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PubMed1994

The P-region and S6 of Kv3.1 contribute to the formation of the ion conduction pathway.

The loop between transmembrane regions S5 and S6 (P-region) of voltage-gated K+ channels has been proposed to form the ion-conducting pore, and the internal part of this segment is reported to be responsible for ion permeation and internal tetraethylammonium (TEA) binding. The two T-cell K+ channels, Kv3.1 and Kv1.3, with widely divergent pore properties, differ by a single residue in this internal P-region, leucine 401 in Kv3.1 corresponding to valine 398 in Kv1.3. The L401V mutation in Kv3.1 was created with the anticipation that the mutant channel would exhibit Kv1.3-like deep-pore properties. Surprisingly, this mutation did not alter single channel conductance and only moderately enhanced internal TEA sensitivity, indicating that residues outside the P-region influence these properties. Our search for additional residues was guided by the model of Durell and Guy, which predicted that the C-terminal end of S6 formed part of the K+ conduction pathway. In this segment, the two channels diverge at only one position, Kv3.1 containing M430 in place of leucine in Kv1.3. The M430L mutant of Kv3.1 exhibited permeant ion- and voltage-dependent flickery outward single channel currents, with no obvious changes in other pore properties. Modification of one or more ion-binding sites located in the electric field and possibly within the channel pore could give rise to this type of channel flicker.

Amino Acid Sequence↗
PubMed2001

Kv3 channels: voltage-gated K+ channels designed for high-frequency repetitive firing.

Analysis of the Kv3 subfamily of K(+) channel subunits has lead to the discovery of a new class of neuronal voltage-gated K(+) channels characterized by positively shifted voltage dependencies and very fast deactivation rates. These properties are adaptations that allow these channels to produce currents that can specifically enable fast repolarization of action potentials without compromising spike initiation or height. The short spike duration and the rapid deactivation of the Kv3 currents after spike repolarization maximize the quick recovery of resting conditions after an action potential. Several neurons in the mammalian CNS have incorporated into their repertoire of voltage-dependent conductances a relatively large number of Kv3 channels to enable repetitive firing at high frequencies - an ability that crucially depends on the special properties of Kv3 channels and their impact on excitability.

Action Potentials↗
PubMed2005

Fast delayed rectifier potassium current is required for circadian neural activity.

In mammals, the precise circadian timing of many biological processes depends on the generation of oscillations in neural activity of pacemaker cells in the suprachiasmatic nucleus (SCN). The ionic mechanisms that underlie these rhythms are largely unknown. Using the mouse brain slice preparation, we show that the magnitude of fast delayed rectifier (FDR) potassium currents has a diurnal rhythm that peaks during the day. Notably, this rhythm continues in constant darkness, providing the first demonstration of the circadian regulation of an intrinsic voltage-gated current in mammalian cells. Blocking this current prevented the daily rhythm in firing rate in SCN neurons. Kv3.1b and Kv3.2 potassium channels were widely distributed within the SCN, with higher expression during the day. We conclude that the FDR is necessary for the circadian modulation of electrical activity in SCN neurons and represents an important part of the ionic basis for the generation of rhythmic output.

Action Potentials↗
PubMed2004

Increased motor drive and sleep loss in mice lacking Kv3-type potassium channels.

The voltage-gated potassium channels Kv3.1 and Kv3.3 are widely expressed in the brain, including areas implicated in the control of motor activity and in areas thought to regulate arousal states. Although Kv3.1 and Kv3.3-single mutants show some physiological changes, previous studies revealed relatively subtle behavioral alterations suggesting that Kv3.1 and Kv3.3 channel subunits may be encoded by a pair of redundant genes. In agreement with this hypothesis, Kv3.1/Kv3.3-deficient mice display a 'strong' mutant phenotype that includes motor dysfunction (ataxia, myoclonus, tremor) and hyperactivity when exposed to a novel environment. In this paper we report that Kv3.1/Kv3.3-deficient mice are also constitutively hyperactive. Compared to wildtype mice, double mutants display 'restlessness' that is particularly prominent during the light period, when mice are normally at rest, characterized by more than a doubling of ambulatory and stereotypic activity, and accompanied by a 40% sleep reduction. When we reinvestigated both single mutants, we observed constitutive increases of ambulatory and stereotypic activity in conjunction with sleep loss in Kv3.1-single mutants but not in Kv3.3-single mutants. These findings indicate that the absence of Kv3.1-channel subunits is primarily responsible for the increased motor drive and the reduction in sleep time.

Animals↗
PubMed2002

Bupivacaine inhibits human neuronal Kv3 channels in SH-SY5Y human neuroblastoma cells.

BACKGROUND: Information on molecular targets that may be involved in the neurotoxicity of bupivacaine is limited. Suppression of Kv3 channels has been demonstrated to result in abnormal patterns in the electroencephalogram and in seizures. Inhibition of Kv3 channels by bupivacaine may consequently contribute to its neuroexcitatory side-effects. Data on the effects of bupivacaine on these potassium channels are lacking. We therefore characterized the effects of bupivacaine on human Kv3 channels natively expressed in SH-SY5Y cells. METHODS: Kv3 channels natively expressed in human SH-SY5Y cells were studied using a standard whole-cell patch-clamp protocol. RESULTS: Bupivacaine reversibly inhibited Kv3 channels in a concentration-dependent manner. The half-maximal inhibitory concentration (IC50) for conductance block was 57 microM and the Hill coefficient was close to unity. Bupivacaine accelerated macroscopic current decline by inducing inactivation-like behaviour. The midpoint of current activation was shifted to depolarized potentials in a concentration-dependent and reversible manner by a maximum of 26 mV. The IC50 was 47 microM and the Hill coefficient was 2.4. The free arterial plasma concentrations of bupivacaine that have been estimated to occur during convulsions in man would inhibit the Kv3 channels by at least 40% and would shift the midpoint of current activation by a minimum of 9 mV. CONCLUSIONS: Both inhibition of potassium channels and a depolarizing shift of their activation midpoint would increase neuronal excitability. The effects of bupivacaine on human Kv3 channels are thus compatible with a contributory role of Kv channel alteration in bupivacaine-induced neuronal excitation.

Anesthetics, Local↗
PubMed2004

Allele-dependent changes of olivocerebellar circuit properties in the absence of the voltage-gated potassium channels Kv3.1 and Kv3.3.

Double-mutant mice (DKO) lacking the two voltage-gated K(+) channels Kv3.1 and Kv3.3 display a series of phenotypic alterations that include ataxia, myoclonus, tremor and alcohol hypersensitivity. The prominent cerebellar expression of mRNAs encoding Kv3.1 and Kv3.3 subunits raised the question as to whether altered electrical activity resulting from the lack of these K(+) channels might be related to the dramatic motor changes. We used the tremorogenic agent harmaline to probe mutant mice lacking different K(+) channel alleles for altered olivocerebellar circuit properties. Harmaline induced the characteristic 13-Hz tremor in wildtype mice (WT); however, no tremor was observed in DKO suggesting that the ensemble properties of the olivocerebellar circuitry are altered in the absence of Kv3.1 and Kv3.3 subunits. Harmaline induced tremor in Kv3.1-single mutants, but it was of smaller amplitude and at a lower frequency indicating the participation of Kv3.1 subunits in normal olivocerebellar system function. In contrast, harmaline tremor was virtually absent in Kv3.3-single mutants indicating an essential role for Kv3.3 subunits in tremor induction by harmaline. Immunohistochemical staining for Kv3.3 showed clear expression in the somata and proximal dendrites of Purkinje cells and in their axonal projections to the deep cerebellar nuclei (DCN). In DCN, both Kv3.1 and Kv3.3 subunits are expressed. Action potential duration is increased by approximately 100% in Purkinje cells from Kv3.3-single mutants compared to WT or Kv3.1-single mutants. We conclude that Kv3.3 channel subunits are essential for the olivocerebellar system to generate and sustain normal harmaline tremor whereas Kv3.1 subunits influence tremor amplitude and frequency.

Action Potentials↗
PubMed2002

Resilient RTN fast spiking in Kv3.1 null mice suggests redundancy in the action potential repolarization mechanism.

Fast spiking (FS), GABAergic neurons of the reticular thalamic nucleus (RTN) are capable of firing high-frequency trains of brief action potentials, with little adaptation. Studies in recombinant systems have shown that high-voltage-activated K(+) channels containing the Kv3.1 and/or Kv3.2 subunits display biophysical properties that may contribute to the FS phenotype. Given that RTN expresses high levels of Kv3.1, with little or no Kv3.2, we tested whether this subunit was required for the fast action potential repolarization mechanism essential to the FS phenotype. Single- and multiple-action potentials were recorded using whole-cell current clamp in RTN neurons from brain slices of wild-type and Kv3.1-deficient mice. At 23 degrees C, action potentials recorded from homozygous Kv3.1 deficient mice (Kv3.1(-/-)) compared with their wild-type (Kv3.1(+/+)) counterparts had reduced amplitudes (-6%) and fast after-hyperpolarizations (-16%). At 34 degrees C, action potentials in Kv3.1(-/-) mice had increased duration (21%) due to a reduced rate of repolarization (-30%) when compared with wild-type controls. Action potential trains in Kv3.1(-/-) were associated with a significantly greater spike decrement and broadening and a diminished firing frequency versus injected current relationship (F/I) at 34 degrees C. There was no change in either spike count or maximum instantaneous frequency during low-threshold Ca(2+) bursts in Kv3.1(-/-) RTN neurons at either temperature tested. Our findings show that Kv3.1 is not solely responsible for fast spikes or high-frequency firing in RTN neurons. This suggests genetic redundancy in the system, possibly in the form of other Kv3 members, which may suffice to maintain the FS phenotype in RTN neurons in the absence of Kv3.1.

Action Potentials↗
PubMed2003

Kv1.5 is an important component of repolarizing K+ current in canine atrial myocytes.

Although the canine atrium has proven useful in several experimental models of atrial fibrillation and for studying the effects of rapid atrial pacing on atrial electrical remodeling, it may not fully represent the human condition because of reported differences in functional ionic currents and ion channel subunit expression. In this study, we reassessed the molecular components underlying one current, the ultrarapid delayed rectifier current in canine atrium [IKur(d)], by evaluating the mRNA, protein, immunofluorescence, and currents of the candidate channels. Using reverse transcriptase-polymerase chain reaction, we found that Kv1.5 mRNA was expressed in canine atrium whereas message for Kv3.1 was not detected. Western analysis on cytosolic and membrane fractions of canine tissues, using selective antibodies, showed that Kv3.1 was only detectable in the brain preparations, whereas Kv1.5 was expressed at high levels in both atrial and ventricular membrane fractions. Confocal imaging performed on isolated canine atrial myocytes clearly demonstrated the presence of Kv1.5 immunostaining, whereas that of Kv3.1 was equivocal. Voltage- and current-clamp studies showed that 0.5 mmol/L tetraethylammonium had variable effects on sustained K+ currents, whereas a compound with demonstrated selectivity for hKv1.5 versus Kv3.1, hERG or the sodium channel, fully suppressed canine atrial IKur tail currents and depressed sustained outward K+ current. This agent also increased action potential plateau potentials and action potential duration at 20% and 50% repolarization. These results suggest that in canine atria, as in other species including human, Kv1.5 protein is highly expressed and contributes to IKur.

Action Potentials↗
PubMed2004

Association of novel single nucleotide polymorphisms in the calcium channel alpha 1 subunit gene (Ca(v)1.1) and thyrotoxic periodic paralysis.

Thyrotoxic (hypokalemic) periodic paralysis (TPP) is a frequent complication of thyrotoxicosis among Chinese men. To determine the genetic association of TPP, we studied 97 male TPP patients, 77 Graves' disease patients without TPP, and 100 normal male subjects. Mutations of the voltage-dependent calcium channel (Ca(v)1.1), sodium channel (Na(v)1.4), and potassium channel (K(v)3.4), and association of the microsatellite markers on chromosome 1 in the region of the Na/K-ATPase subunits alpha1, alpha2, and beta1 were studied. None of the TPP patients carried the known mutations in Ca(v)1.1, Na(v)1.4, and K(v)3.4 genes. There was no association of TPP with the microsatellite markers that mapped to 1p13, 1q21-23, and 1q22-25. We detected 12 single nucleotide polymorphisms (SNPs) in Ca(v)1.1 in our population, of which three were novel. Significant differences in the SNP genotype distribution between TPP compared with Graves' disease controls and normal controls were seen at the 5' flanking region nucleotide (nt) -476 (P = 0.02), intron 2 nt 57 (P < 0.01), and intron 26 nt 67 (P < 0.001). Because these SNPs lie at or near the thyroid hormone responsive element, it is possible that they may affect the binding affinity of the thyroid hormone responsive element and modulate the stimulation of thyroid hormone on the Ca(v)1.1 gene.

5' Flanking Region↗
PubMed1997

Modulation of two cloned potassium channels by 1-alkanols demonstrates different cutoffs.

It is not known whether alcohols modulate ion channels by directly binding to the channel protein or by perturbing the surrounding membrane lipid. Cutoff describes the phenomenon where the potency of 1-alkanols monotonically increases with alkyl chain length until a loss of efficacy occurs. Determination of the cutoff for a variety of channels can be important, because similar and/or dissimilar cutoffs might yield information regarding the nature of ethanol's site of action. In this study, the two-electrode voltage clamp technique was used to determine the cutoffs for the 1-alkanol potentiation of cloned Ca(2+)-activated-K+ (BK) channels and for the inhibition of cloned Shaw2 K+ channels, expressed in Xenopus oocytes. Ethanol, butanol, hexanol, and heptanol reversibly enhanced BK currents, whereas octanol and nonanol had no effect. In contrast, Shaw2 currents were potently inhibited by both octanol and decanol, but not by undecanol. Taken together, data demonstrate that the modulation of K+ channels by long chain alcohols is channel-specific. Interestingly, ethanol was a less potent activator of BK currents in the intact oocyte in comparison with its effect on this channel in excised membrane patches. The decrease in potency could not be attributed to an ethanol-dependent change in Ca2+ influx through endogenous voltage-gated channels, an effect that would alter the concentration of Ca2+ available to activate BK channels.

Alcohols↗
PubMed2000

Endogenously generated spontaneous spiking activities recorded from postnatal spiral ganglion neurons in vitro.

Spontaneous spiking activities in the nervous system play an important role in the neuronal development and the coding of sensory information. Such firings could be initiated by transmitter leaked from the first-order sensory receptors or as a result of the internal operation of voltage-dependent ion channels intrinsic to the neuron. We recorded endogenously-generated spontaneous action potentials (APs) from postnatal spiral ganglion (SG) neurons of mouse in vitro. SG neurons in cultures displayed statistically stable spontaneous firings with no obvious bursting, rhythmic spiking and long silent gaps for as long as the recording configuration could be maintained. Average firing rates ranged from less than 1 to over 10 spikes/s, with most cells fired around 4 spikes/s. Interpulse interval histograms were remarkably similar to those recorded in vivo from the auditory nerve, with characteristics of a Poisson-like distribution. Resting membrane potential greatly altered the AP width and the rate of spontaneous firings. Spontaneous firing rates were also found to be controlled by the availability of the Shaw-like potassium channels. In contrast, matured SG neurons did not display any spontaneous APs, probably due to a large increase in the expression of the whole-cell potassium currents in comparison to their postnatal counterparts. This study provided the first direct evidence that postnatal SG neurons were capable of generating spontaneous APs independent of inputs from hair cells. Intracellular mechanisms for generating the spontaneous random spikes and the possible roles of such spontaneous activities in the postnatal development of SG neurons are discussed.

Action Potentials↗
PubMed2005

Inhibition of the cloned delayed rectifier K+ channels, Kv1.5 and Kv3.1, by riluzole.

The action of riluzole, a neuroprotective drug, on cloned delayed rectifier K+ channels (Kv1.5 and Kv3.1) was examined using the whole-cell patch-clamp technique. Riluzole reversibly inhibited Kv1.5 currents in a concentration-dependent manner with an IC50 of 39.69+/-2.37 microM. G-protein inhibitors (pertussis toxin and GDPbetaS) did not prevent this inhibition of riluzole on Kv1.5. No voltage-dependent inhibition by riluzole was found over the voltage range in which channels are fully activated. Riluzole shifted the steady-state inactivation curves of Kv1.5 in a hyperpolarizing direction in a concentration-dependent manner. It accelerated the deactivation kinetics of Kv1.5 in a concentration dependent-manner, but had no effect on the steady-state activation curve. Riluzole exhibited a use-independent inhibition of Kv1.5. The effects of riluzole on Kv3.1, the Shaw-type K+ channel were also examined. Riluzole caused a concentration-dependent inhibition of Kv3.1 currents with an IC50 of 120.98+/-9.74 microM and also shifted the steady-state inactivation curve of Kv3.1 in the hyperpolarizing direction. Thus, riluzole inhibits both Kv1.5 and Kv3.1 currents in a concentration-dependent manner and interacts directly with Kv1.5 by preferentially binding to the inactivated and to the closed states of the channel.

Animals↗
PubMed2004

Morphology and molecular organization of the adult neuromuscular junction of Drosophila.

While the larval neuromuscular junction (NMJ) of Drosophila has emerged as a model system to study synaptic function and development, little attention has been given to the study of the adult NMJ. Here we report an immunocytochemical and morphological characterization of an adult NMJ preparation of the prothorax. All muscles examined were innervated by small, uniform type II terminals (0.5-1.5 microm), a subset of which contained octopamine. Terminals classified as type I varied in their morphology across different muscles, ranging from strings or clusters of boutons (0.8-5.5 microm) to an elongate terminal (80-100 microm long) with few branches and contiguous swellings (3-15 microm) along its length. Analysis of the molecular composition of the NMJs during the first 5 days after eclosion revealed four major findings: 1) type I boutons increase in size during early adulthood; 2) Fasciclin II-immunoreactivity is not detectable at type I terminals, while DLG-immunoreactivity is observed at the synapse; 3) a Shaker-GFP fusion protein that localizes to all type I boutons in the larva is differentially localized at adult prothoracic NMJs; and 4) while all type I terminals contain glutamate, the glutamate receptor subunits, DGluRIIA and DGluRIIB, are expressed and clustered in only a subset of muscles. These findings suggest that maturation of the adult NMJ occurs during early adulthood and that muscle-specific properties may play a role in organizing synaptic components in the adult. Furthermore, these results demonstrate that there are major differences in the molecular organization of the adult and larval NMJs.

Aging↗
PubMed2004

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.

Animals↗
PubMed2003

Localization of KCNC1 (Kv3.1) potassium channel subunits in the avian auditory nucleus magnocellularis and nucleus laminaris during development.

The KCNC1 (previously Kv3.1) potassium channel, a delayed rectifier with a high threshold of activation, is highly expressed in the time coding nuclei of the adult chicken and barn owl auditory brainstem. The proposed role of KCNC1 currents in auditory neurons is to reduce the width of the action potential and enable neurons to transmit high frequency temporal information with little jitter. Because developmental changes in potassium currents are critical for the maturation of the shape of the action potential, we used immunohistochemical methods to examine the developmental expression of KCNC1 subunits in the avian auditory brainstem. The KCNC1 gene gives rise to two splice variants, a longer KCNC1b and a shorter KCNC1a that differ at the carboxy termini. Two antibodies were used: an antibody to the N-terminus that does not distinguish between KCNC1a and b isoforms, denoted as panKCNC1, and another antibody that specifically recognizes the C terminus of KCNC1b. A comparison of the staining patterns observed with the panKCNC1 and the KCNC1b specific antibodies suggests that KCNC1a and KCNC1b splice variants are differentially regulated during development. Although panKCNC1 immunoreactivity is observed from the earliest time examined in the chicken (E10), a subcellular redistribution of the immunoproduct was apparent over the course of development. KCNC1b specific staining has a late onset with immunostaining first appearing in the regions that map high frequencies in nucleus magnocellularis (NM) and nucleus laminaris (NL). The expression of KCNC1b protein begins around E14 in the chicken and after E21 in the barn owl, relatively late during ontogeny and at the time that synaptic connections mature morphologically and functionally.

Animals↗
PubMed1999

Cortical neurons immunoreactive for the potassium channel Kv3.1b subunit are predominantly surrounded by perineuronal nets presumed as a buffering system for cations.

Perineuronal nets (PNs) are known as chondroitin sulphate-rich, lattice-like coatings of the extracellular matrix. In the cortex of mammalian species investigated so far, they were mainly found around GABAergic neurons, but to a lesser degree also around pyramidal cells. Previous investigations in the rat revealed similar distribution patterns of fast-firing neurons expressing both the Kv3.1b subunit of voltage-gated potassium channels and the calcium-binding protein parvalbumin. In the present study, triple fluorescence labelling was applied for the simultaneous demonstration of PNs with the N-acetylgalactosamine-specific Wisteria floribunda agglutinin (WFA), parvalbumin-immunoreactivity (ir) with a monoclonal antibody and of Kv3.1b-ir with several rabbit antibodies. Subsets of non-pyramidal neurons - enwrapped by PNs and expressing parvalbumin and Kv3.1b - were detected in the rat and monkey neocortex and hippocampus. In the rat, faintly stained PNs were additionally found around several layer II/III and V pyramidal cells immunonegative for Kv3.1b, but contacted by Kv3.1b-containing boutons. In the monkey, more intensely labelled PNs frequently occurred around pyramidal cells which themselves appeared to be Kv3. 1b-immunopositive. We also observed minor Kv3.1b-ir and parvalbumin-ir cortical cell populations which were devoid of PNs; occasionally, nets were detected around neurons lacking both immunoreactivities. By confocal laser scanning microscopy, Kv3.1b-ir and WFA-binding sites were found adjoining at the soma and proximal dendritic surface, while lectin-binding sites usually extended on more distal dendritic segments and the axon initial segments which failed to express detectable Kv3.1b-ir. This spatial relationship of both markers was also confirmed by combined WFA-gold labelling and Kv3.1b-immunoperoxidase staining at the electron microscopic level. The data are used for a critical examination of current hypotheses concerning the functional role of PNs. We conclude that PNs may serve as rapid local buffers of excess cation changes in the extracellular space. Somatic membranes of fast-spiking neurons seem to be a main, but not the only source of such changes.

Animals↗