Biomedical subjects

Leonard K Kaczmarek

Publications and source records attributed to Leonard K Kaczmarek.

At least 19 recordsLinked to original sources

PubMed2006

Functional analysis of a novel potassium channel (KCNA1) mutation in hereditary myokymia.

Myokymia is characterized by spontaneous, involuntary muscle fiber group contraction visible as vermiform movement of the overlying skin. Myokymia with episodic ataxia is a rare, autosomal dominant trait caused by mutations in KCNA1, encoding a voltage-gated potassium channel. In the present study, we report a family with four members affected with myokymia. Additional clinical features included motor delay initially diagnosed as cerebral palsy, worsening with febrile illness, persistent extensor plantar reflex, and absence of epilepsy or episodic ataxia. Mutation analysis revealed a novel c.676C>A substitution in the potassium channel gene KCNA1, resulting in a T226K nonconservative missense mutation in the Kv1.1 subunit in all affected individuals. Electrophysiological studies of the mutant channel expressed in Xenopus oocytes indicated a loss of function. Co-expression of WT and mutant cRNAs significantly reduced whole-oocyte current compared to expression of WT Kv1.1 alone.

Amino Acid Sequence↗

PubMed2006

A store-operated Ca(2+) influx pathway in the bag cell neurons of Aplysia.

Although store-operated Ca(2+) influx has been well-studied in nonneuronal cells, an understanding of its nature in neurons remains poor. In the bag cell neurons of Aplysia californica, prior work has suggested that a Ca(2+) entry pathway can be activated by Ca(2+) store depletion. Using fura-based imaging of intracellular Ca(2+) in cultured bag cell neurons, we now characterize this pathway as store-operated Ca(2+) influx. In the absence of extracellular Ca(2+), the endoplasmic reticulum Ca(2+)-ATPase inhibitors, cyclopiazonic acid (CPA) or thapsigargin, depleted intracellular stores and elevated intracellular free Ca(2+). With the subsequent addition of extracellular Ca(2+), a prominent Ca(2+) influx was observed. The ryanodine receptor agonist, chloroethylphenol (CEP), also increased intracellular Ca(2+) but did not initiate store-operated Ca(2+) influx, despite overlap between CEP- and CPA-sensitive stores. Bafilomycin A, a vesicular H(+)-ATPase inhibitor, liberated intracellular Ca(2+) from acidic stores and attenuated subsequent Ca(2+) influx, presumably by replenishing CPA-depleted stores. Store-operated Ca(2+) influx was partially blocked by low concentrations of La(3+) or BTP2, and strongly inhibited by either 1-[b-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole (SKF-96365) or a high concentration of Ni(2+). Regarding IP(3) receptor blockers, 2-aminoethyldiphenyl borate, but not xestospongin C, prevented store-operated Ca(2+) influx. However, jasplakinolide, an actin stabilizer reported to inhibit this pathway in smooth muscle cell lines, was ineffective. The bag cell neurons initiate reproductive behavior through a prolonged afterdischarge associated with intracellular Ca(2+) release and neuropeptide secretion. Store-operated Ca(2+) influx may serve to replenish stores depleted during the afterdischarge or participate in the release of peptide that triggers behavior.

Anilides↗

PubMed2006

Pharmacological activation and inhibition of Slack (Slo2.2) channels.

The Slack (Sequence like a calcium-activated K channel) (Slo2.2) gene is abundantly expressed in the mammalian brain and encodes a sodium-activated K+ (KNa) channel. Although the specific roles of Slack channel subunits in neurons remain to be identified, they may play a role in the adaptation of firing rate and in protection against ischemic injury. In the present study, we have generated a stable cell line expressing the Slack channel, and have analyzed the pharmacological properties of these channels in these cells and in Xenopus oocytes. Two known blockers of KNa channels, bepridil and quinidine, inhibited Slack currents in a concentration-dependent manner and decreased channel activity in excised membrane patches. The inhibition by bepridil was potent, with an IC50 of 1.0 microM for inhibition of Slack currents in HEK cells. In contrast, bithionol was found to be a robust activator of Slack currents. When applied to the extracellular face of excised patches, bithionol rapidly induced a reversible increase in channel opening, suggesting that it acts on Slack channels relatively directly. These data establish an important early characterization of agents that modulate Slack channels, a process essential for the experimental manipulation of Slack currents in neurons.

Animals↗

PubMed2006

Opposite regulation of Slick and Slack K+ channels by neuromodulators.

Slick (Slo2.1) and Slack (Slo2.2) are two novel members of the mammalian Slo potassium channel gene family that may contribute to the resting potentials of cells and control their basal level of excitability. Slo2 channels have sensors that couple channel activity to the intracellular concentrations of Na+ and Cl- ions (Yuan et al., 2003). We now report that activity of both Slo2 channels is controlled by neuromodulators through Galphaq-protein coupled receptors (GqPCRs) (the M1 muscarinic receptor and the mGluR1 metabotropic glutamate receptor). Experiments coexpressing channels and receptors in Xenopus oocytes show that Slo2.1 and Slo2.2 channels are modulated in opposite ways: Slo2.1 is strongly inhibited, whereas Slo2.2 currents are strongly activated through GqPCR stimulation. Differential regulation involves protein kinase C (PKC); application of the PKC activator PMA, to cells expressing channels but not receptors, inhibits Slo2.1 whole-cell currents and increases Slo2.2 currents. Synthesis of a chimera showed that the distal carboxyl region of Slo2.1 controls the sensitivity of Slo2.1 to PMA. Slo2 channels have widespread expression in brain (Bhattacharjee et al., 2002, 2005). Using immunocytochemical techniques, we show coexpression of Slo2 channels with the GqPCRs in cortical and hippocampal brain sections and in cultured hippocampal neurons. The differential control of these novel channels by neurotransmitters may elicit long-lasting increases or decreases in neuronal excitability and, because of their widespread distribution, may provide a mechanism to activate or repress electrical activity in many systems of the brain.

Animals↗

PubMed2006

Modulation of Kv3.1b potassium channel phosphorylation in auditory neurons by conventional and novel protein kinase C isozymes.

In fast-spiking neurons such as those in the medial nucleus of the trapezoid body (MNTB) in the auditory brainstem, Kv3.1 potassium channels are required for high frequency firing. The Kv3.1b splice variant of this channel predominates in the mature nervous system and is a substrate for phosphorylation by protein kinase C (PKC) at Ser-503. In resting neurons, basal phosphorylation at this site decreases Kv3.1 current, reducing neuronal ability to follow high frequency stimulation. We used a phospho-specific antibody to determine which PKC isozymes control serine 503 phosphorylation in Kv3.1b-tranfected cells and in auditory neurons in brainstem slices. By using isozyme-specific inhibitors, we found that the novel PKC-delta isozyme, together with the novel PKC-epsilon and conventional PKCs, contributed to the basal phosphorylation of Kv3.1b in MNTB neurons. In contrast, only PKC-epsilon and conventional PKCs mediate increases in phosphorylation produced by pharmacological activation of PKC in MNTB neurons or by metabotropic glutamate receptor activation in Kv3.1/mGluR1-cotransfected cells. We also measured the time course of dephosphorylation and recovery of basal phosphorylation of Kv3.1b following brief high frequency electrical stimulation of the trapezoid body, and we determined that the recovery process is mediated by both novel PKC-delta and PKC-epsilon isozymes and by conventional PKCs. The association between Kv3.1b and PKC isozymes was confirmed by reciprocal coimmunoprecipitation of Kv3.1b with multiple PKC isozymes. Our results suggest that the Kv3.1b channel is regulated by both conventional and novel PKC isozymes and that novel PKC-delta contributes specifically to the maintenance of basal phosphorylation in auditory neurons.

Animals↗

PubMed2006

Policing the ball: a new potassium channel subunit determines inactivation rate.

Inactivation of potassium currents during maintained firing results in a progressive increase in action potential width and neuronal excitability. In Kv1.1 channels, inactivation has attributed to a beta subunit that blocks the pore of the channel shortly after channel opening. In this issue of Neuron, Shulte and colleagues have identified a novel channel subunit whose interaction with Kv1.1 and the beta subunit prevents such inactivation. Mutations in this subunit lead to temporal lobe epilepsy.

Animals↗

PubMed2006

Non-conducting functions of voltage-gated ion channels.

Various studies, mostly in the past 5 years, have demonstrated that, in addition to their well-described function in regulating electrical excitability, voltage-dependent ion channels participate in intracellular signalling pathways. Channels can directly activate enzymes linked to cellular signalling pathways, serve as cell adhesion molecules or components of the cytoskeleton, and their activity can alter the expression of specific genes. Here, I review these findings and discuss the extent to which the molecular mechanisms of such signalling are understood.

Animals↗

PubMed2005

Retraction.

Explore the source record for details and available documents.

Retraction Notice↗

PubMed2005

Association/dissociation of a channel-kinase complex underlies state-dependent modulation.

Although ion channels are regulated by protein kinases, it has yet to be established whether the behavioral state of an animal may dictate whether or not modulation by a kinase can occur. Here, we describe behaviorally relevant changes in the ability of a nonselective cation channel from Aplysia bag cell neurons to be regulated by protein kinase C (PKC). This channel drives a prolonged afterdischarge that triggers the release of egg-laying hormone and a series of reproductive behaviors. The afterdischarge is followed by a lengthy refractory period, during which additional bursting cannot be elicited. Previously, we reported that, in excised inside-out patches, the cation channel is closely associated with PKC, which increases channel activity. We now show that this channel-kinase association is plastic, because channels excised from certain neurons lack PKC-dependent modulation. Although direct application of PKC-activating phorbol ester to these patches had no effect, exposing the neurons themselves to phorbol ester reinstated modulation, suggesting that an absence of modulation was attributable to a lack of associated kinase. Furthermore, modulation was restored by pretreating neurons with either PP1 [4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine] or SU6656, inhibitors of Src tyrosine kinase, an enzyme whose Src homology 3 domain is required for channel-PKC association. Neurons that were stimulated to afterdischarge and had entered the prolonged refractory period were found to have more phosphotyrosine staining and less channel-PKC association than unstimulated neurons. These findings suggest that Src-dependent regulation of the association between the cation channel and PKC controls both the long-term excitability of these neurons and their ability to induce reproduction.

Adenosine Triphosphate↗

PubMed2005

Acoustic environment determines phosphorylation state of the Kv3.1 potassium channel in auditory neurons.

Sound localization by auditory brainstem nuclei relies on the detection of microsecond interaural differences in action potentials that encode sound volume and timing. Neurons in these nuclei express high amounts of the Kv3.1 potassium channel, which allows them to fire at high frequencies with short-duration action potentials. Using computational modeling, we show that high amounts of Kv3.1 current decrease the timing accuracy of action potentials but enable neurons to follow high-frequency stimuli. The Kv3.1b channel is regulated by protein kinase C (PKC), which decreases current amplitude. Here we show that in a quiet environment, Kv3.1b is basally phosphorylated in rat brainstem neurons but is rapidly dephosphorylated in response to high-frequency auditory or synaptic stimulation. Dephosphorylation of the channel produced an increase in Kv3.1 current, facilitating high-frequency spiking. Our results indicate that the intrinsic electrical properties of auditory neurons are rapidly modified to adjust to the ambient acoustic environment.

Acoustic Stimulation↗

PubMed2005

Localization of the Na+-activated K+ channel Slick in the rat central nervous system.

Na+-activated K+ currents (K(Na)) have been reported in multiple neuronal nuclei and the properties of K(Na) vary in different cell types. We have described previously the distribution of Slack, a Na+-activated K+ channel subunit. Another recently cloned Na+-activated K+ channel is Slick, which differs from Slack in its rapid activation and its sensitivity to intracellular ATP levels. We now report the localization of Slick in the rat central nervous system using in situ and immunohistochemical techniques. As for Slack, we find that Slick is widely distributed in the brain. Specifically, strong hybridization signals and immunoreactivity were found in the brainstem, including auditory neurons such as the medial nucleus of the trapezoid body. As has also been shown for Slack, Slick is expressed in the olfactory bulb, red nucleus, facial nucleus, pontine nucleus, oculomotor nucleus, substantia nigra, deep cerebellar nuclei, vestibular nucleus, and the thalamus. Slick mRNA and protein, however, also are found in certain neurons that do not express Slack. These neurons include those of the hippocampal CA1, CA2, and CA3 regions, the dentate gyrus, supraoptic nucleus, hypothalamus, and cortical layers II, III, and V. These data suggest that Slick may function independently of Slack in these regions. Computer simulations indicate that Slick currents can cause adaptation during prolonged stimuli. Such adaptation allows a neuron to respond to high-frequency stimulation with lower-frequency firing that remains temporally locked to individual stimuli, a property seen in many auditory neurons. Although it is not yet known if Slick and Slack subunits heteromultimerize, the existence of two genes that encode K(Na), that are widely expressed in the nervous system, with both overlapping and nonoverlapping distributions, provides the basis for the reported heterogeneity in the properties of K(Na) from various neurons.

Animals↗

PubMed2005

RETRACTED: Requirement of voltage-gated calcium channel beta4 subunit for T lymphocyte functions.

Calcium is known to play vital roles in diverse physiological processes, and it is known that voltage-gated calcium channels (Cav) mediate calcium influx in excitable cells. However, no consensus exists on the molecular identity of the calcium channels present in nonexcitable cells such as T lymphocytes. Here, we demonstrate that T lymphocytes express both regulatory beta4 and poreforming Cav1 alpha1 subunits of Cav channels. Cav beta4-mutant T lymphocytes fail to acquire normal functions and display impairment in the calcium response, activation of the transcription factor NFAT, and cytokine production. Although Cav1 channels of lymphocytes retain their voltage dependency, T cell receptor stimulation dramatically increases channel opening, providing a new mechanism for calcium entry in lymphocytes.

Animals↗

PubMed2005

Aminoglycosides block the Kv3.1 potassium channel and reduce the ability of inferior colliculus neurons to fire at high frequencies.

The Kv3.1 potassium channel is expressed at high levels in auditory nuclei and contributes to the ability of auditory neurons to fire at high frequencies. We have tested the effects of streptomycin, an agent that produces progressive hearing loss, on the firing properties of inferior colliculus neurons and on Kv3.1 currents in transfected cells. We found that in inferior colliculus neurons, intracellular streptomycin decreased the current density of a high threshold, noninactivating outward current and reduced the rate of repolarization of action potentials and the ability of these neurons to fire at high frequencies. Furthermore, potassium current in CHO cells transfected with the Kv3.1 gene was reduced by 50% when cells were cultured in the presence of streptomycin or when streptomycin was introduced intracellularly in the pipette solution. In the presence of intracellular streptomycin, the activation rate of Kv3.1 current increased and inhibition by extracellular TEA become voltage-dependent. The data indicate that streptomycin inhibits Kv3.1 currents by inducing a conformational change in the Kv3.1 channel. The hearing loss caused by aminoglycoside antibiotics may be partially mediated by their inhibition of Kv3.1 current in auditory neurons.

Action Potentials↗

PubMed2005

Regulation of the timing of MNTB neurons by short-term and long-term modulation of potassium channels.

The firing patterns of neurons in central auditory pathways encode specific features of sound stimuli, such as frequency, intensity and localization in space. The generation of the appropriate pattern depends, to a major extent, on the properties of the voltage-dependent potassium channels in these neurons. The mammalian auditory pathways that compute the direction of a sound source are located in the brainstem and include the connection from bushy cells in the anteroventral cochlear nucleus (AVCN) to the principal neurons of the medial nucleus of the trapezoid body (MNTB). To preserve the fidelity of timing of action potentials that is required for sound localization, these neurons express several types of potassium channels, including the Kv3 and Kv1 families of voltage-dependent channels and the Slick and Slack sodium-dependent channels. These channels determine the pattern of action potentials and the amount of neurotransmitter released during repeated stimulation. The amplitude of currents carried by one of these channels, the Kv3.1b channel, is regulated in the short term by protein phosphorylation, and in the long term, by changes in gene expression, such that the intrinsic excitability of the neurons is constantly being regulated by the ambient auditory environment.

Action Potentials↗

PubMed2005

For K+ channels, Na+ is the new Ca2+.

Although K+ channels activated by Ca2+ have long been known to shape neuronal excitability, evidence is accumulating that K+ channels sensitive to intracellular Na+, termed K(Na) channels, have an equally significant role. K(Na) channels contribute to adaptation of firing rate and to slow afterhyperpolarizations that follow repetitive firing. In certain neurons, they also appear to be activated by Na+ influx accompanying a single spike. Two genes encoding these channels, Slick and Slack, are expressed throughout the brain. The spatial localization of K(Na) channels along axons, dendrites and somata appears to be highly cell-type specific. Their molecular properties also suggest that these channels contribute to the response of neurons to hypoxia.

Action Potentials↗

PubMed2004

Exposure to hypoxia rapidly induces mitochondrial channel activity within a living synapse.

One of the earliest effects of hypoxia on neuronal function is to produce a run-down of synaptic transmission, and more prolonged hypoxia results in neuronal death. An increase in the permeability of the outer mitochondrial membrane, controlled by BCL-2 family proteins, occurs in response to stimuli that trigger cell death. By patch clamping mitochondrial membranes inside the presynaptic terminal of a squid giant synapse, we have now found that several minutes of hypoxia trigger the opening of large multiconductance channels. The channel activity is induced concurrently with the attenuation of synaptic responses that occurs under hypoxic conditions. Hypoxia-induced channels are inhibited by NADH, an agent that inhibits large conductance channels produced by a pro-apoptotic fragment of BCL-xL in these synaptic mitochondria. The appearance of hypoxia-induced channels was also prevented by the caspase/cysteine protease inhibitor benzyloxycarbonyl-VAD-fluoromethyl ketone (Z-VAD-fmk), which inhibits proteolysis of BCL-xL during hypoxia. Both NADH and Z-VAD-fmk reduced significantly the rate of decline of synaptic responses during hypoxia. Our results indicate that an increase in outer mitochondrial channel activity is a very early event in the response of neurons to hypoxia and suggest that this increase in activity may contribute to the decline in synaptic function during hypoxia.

Amino Acid Chloromethyl Ketones↗

PubMed2004

The appearance of a protein kinase A-regulated splice isoform of slo is associated with the maturation of neurons that control reproductive behavior.

In response to brief synaptic stimulation that activates protein kinase A (PKA), the bag cell neurons of Aplysia trigger the onset of reproductive behaviors by generating a prolonged afterdischarge. In juvenile animals, such afterdischarges are inhibited by a high density of Ca2+ -activated K+ (BK) channels, encoded by the slo gene. An increase in this current also follows an afterdischarge in mature animals, contributing to a subsequent refractory state that limits reproductive behaviors. Using a bag cell cDNA library, we have isolated two alternative transcripts of the slo gene, differing in the presence (slo-a) or absence (slo-b) of a consensus phosphorylation site for PKA. Expression of either isoform in Chinese hamster ovary cells produced Ca2+ - and voltage-dependent channels with macroscopic and unitary properties matching those in bag cell neurons. The isoforms differed, however, in their response to application of the catalytic subunit of PKA, which reduced the open probability of Slo-a, an effect that was reversed by a PKA inhibitor. In contrast, PKA had no effect on Slo-b. By immunocytochemistry, we determined that the PKA-regulated Slo-a subunit is present in adult, but not juvenile, bag cell neurons. Patch clamp recordings from adult and juvenile bag cell neurons confirmed that PKA decreases BK channel activity only in adults. Our findings suggest that a change in the identity of Slo isoforms expressed during development allows mature neurons to generate afterdischarges that are required for reproduction.

Alternative Splicing↗

PubMed2004

Activation of a calcium entry pathway by sodium pyrithione in the bag cell neurons of Aplysia.

The ability of sodium pyrithione (NaP), an agent that produces delayed neuropathy in some species, to alter neuronal physiology was accessed using ratiometric imaging of cytosolic free Ca(2+) concentration ([Ca(2+)](i)) in fura PE-filled cultured Aplysia bag cell neurons. Bath-application of NaP evoked a [Ca(2+)](i) elevation in both somata and neurites with an EC(50) of approximately 300 nM and a Hill coefficient of approximately 1. The response required the presence of external Ca(2+), had an onset of 3-5 min, and generally reached a maximum within 30 min. 2-Methyl-sulfonylpyridine, a metabolite and close structural analog of NaP, did not elevate [Ca(2+)](i). Under whole-cell current-clamp recording, NaP produced a approximately 14 mV depolarization of resting membrane potential that was dependent on external Ca(2+). These data suggested that NaP stimulates Ca(2+) entry across the plasma membrane. To minimize the possibility that a change in cytosolic pH was the basis for NaP-induced Ca(2+) entry, bag cell neuron intracellular pH was estimated with the dye 2',7'-bis(carboxyethyl-5(6)-carboxy-fluorescein acetoxy methylester. Exposure of the neurons to NaP did not alter intracellular pH. The slow onset and sustained nature of the NaP response suggested that a cation exchange mechanism coupled either directly or indirectly to Ca(2+) entry could underlie the phenomenon. However, neither ouabain, a Na(+)/K(+) ATPase inhibitor, nor removal of extracellular Na(+), which eliminates Na(+)/Ca(2+) exchanger activity, altered the NaP-induced [Ca(2+)](i) elevation. Finally, the possibility that NaP gates a Ca(2+)-permeable ion channel in the plasma membrane was examined. NaP did not appear to activate two major forms of bag cell neuron Ca(2+)-permeable ion channels, as Ca(2+) entry was unaffected by inhibition of voltage-gated Ca(2+) channels using nifedipine or by inhibition of a voltage-dependent, nonselective cation channel using a high concentration of tetrodotoxin. In contrast, two potential store-operated Ca(2+) entry current inhibitors, SKF-96365 and Ni(2+), attenuated NaP-induced Ca(2+) entry. We conclude that NaP activates a slow, persistent Ca(2+) influx in Aplysia bag cell neurons.

Animals↗