Kv1.2蛋白表达。结果模型组大鼠海马CA3区Kv1.2蛋白表达水平低于空白对照组(P<0.05);OST组大鼠海马CA3区Kv1.2蛋白表达水平高于模型组(P<0.05);且与空白对照组比较无明显差异(P>0.05)。结论大鼠海马CA3区神经元Kv1.2表达减少与KA导致大鼠痫样发作有关;OST对KA致痫大鼠神经元有保护作用,其作用的发挥可能与OST可增加海马CA3区神经元Kv1.2的表达有关。     

Chemical modification of sodium channel inactivation: separate sites for the action of grayanotoxin and tetramethrin

The gating mechanisms of the sodium channel are known to be modified by grayanotoxin and the pyrethroid tetramethrin. Voltage clamp experiments with internally perfused squid giant axons were performed to determine whether or not these two chemicals shared a common site of action in exerting their effects. An additive effect of the two drugs in prolonging sodium currents was observed. Additionally, the characteristic tetramethrin-induced sodium tail current and the grayanotoxin-induced hyperpolarizing shift in the voltage that activated the sodium current were observed simultaneously and independently of the order of drug introduction. Inactive stereoisomers of tetramethrin, which are known to prevent the active tetramethrin stereoisomers from exerting their effect, had no effect on the development of the grayanotoxin-induced modifications of sodium current. It was concluded that tetramethrin and grayanotoxin act at separate sites of action in modifying the sodium channel gating mechanisms in the squid axon membrane.