Biomedical subjects

L K Kaczmarek

Publications and source records attributed to L K Kaczmarek.

At least 55 records · Page 3Linked to original sources

PubMed1997

Giga-ohm seals on intracellular membranes: a technique for studying intracellular ion channels in intact cells.

A method is outlined for obtaining giga-ohm seals on intracellular membranes in intact cells. The technique employs a variant of the patch-clamp technique: a concentric electrode arrangement protects an inner patch pipette during penetration of the plasma membrane, after which a seal can be formed on an internal organelle membrane. Using this technique, successful recordings can be obtained with the same frequency as with conventional patch clamping. To localize the position of the pipette within cells, lipophilic fluorescent dyes are included in the pipette solution. These dyes stain the membrane of internal organelles during seal formation and can then be visualized by video-enhanced or confocal imaging. The method can detect channels activated by inositol trisphosphate, as well as other types of intracellular membrane ion channel activity, and should facilitate studies of internal membranes in intact neurons and other cell types.

Animals↗

PubMed1997

Properties and regulation of the minK potassium channel protein.

The minK gene encodes a protein of 130 amino acids that has a single transmembrane segment and is expressed in many tissues including heart, uterus, and kidney. When Xenopus oocytes are injected with minK mRNA, a very slowly activating voltage-dependent potassium current is induced in these cells. The induced channels appear to result from the interaction of the minK protein with other channel-forming subunits such as the KvLQT1 channel. The minK protein is intimately associated with the structure of the resultant channels, and mutations in minK can alter ion selectivity and modulation by second messengers. Strong candidates for native currents regulated by the minK protein include the slow component of the cardiac delayed rectifier and potassium currents recorded across epithelial cells in vestibular organs and cochlea.

Amino Acid Sequence↗

PubMed1996

Ionic currents underlying developmental regulation of repetitive firing in Aplysia bag cell neurons.

We have investigated the developmental regulation of the ability to fire repetitively in the bag cell neurons of Aplysia californica, a neuronal system in which the behavioral effects of repetitive firing are well characterized. Adult bag cell neurons exhibit an afterdischarge, consisting of prolonged depolarization and repetitive firing, which causes the release of several peptides from these neurons that induce egg-laying behaviors. Afterdischarge can be triggered in vitro by a variety of stimuli, including electrical stimulation and exposure to the potassium channel blocker tetraethyl ammonium chloride (TEA). In contrast to adults, juvenile neurons did not exhibit afterdischarge in response to pleural-abdominal connective shock or TEA. Juvenile neurons did exhibit, however, prolonged depolarizations in the presence of TEA, perhaps reflecting the anlage of the mechanism responsible for afterdischarge in the adult. To investigate developmental mechanisms underlying the regulation of repetitive firing, we compared ionic currents in adult and juvenile bag cell neurons. We found that during the period in which these neurons acquire the capacity to fire repetitively, a number of currents are regulated: (1) three K+ currents decrease (Ca2+)-dependent K+ and two components of voltage-dependent delayed-rectifier K+ current); (2) A-type K+ current increases; and (3) two Ca2+ currents increase (basal and PKC-activated). This pattern is consistent with the increase in the ability to fire repetitively that we observe during maturation: our results indicate that developmental control of repetitive firing in this system is accompanied by selective regulation of specific ionic currents which, after maturation, play important roles in generating the afterdischarge and triggering egg-laying behaviors.

Animals↗

PubMed1996

Ca2+ influx and activation of a cation current are coupled to intracellular Ca2+ release in peptidergic neurons of Aplysia californica.

1. Stimulation of inputs to bag cell neurons in the abdominal ganglion of Aplysia californica causes an increase in their intracellular Ca2+ concentration ([Ca2+]i). We have used thapsigargin, a specific inhibitor of the endoplasmic reticulum Ca2+ pump, to analyse the effects of Ca2+ released from intracellular stores on the electrophysiological responses of bag cell neurons. 2. Using digital imaging of fura-2-loaded isolated bag cell neurons we found that thapsigargin rapidly evoked an increase in [Ca2+]i in somata, with smaller increases in neurites. Thapsigargin-induced elevation of [Ca2+]i peaked at about 1 microM within 5-10 min and then decayed to basal levels by 30 min. 3. Placement of an extracellular vibrating Ca(2+)-selective microelectrode to within 1 micron of somata revealed a relatively large steady-state Ca2+ efflux. Thapsigargin produced a rapid increase in Ca2+ influx. Changes in Ca2+ flux were not detected at neurites. 4. Thapsigargin produced a small depolarization in isolated bag cell neurons in artificial sea water (ASW). Sometimes enhanced depolarizations were observed when extracellular Na+ was replaced by TEA or Tris, but not N-methyl-D-glucamine (NMDG). The depolarization was not blocked by 100 microM tetrodotoxin (TTX), removal of extracellular Ca2+ (0.5 mM EGTA) or addition of 10 mM Co2+ to the bath solution. 5. In voltage-clamp experiments, thapsigargin induced an inward current (ITg) that was recorded in Ca(2+)-free media containing TEA or Tris substituted for Na+. The apparent reversal potential of ITg was -16.8 +/- 1.2 mV in TEA-ASW. Induction of ITg was inhibited in neurons that were microinjected with the Ca2+ chelator BAPTA-Dextran70 or treated with the membrane-permeant analogue BAPTA AM. Activation of ITg was not observed when Na+ was replaced with NMDG. Manipulation of [Na+]o and [K+]o produced shifts in the reversal potential of ITg consistent with the underlying channels being permeable to both Na+ and K+. 6. Thapsigargin did not alter the amplitude or kinetics of voltage-activated Ba2+ currents, but in some experiments it did increase the amplitude of a component of outward K+ current. 7. Thapsigargin neither induced bag cell neurons within the intact ganglion to depolarize and fire spontaneously, nor did it alter the frequency or duration of firing of an electrically stimulated bag cell after-discharge. 8. We conclude that thapsigargin-sensitive Ca2+ pools are present predominantly in the somata of bag cell neurons. Ca2+ that is released from thapsigargin-sensitive Ca2+ stores activates a non-selective cation current that may help sustain depolarization of the somata, but does not by itself trigger an after-discharge.

Animals↗

PubMed1996

Identification and characterization of a Ca(2+)-sensitive nonspecific cation channel underlying prolonged repetitive firing in Aplysia neurons.

The afterdischarge of Aplysia bag cell neurons has served as a model system for the study of phosphorylation-mediated changes in neuronal excitability. The nature of the depolarization generating the afterdischarge, however, has remained unclear. We now have found that venom from Conus textile triggers a similar prolonged discharge, and we have identified a slow inward current and corresponding channel, the activation of which seems to contribute to the onset of the discharge. The slow inward current is voltage-dependent and Ca(2+)-sensitive, reverses at potentials slightly positive to O mV, exhibits a selectivity of K approximately equal to Na >> Tris > N-methyl-D-glucamine (NMDG), and is blocked by high concentrations of tetrodotoxin. Comparison of these features with those observed in channel recordings provides evidence that a Ca(2+)-sensitive, nonspecific cation channel is responsible for a slow inward current that regulates spontaneous repetitive firing and suggests that modulation of the cation channel underlies prolonged changes in neuronal response properties.

Animals↗

PubMed1996

Cloning and characterization of the promoter for a potassium channel expressed in high frequency firing neurons.

The Kv3.1 potassium channel is expressed in neurons that generate trains of high frequency action potentials in response to synaptic inputs. To understand the mechanisms underlying the regulation and restricted expression pattern of the Kv3.1 gene, we have cloned and characterized its promoter. We first isolated a 5.3-kilobase pair fragment of the Kv3.1 5'-flanking region. When linked to the chloramphenicol acetyltransferase reporter gene, this fragment was found to be active in the undifferentiated PC12 cell line, a neuron-like cell line, but not in a fibroblast cell line. By carrying out a series of deletion analyses in undifferentiated PC12 cells, we have localized the essential promoter region to a highly GC-rich region containing four Sp-1 binding sites. Similar deletion analysis in NIH3T3 cells suggests that multiple silencing elements and enhancing element(s) are involved in the cell type-specific expression of this gene. Further regulatory elements, including one cyclic AMP/calcium response element (CRE) and one Ap-1 element were found in the upstream region of the promoter. Using a stable undifferentiated PC12 cell line transfected with the Kv3.1 5'-flanking region, we determined that promoter activity is enhanced by a cAMP analog and a calcium ionophore. Deletion of the CRE-like element at position -252 eliminated the enhancement of promoter activity by cAMP, and mobility shift assays confirmed that the Kv3.1 CRE sequence binds both a nuclear factor in undifferentiated PC12 cells and recombinant CRE binding protein. Our results suggest that the transcription of the Kv3.1 channel may be regulated by neurotransmitters that elevate cAMP levels in neurons.

3T3 Cells↗

PubMed1996

Insulin receptor in Aplysia neurons: characterization, molecular cloning, and modulation of ion currents.

We have isolated the cDNA for a tyrosine kinase receptor that is expressed in the nervous system of Aplysia californica and that is similar to the vertebrate insulin receptor. Binding studies and immunocytochemical staining show that the receptor is abundant in the bag cell neurons. Application of vertebrate insulin to clusters of bag cell neurons stimulates the phosphorylation of the receptor on tyrosine residues, and exposure of isolated bag cell neurons to insulin produces an increase in height and a decrease in duration of the action potentials that can be detected within 15-30 min. These effects were not seen with insulin-like growth factor-1. In voltage-clamped neurons, insulin produces an increase in the amplitude of the voltage-dependent Ca2+ current that can be blocked by preincubation with herbimycin A, an inhibitor of tyrosine kinases. Insulin also enhances a delayed K+ current. We suggest that insulin-like peptides regulate the excitability of the bag cell neurons.

Action Potentials↗

PubMed1996

Manipulation of the delayed rectifier Kv1.5 potassium channel in glial cells by antisense oligodeoxynucleotides.

Glial cells have been shown to express several biophysically and pharmacology distinct potassium channel types. However, the molecular identity of most glial K+ channels is unknown. We have developed an antibody specific for the Shaker type potassium channel Kv1.5 protein, and demonstrate by immunohistochemistry the presence of this channel in glial cells of adult rat hippocampal and cerebellar slices, as well as in cultured spinal cord astrocytes. Immunoreactivity was particularly intense in the endfoot processes of astrocytes surrounding the microvasculature of the hippocampus. The specific contribution of this channel protein to the delayed rectifying K+ current of spinal cord astrocytes was determined by incubating these cells with antisense oligodeoxynucleotides complementary to the mRNA coding for Kv1.5 protein. Such treatment reduced delayed rectifier current density and shifted the potassium current steadystate inactivation, without altering current activation, cell capacitance, or cell resting potential. The tetraethylammonium acetate (TEA) sensitivity of astrocytic delayed rectifier current was enhanced following antisense oligodeoxynucleotide treatment, suggesting that Kv1.5 channel protein may provide a significant component of the TEA-insensitive current in this preparation. Our results suggest that Kv1.5 is widely expressed in glial cells of brain and spinal cord and that delayed rectifying K+ currents in astrocytes are largely mediated by Kv1.5 channel protein.

Animals↗

PubMed1996

Inhibition by nystatin of Kv1.3 channels expressed in Chinese hamster ovary cells.

The patch-clamp technique was used to study the effects of nystatin on a cloned delayed rectifier potassium channel (Kv1.3) expressed in Chinese hamster ovary (CHO) cells. Kv1.3 currents recorded in the whole-cell configuration, using an intracellular solution containing nystatin, were subjected to a time- and concentration-dependent reduction in their amplitude and in the time constants of apparent inactivation. Direct application of nystatin to the cytoplasmic side of excised inside-out patches inhibited Kv1.3 currents and this inhibition was immediately reversible upon washout of the drug. In contrast, currents mediated by another delayed rectifier (Kv3.1) were not affected by this drug. The concentrations for nystatin and its structural analog, amphotericin B, required to produce half maximal inhibition (IC50) of the current were estimated to be about 3 and 60 microM, respectively. The effects of nystatin on the amplitude and inactivation of Kv1.3 currents were not voltage-dependent. In inside-out patches, tetraethylammonium (TEA) produced a rapid block of Kv1.3 currents upon the onset of a voltage pulse, while the inhibition by nystatin developed slowly. When co-applied with TEA, nystatin potentiated the extent of the TEA-dependent block, and the kinetic effect of nystatin was slowed by TEA. In summary, nystatin, a compound frequently used in perforated patch recordings to preserve intracellular dialyzable components, specifically inhibited the potassium channel Kv1.3 at concentrations well below those required for perforation. The site of this inhibition may be different from that for TEA and is readily accessible from the cytoplasmic side of the membrane.

Amphotericin B↗

PubMed1996

Regulation of potassium channels by protein kinases.

Studies of the role of protein phosphorylation in the modulation of neuronal excitability are beginning to identify specific sites on ion channels that are substrates for serine/threonine kinases and that contribute to short-term and long-term regulation of current amplitude and kinetics. In addition, it is becoming apparent that phosphorylation of tyrosine residues may produce acute changes in the characteristics of ion channels. These recent findings are best illustrated by examining the Shaker superfamily of potassium channels.

Animals↗

PubMed1996

Developmental dissociation of excitability and secretory ability in Aplysia bag cell neurons.

1. Despite the considerable progress made in understanding the role of electrical activity in triggering secretion, the developmental relationships between excitability and secretion are not well understood. The well-characterized bag cell neurons of Aplysia provide an advantageous system in which to investigate developmental interactions of these two key properties of neurons. 2. A prolonged afterdischarge triggers egg laying hormone (ELH) secretion in mature bag cell neurons. To investigate secretion in the developmental framework of excitability, we first examined whether immature neurons, which are incapable of the mature form of excitability (afterdischarge), contain ELH and whether this hormone is packaged in vesicles. We used immunoelectron microscopy to compare vesicular localization of ELH and to compare the size and density of ELH-containing vesicles in neurons from adult and juvenile Aplysia. This comparison revealed that immature neurons contain ELH in vesicles in the size range of secretory vesicles. However, they lack a class of large vesicles (> 250 nm in diameter) that is characteristic of mature neurons. 3. To investigate whether the ELH contained in immature bag cell neurons could be secreted in response to electrical activity, we used the potassium channel blocker tetraethylammonium (TEA) combined with nerve stimulation to depolarize neurons from both juvenile animals (ovotestes do not contain eggs) and from adult Aplysia (ovotestes contain eggs). Using radioimmunoassay, we have found that the duration and amount of ELH secreted from bag cell neurons from juvenile Aplysia in response to TEA does not depend on whether or not the cells can be induced to afterdischarge, and the amount and duration of ELH secreted from bag cell neurons of juvenile Aplysia (whether or not they afterdischarged) differed from those secreted by adult neurons. However, by normalizing for body size, we found that the final estimated hemolymph concentration of ELH would be similar in juvenile and adult animals. 4. We investigated the potential functional significance of secretion of bag cell hormones in juvenile Aplysia by attempting to bypass the bag cell neurons and directly activate downstream elements with extract from adult bag cell neurons (BCE), known to contain ELH and other peptides. We found that juvenile Aplysia exhibit at least one component of egg-laying behavior, cessation of locomotion, in response to BCE during a developmental period (as measured by weight) in which they normally would possess neurons incapable of afterdischarge. Thus developmental regulation of excitability in the bag cell neurons may prevent inappropriate hormone release and subsequent premature expression of reproductive behaviors.

Animals↗

PubMed1995

A new family of outwardly rectifying potassium channel proteins with two pore domains in tandem.

Potassium channels catalyse the permeation of K+ ions across cellular membranes and are identified by a common structural motif, a highly conserved signature sequence of eight amino acids in the P domain of each channel's pore-forming alpha-subunit. Here we describe a novel K+ channel (TOK1) from Saccharomyces cerevisiae that contains two P domains within one continuous polypeptide. Xenopus laevis oocytes expressing the channel exhibit a unique, outwardly rectifying, K(+)-selective current. The channel is permeable to outward flow of ions at membrane potentials above the K+ equilibrium potential; its conduction-voltage relationship is thus sensitive to extracellular K+ ion concentration. In excised membrane patches, external divalent cations block the channel in a voltage-dependent manner, and their removal in this configuration allows inward channel current. These attributes are similar to those described for inwardly rectifying K+ channels, but in the opposite direction, a previously unrecognized channel behaviour. Our results identify a new class of K+ channel which is distinctive in both its primary structure and functional properties. Structural homologues of the channel are present in the genome of Caenorhabditis elegans.

Amino Acid Sequence↗

PubMed1995

Elimination of potassium channel expression by antisense oligonucleotides in a pituitary cell line.

The clonal rat pituitary cell line GH4C1 expresses the genes for several voltage-dependent potassium channels including Kv1.5 and Kv1.4. Dexamethasone, a glucocorticoid agonist, induces a slowly inactivating potassium current in these cells but does not alter the amplitude of a rapidly inactivating component of potassium current. We have found that the induction of the slowly inactivating current can be blocked by an antisense phosphorothioate deoxyoligonucleotide to the Kv1.5 mRNA sequence. In contrast, antisense deoxyoligonucleotides against Kv1.4 mRNA specifically decrease the expression of the dexamethasone-insensitive rapidly inactivating current. These results demonstrate the usefulness of antisense oligonucleotides in correlating potassium currents with specific potassium channel proteins in the cell types in which they are naturally expressed.

Animals↗

PubMed1995

Electrophysiological and pharmacological characterization of a mammalian Shaw channel expressed in NIH 3T3 fibroblasts.

1. The Shaw-like voltage-activated potassium channel Kv3.1 is expressed in neurons that generate rapid trains of action potentials. By expressing this channel in a mammalian cell line and by simulating its activation, we tested the potential role of this channel in action potential repolarization. 2. NIH 3T3 fibroblasts were stably transfected with Kv3.1 DNA. Currents recorded in these cells had a threshold of activation at approximately -10 mV, showed little inactivation, and were very sensitive to blockade by 4-aminopyridine and tetraethylammonium. 3. Kv3.1 currents activated rapidly at the onset of depolarizing voltage pulses. After an initial rapid phase of activation, which could be fit by an n4 Hodgkin-Huxley model, Kv3.1 currents expressed in fibroblasts had a second, slower phase of activation, and, in some cells, a slower phase of partial inactivation, both of which could be fit with modified n4p models. 4. Cell-attached single-channel recordings indicated that the Kv3.1 channel displays two gating behaviors, a short-open-time pattern, which occurs only at the onset of depolarization, and a long-open-time pattern, which predominates during prolonged depolarizations. 5. The amplitude of Kv3.1 currents, and the probability of channel openings, was reduced by a phorbol ester activator of protein kinase C, and the action of this agent was blocked by preincubation with the protein kinase inhibitor H7 (1-[5-isoquinolinesulfonyl]-2-methyl piperazine). In contrast, the effects of dioctanoyl glycerol, which also attenuated the currents, could not be completely blocked by H7, suggesting that diacylglycerols may act on the channel by a kinase-independent pathway. 6. Incorporation of a current with the kinetics and voltage dependence of Kv3.1 currents into a model cell with a sustained inward current showed that, in contrast to other delayed-rectifier currents such as the Shaker-like Kv1.1 and Kv1.6 channels, the level of expression of Kv3.1 currents could be varied over a wide range without attenuation of action potential height. Our results suggest that the Kv3.1 channel may provide rapidly firing neurons with a high safety factor for impulse propagation.

Action Potentials↗

PubMed1995

Modulation of the inactivation of voltage-dependent potassium channels by cAMP.

Modulation of voltage-dependent potassium currents can alter the shape and timing of action potentials, thereby altering neurotransmitter release. To examine the effect of a cAMP analog on potassium currents in metabolically intact cells, perforated-patch and cell-attached patch recordings were carried out using the GH4C1 pituitary cell line. A major component of voltage-dependent potassium current in these cells inactivates slowly, with a time constant of several seconds. Application of dibutyryl cAMP decreased this current at voltages positive to -10 mV and increased the rate of inactivation by approximately twofold. Single channel recordings revealed two channel types whose voltage dependence and kinetics of inactivation match those of the macroscopic current. One of these, the smaller conductance (7.5 pS) channel, was sensitive to the cAMP analog, which decreased the latency of the channel to enter a long-lasting inactivated state. Ensemble averages of the activity of this channel showed that, consistent with its effect on the macroscopic current, dibutyryl cAMP increased its rate of inactivation. Somatostatin, an agent that is known to activate a serine/threonine phosphatase in these cells, completely reversed the effect of dibutyryl cAMP on the channel, while the cyclic GMP analog, dibutyryl cyclic GMP was without effect. In contrast, the rate of inactivation of the larger conductance (approximately 19 pS) channel was not accelerated by dibutyryl cAMP. These studies indicate that different channel subtypes expressed in a single cell respond differently to elevations of cAMP, and suggest that the overall response of potassium currents to second messengers may be determined by the ratio of different channel subtypes.

Animals↗

PubMed1994

A Shab potassium channel contributes to action potential broadening in peptidergic neurons.

We have cloned the gene for a potassium channel, Aplysia Shab, that is expressed in the bag cell neurons of Aplysia. The voltage dependence and kinetics of the Aplysia Shab current in oocytes match those of IK2, one of the two delayed rectifiers in these neurons. Like IK2, but in contrast with other members of the Shab subfamily, the Aplysia Shab current inactivates within several hundred milliseconds. This inactivation occurs by a process whose properties do not match those previously described for C-type and N-type mechanisms. Neither truncation of the N-terminus nor block by tetraethylammonium alters the time course of inactivation. By incorporating the characteristics of Aplysia Shab into a computational model, we have shown how this current contributes to the normal enhancement of action potentials that occurs in the bag cell neurons at the onset of neuropeptide secretion.

Action Potentials↗

PubMed1994

Cyclic AMP modulates fast axonal transport in Aplysia bag cell neurons by increasing the probability of single organelle movement.

Stimulation of Aplysia bag cell neurons triggers elevation of cAMP and prolonged secretion of ELH neuropeptide. Using video-enhanced microscopy to track individual organelle movements in bag cell neurons, we find that organelle translocation consists of periods of movement interrupted by stationary episodes. cAMP elevation leads to a 2- to 3-fold enhancement of the average rate of organelle transport in both anterograde and retrograde directions. This effect does not result from alteration of the instantaneous velocity of organelle transport along microtubules, but rather from an increase in the proportion of time individual organelles spend in motion. Biochemical measurements also provided evidence that cAMP elevation promotes ELH peptide translocation from the somata into axons. Enhanced transport of ELH as a result of these effects may contribute to the replenishment of neuropeptide-containing vesicles at release sites during prolonged periods of secretion.

Animals↗

PubMed1994

Autoactive peptides act at three distinct receptors to depolarize the bag cell neurons of Aplysia.

1. In response to brief stimulation of an afferent input the bag cell neurons of Aplysia depolarize by 15-20 mV and generate an afterdischarge that, in vitro, lasts for approximately 30 min. During the discharge these neurons secrete three small peptides [bag cell peptides (BCPs)], Ala-Pro-Arg-Leu-Arg-Phe-Tyr-Ser-Leu (alpha-BCP), Arg-Leu-Arg-Phe-His (beta-BCP), and Arg-Leu-Arg-Phe-Asp (gamma-BCP), that share a common core sequence and that have electrophysiological effects on the bag cell neurons themselves. We have now studied the action of these three peptides on bag cell neurons isolated in culture. All three peptides were found to be capable of producing a depolarization of these cells. 2. The ability of alpha-, beta-, and gamma-BCP to induce a depolarization in isolated bag cell neurons exhibits a seasonal variability. The response to the peptides is maximal from early summer through late fall and parallels the frequency of egg-laying in vivo. 3. The depolarization induced by alpha-, beta-, and gamma-BCP desensitizes with repeated application of peptide. Desensitization of the response to one peptide does not, however, prevent the response to application of one of the other two peptides. This suggests that separate autoreceptor populations exist for alpha-, beta-, and gamma-BCP. 4. As reported previously, desensitization of the depolarizing response to the peptides was also observed after preincubation of intact clusters of bag cell neurons with a high concentration of all three peptides.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗