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L K Kaczmarek

Publications and source records attributed to L K Kaczmarek.

At least 37 records · Page 2Linked to original sources
PubMed1999

Cell type-specific expression of the Kv3.1 gene is mediated by a negative element in the 5' untranslated region of the Kv3.1 promoter.

The Kv3.1 potassium channel gene is restrictively expressed in the CNS, and its expression level is especially high in neurons that are able to follow synaptic inputs at high frequencies. To understand the transcriptional mechanisms controlling Kv3.1 expression, we have conducted a functional analysis of the Kv3.1 promoter in various cell lines of different tissue origins and in transgenic mice. Our results suggest that an upstream regulatory fragment coupled with the 5' untranslated region (UTR) is able to confer tissue-specific expression in both cell lines and in transgenic mice. Deletion analysis of the regulatory region carried out in cell lines reveals that a strong negatively acting element, uniquely residing in the 5' UTR (+350 to +158), appears able to confer cell type specificity on both the Kv3.1 promoter and the thymidine kinase promoter in transient transfection assays. A weak cell type-specific enhancer in the proximal region of the promoter (-123 to -71) also contributes to cell type-specific expression of the Kv3.1 gene.

3T3 Cells↗
PubMed1998

Expression of a foreign G-protein coupled receptor modulates the excitability of the peptidergic bag cell neurons of Aplysia.

Brief afferent stimulation of the bag cell neurons can trigger a sustained period of spontaneous firing (the 'afterdischarge'). Pharmacological activation of a number of second messenger pathways has previously been shown to partially replicate this behavior. One strategy to identify additional pathways may be to heterologously express a variety of G-protein coupled receptors in bag cell neurons, then test whether receptor activation can induce an afterdischarge. We find that expression of the 1alpha metabotropic glutamate receptor in single bag cell neurons mimics some features of the afterdischarge. This approach is thus feasible and may allow the reconstruction of different components of afterdischarge in the absence of the afferent input.

Action Potentials↗
PubMed1998

Heterologous expression of the Kv3.1 potassium channel eliminates spike broadening and the induction of a depolarizing afterpotential in the peptidergic bag cell neurons.

The bag cell neurons of Aplysia are a cluster of cells that control egg laying behavior. After brief synaptic stimulation, they depolarize and fire spontaneously for up to 30 min. During the first few seconds of this afterdischarge, the action potentials of the bag cell neurons undergo pronounced broadening. Single bag cell neurons in culture also show spike broadening in response to repeated depolarizations. This broadening is frequency-dependent and associated with the induction of a depolarizing afterpotential lasting minutes. In some neurons the depolarizing afterpotential is sufficient to trigger spontaneous firing. To test the possibility that spike broadening during stimulation is required to trigger the depolarizing afterpotential, we eliminated frequency-dependent broadening by heterologous expression of the Kv3.1 potassium channel. This channel has rapid activation and deactivation kinetics and no use-dependent inactivation. Expression of Kv3.1 prevented spike broadening and also eliminated the depolarizing afterpotential. Measurements of the integral of calcium current during voltage commands, which simulated the action potentials of the control neurons and those expressing Kv3.1, indicate that spike broadening produces up to a fivefold increase in calcium entry. Manipulations that limit calcium entry during action potentials or chelation of intracellular calcium using BAPTA AM prevented the induction of the depolarizing afterpotential. We conclude that spike broadening is essential for the induction of the depolarizing afterpotential probably by regulating calcium influx and suggest that one of the physiological roles of spike broadening may be to regulate long-term changes in neuronal excitability.

Action Potentials↗
PubMed1998

Depolarization selectively increases the expression of the Kv3.1 potassium channel in developing inferior colliculus neurons.

The Kv3.1 channel subunit, when expressed heterologously, gives rise to a high-threshold noninactivating potassium current. Experiments with auditory neurons have suggested that the presence of this channel subunit enables them to fire action potentials at high frequencies. We have found that the expression levels of Kv3.1 transcripts increase in inferior colliculus neurons before the onset of hearing and then remain relatively constant. Because spontaneous neuronal activity plays an important role in modulating neuronal excitability during development, we examined the effects of depolarization with an elevated concentration of external potassium ions on the expression of Kv3.1 channel subunits in immature inferior colliculus neurons. Elevated potassium produced a marked increase in Kv3.1 mRNA levels and in the amplitude of a high-threshold, noninactivating current before the onset of hearing. This increase was prevented by the presence of a calcium channel blocker, indicating that calcium influx mediated the depolarization-induced increase in this current. In contrast, treatment with an elevated external potassium concentration caused only a moderate increase in the peak amplitude of a lower-threshold inactivating current. The repolarization of action potentials in the high-potassium-treated cells was more rapid and complete than in the control cells. Computer simulations confirmed that this change could be explained by a change in Kv3.1-like current of the same magnitude as recorded in voltage-clamp experiments. Thus, depolarization and calcium influx may alter the excitability of immature inferior colliculus neurons by selectively increasing the levels of a Kv3. 1-like potassium current.

Aging↗
PubMed1998

Modulation of a calcium-sensitive nonspecific cation channel by closely associated protein kinase and phosphatase activities.

Regulation of nonspecific cation channels often underlies neuronal bursting and other prolonged changes in neuronal activity. In bag cell neurons of Aplysia, it recently has been suggested that an intracellular messenger-induced increase in the activity of a nonspecific cation channel may underlie the onset of a 30-min period of spontaneous action potentials referred to as the "afterdischarge. " In patch clamp studies of the channel, we show that the open probability of the channel can be increased by an average of 10. 7-fold by application of ATP to the cytoplasmic side of patches. Duration histograms indicate that the increase is primarily a result of a reduction in the duration and percentage of channel closures described by the slowest time constant. The increase in open probability was not observed using 5'-adenylylimidodiphosphate, a nonhydrolyzable ATP analog, and was blocked in the presence of H7 or the more specific calcium/phospholipid-dependent protein kinase C (PKC) inhibitor peptide(19-36). Because the increase in activity observed in response to ATP occurred without application of protein kinase, our results indicate that a kinase endogenous to excised patches mediates the effect. The effect of ATP could be reversed by exogenously applied protein phosphatase 1 or by a microcystin-sensitive phosphatase also endogenous to excised patches. These results, together with work demonstrating the presence of a protein tyrosine phosphatase in these patches, suggest that the cation channel is part of a regulatory complex including at least three enzymes. This complex may act as a molecular switch to activate the cation channel and, thereby, trigger the afterdischarge.

Adenine Nucleotides↗
PubMed1998

High-frequency firing helps replenish the readily releasable pool of synaptic vesicles.

Synapses in the central nervous system undergo various short- and long-term changes in their strength, but it is often difficult to distinguish whether presynaptic or postsynaptic mechanisms are responsible for these changes. Using patch-clamp recording from giant synapses in the mouse auditory brainstem, we show here that short-term synaptic depression can be largely attributed to rapid depletion of a readily releasable pool of vesicles. Replenishment of this pool is highly dependent on the recent history of synaptic activity. High-frequency stimulation of presynaptic terminals significantly enhances the rate of replenishment. Broadening the presynaptic action potential with the potassium-channel blocker tetraethylammonium, which increases Ca2+ entry, further enhances the rate of replenishment. As this increase can be suppressed by the Ca2+-channel blocker Cd2+ or by the Ca2+ buffer EGTA, we conclude that Ca2+ influx through voltage-gated Ca2+ channels is the key signal that dynamically regulates the refilling of the releasable pool of synaptic vesicles in response to different patterns of inputs.

Action Potentials↗
PubMed1998

Contribution of the Kv3.1 potassium channel to high-frequency firing in mouse auditory neurones.

1. Using a combination of patch-clamp, in situ hybridization and computer simulation techniques, we have analysed the contribution of potassium channels to the ability of a subset of mouse auditory neurones to fire at high frequencies. 2. Voltage-clamp recordings from the principal neurones of the medial nucleus of the trapezoid body (MNTB) revealed a low-threshold dendrotoxin (DTX)-sensitive current (ILT) and a high-threshold DTX-insensitive current (IHT). 3. IHT displayed rapid activation and deactivation kinetics, and was selectively blocked by a low concentration of tetraethylammonium (TEA; 1 mM). 4. The physiological and pharmacological properties of IHT very closely matched those of the Shaw family potassium channel Kv3.1 stably expressed in a CHO cell line. 5. An mRNA probe corresponding to the C-terminus of the Kv3.1 channel strongly labelled MNTB neurones, suggesting that this channel is expressed in these neurones. 6. TEA did not alter the ability of MNTB neurones to follow stimulation up to 200 Hz, but specifically reduced their ability to follow higher frequency impulses. 7. A computer simulation, using a model cell in which an outward current with the kinetics and voltage dependence of the Kv3.1 channel was incorporated, also confirmed that the Kv3.1- like current is essential for cells to respond to a sustained train of high-frequency stimuli. 8. We conclude that in mouse MNTB neurones the Kv3.1 channel contributes to the ability of these cells to lock their firing to high-frequency inputs.

Animals↗
PubMed1998

The expression of two splice variants of the Kv3.1 potassium channel gene is regulated by different signaling pathways.

The Kv3.1 potassium channel gene gives rise to two different channel proteins, Kv3.1a and Kv3.1b, by alternative splicing of nuclear RNA. During development the levels of Kv3.1b mRNA (but not Kv3.1a) substantially increase in rat cerebellum after postnatal day 8. The molecular mechanism underlying the differential regulation of the two transcripts is not known. Using in vitro slices of cerebellum, we have found that basic fibroblast growth factor (bFGF) upregulates both Kv3.1a and Kv3.1b at this developmental stage, but that depolarization by elevated potassium concentrations is without effect. Combined treatment with bFGF and depolarization, however, prevents the increase in Kv3.1a transcripts and selectively increases Kv3.1b mRNA levels. A protein kinase C (PKC) inhibitor blocks the increase in Kv3.1a mRNA levels induced by bFGF alone but does not affect the increase in Kv3.1b mRNA. Measurement of nuclear protein kinase C activity shows that bFGF activates this enzyme and that depolarization blocks this activation. In contrast to these findings at postnatal day 8, bFGF fails to alter Kv3.1 transcripts in slices from adult animals, and PKC activity is enhanced rather than suppressed by depolarization. Our results indicate that different signaling pathways regulate Kv3.1a and Kv3.1b expression and suggest that Kv3.1a mRNA levels may be modulated by neuronal activity.

Alternative Splicing↗
PubMed1998

Activation of Kv3.1 channels in neuronal spine-like structures may induce local potassium ion depletion.

Spines are specialized neuronal membrane structures, often localized at sites where synaptic information is relayed from one cell to another in the central nervous system. By electron immunomicroscopy we have found that the mammalian Shaw family potassium channel Kv3.1 is localized on spine-like protrusions, adjacent to postsynaptic membranes of bushy cells in the cochlear nucleus. As direct characterization of the electrophysiological behavior of ion channels in such structures is difficult, we have used Kv3. 1-transfected CHO cells to create artificial spine-like membrane compartments. Membrane patches were sucked into microelectrodes to form small, cell-attached vesicles with dimensions comparable to those of the neuronal structures. Currents mediated by the Kv3.1 channel in these vesicles undergo rapid and complete inactivation, in contrast to their noninactivating behavior in whole-cell recordings. This apparent inactivation is caused by the rapid depletion of K+ from the vesicle and the slow refilling of K+ into the vesicle compartment from the bulk cytoplasm. Our data provide evidence that compartmentalized ionic transients can be generated in spine-like membrane structures and support the view that the localization of ion channels in spine-like structures may influence responses to synaptic stimulation.

Animals↗
PubMed1998

When, where, and how much? Expression of the Kv3.1 potassium channel in high-frequency firing neurons.

The Kv3.1 potassium channel gene is expressed in neurons that fire action potentials at high frequencies. Neurons that express this gene, such as auditory brain stem neurons, have high-threshold voltage-dependent potassium currents that activate and deactivate unusually rapidly, and whose characteristics match those of the Kv3.1 subunit expressed heterologously. The level of Kv3.1 expression in neurons is regulated during development and by environmental stimuli. Pharmacological and computer modeling studies indicate that changes in the level of this channel alter the ability of a neuron to follow synaptic inputs at high frequencies. To understand the transcriptional mechanisms that control Kv3.1 expression, an initial characterization of the primary promoter for the Kv3.1 gene was carried out. This review summarizes current knowledge regarding Kv3.1 gene transcription and the roles of upstream regulatory elements in conferring cell-type specificity and long-term regulation by extrinsic factors.

Animals↗
PubMed1998

Formation of intermediate-conductance calcium-activated potassium channels by interaction of Slack and Slo subunits.

Large-conductance calcium-activated potassium channels (maxi-K channels) have an essential role in the control of excitability and secretion. Only one gene Slo is known to encode maxi-K channels, which are sensitive to both membrane potential and intracellular calcium. We have isolated a potassium channel gene called Slack that is abundantly expressed in the nervous system. Slack channels rectify outwardly with a unitary conductance of about 25-65 pS and are inhibited by intracellular calcium. However, when Slack is co-expressed with Slo, channels with pharmacological properties and single-channel conductances that do not match either Slack or Slo are formed. The Slack/Slo channels have intermediate conductances of about 60-180 pS and are activated by cytoplasmic calcium. Our findings indicate that some intermediate-conductance channels in the nervous system may result from an interaction between Slack and Slo channel subunits.

Amino Acid Sequence↗
PubMed1998

Protein kinase C regulates a vesicular class of calcium channels in the bag cell neurons of aplysia.

Protein kinase C (PKC) acutely increases calcium currents in Aplysia bag cell neurons by recruiting calcium channels different from those constitutively active in the plasma membrane. To study the mechanism of PKC regulation we previously identified two calcium channel alpha1-subunits expressed in bag cell neurons. One of these, BC-alpha1A, is localized to vesicles concentrated primarily in somata and growth cones. We used antibodies to BC-alpha1A to analyze its expression in the bag cell neurons of juvenile Aplysia at a developmental stage at which PKC-sensitive calcium currents have previously been shown to be low. We find that vesicular BC-alpha1A staining is generally reduced in juvenile bag cell neurons but that its expression level can vary among juvenile animals. In 17 bag cell clusters examined, the percentage of neurons that displayed punctate alphaBC-alpha1A staining ranged from 0 to 85%. Sampling of calcium currents from cells of the same clusters by whole cell patch-clamp techniques revealed that the PKC-sensitive calcium current density is significantly correlated with the degree of vesicular staining. In contrast, no correlation of basal calcium current levels with aBC-alpha1A staining was found. These results strongly suggest that BC-alpha1A, a member of the ABE-subfamily of calcium channels, carries the PKC-sensitive calcium current in bag cell neurons. They are consistent with a model in which PKC recruits channels from the vesicular pool to the plasma membrane.

Animals↗
PubMed1997

hSK4, a member of a novel subfamily of calcium-activated potassium channels.

The gene for hSK4, a novel human small conductance calcium-activated potassium channel, or SK channel, has been identified and expressed in Chinese hamster ovary cells. In physiological saline hSK4 generates a conductance of approximately 12 pS, a value in close agreement with that of other cloned SK channels. Like other members of this family, the polypeptide encoded by hSK4 contains a previously unnoted leucine zipper-like domain in its C terminus of unknown function. hSK4 appears unique, however, in its very high affinity for Ca2+ (EC50 of 95 nM) and its predominant expression in nonexcitable tissues of adult animals. Together with the relatively low homology of hSK4 to other SK channel polypeptides (approximately 40% identical), these data suggest that hSK4 belongs to a novel subfamily of SK channels.

Amino Acid Sequence↗
PubMed1997

Localization of a high threshold potassium channel in the rat cochlear nucleus.

Potassium channels play a major role in determining the pattern and frequency of neuronal firing. In the cochlear nucleus (CN), various morphologically defined types of neurons have different responses to a sound. We have previously identified one type of cloned K+ channel, termed Kv3.1, which is highly expressed in many auditory neurons. Expression studies indicate that Kv3.1 channels have an unusually high threshold for activation. In this study, we used both in situ hybridization and immunohistochemistry to examine the expression patterns of the Kv3.1 channel in the CN. In the ventral CN, bushy cells hybridized strongly with Kv3.1 specific probes and a subpopulation of stellate/multipolar cells hybridized with Kv3.1 probes. In the dorsal CN, pyramidal and large multipolar/giant cells expressed Kv3.1 mRNA. Abundant Kv3.1 immunolabeling was also observed in the CN. The pattern of immunolabeling revealed that the Kv3.1 protein is distributed along the soma, proximal dendrites, unmyelinated axons, and axon terminals of stained neurons. In the case of pyramidal and octopus cells, no immunolabeling was detected at the somata, even though these cells expressed Kv3.1 mRNA. Computer simulations were used to explore the functional role of the Kv3.1 channel. The simulations indicate that Kv3.1 conductances may contribute to repolarization of large synaptic potentials. When stimulated at high frequencies, the presence of Kv3.1 enhances the ability of a model cell with some of the features of bushy cells to follow high frequency input with temporal precision.

Amino Acid Sequence↗
PubMed1997

The secretion of classical and peptide cotransmitters from a single presynaptic neuron involves a synaptobrevin-like molecule.

It is not yet understood how the molecular mechanisms controlling the release of neuropeptides differ from those controlling the release of classical transmitters, mainly because there are few peptidergic synapses in which the environment at the presynaptic release sites can be manipulated. Using Aplysia californica neuron B2, which synthesizes both peptide and classical transmitters, we have established two synaptic types. When B2 is cocultured with a sensory neuron, a peptidergic synapse is formed. In contrast, when B2 is cocultured with neuron B6, a classical synapse is formed. In contrast to a common assumption, single action potentials can release both types of transmitters. The secretion of peptide and classical transmitters by B2 is inhibited by the presynaptic injection of tetanus toxin, but not by an inactive mutant. Thus a synaptobrevin-like molecule is involved in the secretion of these two types of transmitters.

Acetylcholine↗
PubMed1997

Identification of a vesicular pool of calcium channels in the bag cell neurons of Aplysia californica.

To study the molecular mechanism of calcium current modulation in the bag cell neurons of Aplysia californica, we have identified calcium channel subtypes expressed in these cells and analyzed their distribution using channel-specific antibodies. Using PCR to amplify reverse-transcribed RNA from bag cell clusters, we identified two classes of calcium channel alpha1 subunit. One, BCCa-I, belongs to the ABE subfamily of calcium channels, whereas the other, BCCa-II, belongs to the SCD subfamily. Antibodies generated against the bag cell calcium channels recognize membrane proteins of approximately 210 and 280 kDa on immunoblots. Both channels are expressed in the bag cell clusters as well as in other parts of the Aplysia nervous system. BCCa-II also localizes to glia and muscle. The subcellular distribution of the two channel types is strikingly different. Antibody staining of bag cell neurons in primary culture shows that BCCa-II is present on the plasma membrane, whereas BCCa-I has a punctate, intracellular distribution consistent with a vesicular localization. The BCCa-I-containing vesicles are found in bag cell neuron somata and growth cones and occasionally in neuritic hotspots. Their distribution is similar but not identical to that of LysoTracker Red, a marker for acidic organelles, but unlike that of dense-core vesicles containing egg-laying hormone. The vesicular channels may represent the protein kinase C-sensitive calcium channels of bag cell neurons that are believed to enhance hormonal release during electrical activity.

Amino Acid Sequence↗
PubMed1997

Regulation by insulin of a unique neuronal Ca2+ pool and of neuropeptide secretion.

The insulin receptor is a tyrosine kinase receptor that is found in mammalian brain and at high concentrations in the bag cell neurons of Aplysia. We show here that insulin causes an acute rise in intracellular Ca2+ concentration ([Ca2+]i) in these neurons and triggers release of neuropeptide. The insulin-sensitive intracellular Ca2+ pool differs pharmacologically from previously described Ca2+ stores that are sensitive to inositol trisphosphate and from mitochondrial Ca2+ stores. Insulin, but not thapsigargin, stimulates Ca2+ release at the distal tips of neurites, the presumed site of neuropeptide secretion. The effects of insulin on intracellular Ca2+ release and neuropeptide secretion occur without triggering spontaneous action potentials. The insulin-sensitive rise in [Ca2+]i moves into the distal tips of neurites after exposure to a cyclic AMP analogue, a treatment that causes a similar translocation of neuronal vesicles. Our data indicate that Ca2+ release from a distinct intracellular pool associated with secretory vesicles may contribute to secretion of neuropeptide in the absence of neuronal discharge.

Animals↗