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L K Kaczmarek

Publications and source records attributed to L K Kaczmarek.

At least 91 records · Page 5Linked to original sources
PubMed1991

Voltage-dependent potassium channels: minK and Shaker families.

In the last 4 years, the molecular identity of several types of voltage-dependent potassium channels has been discovered. These include channels that terminate action potentials and control repetitive neuronal firing, as well as channels whose biological role is not yet understood. The majority of these are encoded by genes related to the Drosophila Shaker gene. The large number of genes comprising the Shaker gene family, coupled with the existence of different channels that result from alternatively spliced messages from the same gene, provide both vertebrates and invertebrates with a wide selection of channels whose voltage-dependence and kinetics can be tailored to the needs of a specific cell. Mutagenesis experiments on such channels are providing new information on those regions of the protein that govern essential aspects of channel activity, such as gating by voltage and ion permeation. Another gene, unrelated to the Shaker family, encodes a voltage-dependent potassium channel that activates much more slowly than the Shaker channels. This has been termed the MinK channel.

Animals↗
PubMed1990

Control of potassium currents and cyclic AMP levels by autoactive neuropeptides in Aplysia neurons.

The bag cell neurons of Aplysia are capable of generating an afterdischarge, which, in vivo, triggers egg-laying behavior. Pharmacologic elevation of cyclic AMP levels in isolated bag cell neurons has been shown to initiate repetitive firming similar to that seen during an afterdischarge, and to decrease outward currents measured under voltage-clamp. We have now examined the effects of three autoactive neuropeptides, alpha-, beta-, and gamma-bag cell peptide (BCP), on cyclic AMP levels and voltage-dependent potassium currents in these neurons. Previous work has shown that alpha-BCP lowers cyclic AMP levels in intact clusters of bag cell neurons. We have found that beta-BCP elevates cyclic AMP levels, whereas gamma-BCP, like alpha-BCP, lowers cyclic AMP levels. We used whole cell patch clamp technique to determine the effects of the peptides on the delayed voltage-dependent potassium currents in isolated bag cell neurons. As one would predict from their effects on cyclic AMP levels, beta-BCP decreased the amplitude of the delayed potassium currents whereas both alpha- and gamma-BCP increased the amplitude of these currents. In contrast, no consistent effects of these peptides on the transient voltage-dependent potassium current (A-current) were seen in these cells. Our results suggest that these three autoactive peptides may contribute to changes in second messengers and ionic currents during a bag cell afterdischarge.

Amino Acid Sequence↗
PubMed1990

Transfection of activated ras into an excitable cell line (AtT-20) alters tetrodotoxin sensitivity of voltage-dependent sodium current.

The sensitivity of voltage-dependent sodium current to the sodium channel blocker tetrodotoxin (TTX) is altered by transfection of a c-Ha-ras oncogene into an excitable cell line. Control AtT-20 cells, a cell line derived from a mouse anterior pituitary tumor, were found to express both a TTX-sensitive and a TTX-resistant sodium current. AtT-20 cells transfected with the c-Ha-ras gene expressed only a TTX-sensitive current. Properties of TTX-sensitive and -resistant currents were also examined. No differences in voltage dependence of activation or inactivation between the TTX-sensitive and -resistant currents were observed. The rate of inactivation of the TTX-resistant current in control cells was slower, than that of the TTX-sensitive current in either control or ras-transfected AtT-20 cells.

Dose-Response Relationship, Drug↗
PubMed1990

Cyclic AMP regulates processing of neuropeptide precursor in bag cell neurons of Aplysia.

The stimulation of a prolonged afterdischarge of action potentials in the bag cell neurons of Aplysia is accompanied by an elevation of cAMP levels in these cells. Such a discharge causes the release of egg-laying hormone (ELH) and several other neuroactive peptides, which are derived from a 32-kDa protein prohormone. We have examined the relationship between the elevation of cAMP levels and the processing of the 32-kDa ELH prohormone. The ELH prohormone was radiolabeled in bag cell clusters by incubation of abdominal ganglia in [3H]leucine and identified on SDS-PAGE by its specific localization to bag cell neurons and its immunoreactivity with antisera to ELH. After labeling the prohormone, further incorporation of [3H]leucine was blocked using either the protein synthesis inhibitor anisomycin or an excess of unlabeled leucine. The stimulation of an afterdischarge, or treatment of cells with the adenylate cyclase activator forskolin or a membrane permeant cAMP analog, resulted in the loss of radiolabeled 32-kDa ELH prohormone relative to that in control clusters. In the presence of tetrodotoxin (TTX), which prevents discharges and stimulation-evoked secretion in the bag cell neurons, forskolin also caused the depletion of labeled ELH prohormone, suggesting that secretion per se is not likely to be required for this effect. The decrease in intensity of the 32-kDa band was accompanied by an increase in a 29-kDa band within the somata. Occasionally, an increase in a group of faint bands with approximate Mr of 26-kDa was observed. Comparative peptide mapping indicated that the 29-kDa protein is likely to be derived from the 32-kDa ELH prohormone. Our findings suggest that elevations of cAMP accelerate and possibly alter the pattern of, processing of the 32-kDa ELH prohormone.

Action Potentials↗
PubMed1990

Estrogen induction of a small, putative K+ channel mRNA in rat uterus.

Estrogen causes dramatic long-term changes in the activity of the uterus. Here we report the molecular cloning of a small (700 base) uterine mRNA species capable of inducing a slow K+ current in Xenopus oocytes. The 130 amino acid protein encoded by this mRNA species has a predicted structure that does not resemble that of previously described voltage-dependent channels from mammalian sources. It is, however, similar to structural motifs found in certain prokaryotic ion channels. The induction of this mRNA by estrogen is rapid; this uterine mRNA species is not detectable in uteri from estrogen-deprived rats, but is substantially induced after 3 hr of estrogen treatment. These results support a critical role for regulation of ion channel expression by estrogen in the uterus.

Amino Acid Sequence↗
PubMed1990

Progressive potentiation of peptide release during a neuronal discharge.

1. In response to electrical stimulation, the bag cell neurons of Aplysia generate an afterdischarge that lasts 20-40 min. During this afterdischarge several neuroactive peptides are released. We have now studied the time course of release of two of these peptides, egg-laying hormone (ELH) and acidic peptide (AP). For the collection of released peptides, the artery to the bag cell clusters was perfused. The medium surrounding the clusters (artificial seawater, ASW) was completely exchanged at 5-min intervals before, during, and after stimulation of an afterdischarge. Peptides released into the external medium were analyzed with the use of high-pressure liquid chromatography. 2. Before stimulation, no detectable ELH and AP were found in the external medium. After the onset of an afterdischarge, the amount of ELH and AP released increased progressively until 15-20 min of firing. Toward the conclusion of an afterdischarge, the release of ELH and AP returned to control levels. 3. In contrast to the pattern of release of the peptides, the firing rate of the bag cell neurons is maximal within the first minute of afterdischarge and thereafter declines. 4. Release of the peptides from axonal varicosities occurs within the vascularized connective-tissue sheath that covers the clusters of bag cell neurons. Experiments were therefore carried out to establish whether the observed time course of release is affected by diffusion of the peptides through the vasculature into the external medium and, in particular, to determine whether the maximal rate of release at 15-20 min into the afterdischarge could be accounted for by a delay in transport of peptides from the neurites.(ABSTRACT TRUNCATED AT 250 WORDS)

Action Potentials↗
PubMed1989

The bag cell neurons of Aplysia. A model for the study of the molecular mechanisms involved in the control of prolonged animal behaviors.

Egg laying in Aplysia involves a well-characterized series of behaviors that can last for several hours. The behaviors are controlled by two bilateral clusters of peptidergic neurons in the abdominal ganglion. Following brief stimulation, these neurons, which have been termed the bag cell neurons, undergo a sequence of changes in their excitability lasting many hours. The bag cell neurons have served as a model system for studying the molecular mechanisms involved in the synthesis, processing, and release of neuroactive peptides and in the regulation of prolonged changes in neuronal excitability.

Animals↗
PubMed1989

Protein kinase inhibitors selectively block phorbol ester- or forskolin-induced changes in excitability of Aplysia neurons.

Exposure of the bag cell neurons of Aplysia to activators of protein kinase C, such as phorbol esters, enhances electrically evoked action potentials by increasing the voltage-dependent calcium current. We have hypothesized that this effect is mediated by the activation of protein kinase C (PKC). An important prediction of this hypothesis is that inhibitors of PKC should inhibit these phorbol ester-induced changes in bag cell neuronal excitability. We have now found that treatment of bag cell neurons with the protein kinase inhibitor 1-[5-isoquinolinesulfonyl]-2-methyl piperazine (H-7) inhibits the phorbol ester-induced enhancement of bag cell action potentials and prevents the enhancement of calcium current by phorbol esters. The height and width of electrically evoked action potentials in bag cell neurons can also be enhanced by cAMP analogs or agents that elevate cAMP. These agents do not influence the major voltage-dependent calcium current in the bag cell neurons but may act by modulating potassium currents and other voltage-dependent currents. We have found that microinjection of a protein inhibitor of cAMP-PK (PKA-I) into isolated bag cell neurons prevents and reverses the effect of the adenylate cyclase activator forskolin on action potentials of these cells. In contrast, H-7 does not inhibit the effects of forskolin on a variety of responses in these cells, including its effects on action potentials, granule movement, and 32P incorporation into phosphoproteins. This suggests that H-7 is selective for PKC relative to cAMP-PK in intact bag cell neurons.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1989

Inhibitors of protein kinase C prevent enhancement of calcium current and action potentials in peptidergic neurons of Aplysia.

Following brief stimulation of an afferent pathway, the bag cell neurons of Aplysia undergo a dramatic change in excitability, resulting in a prolonged discharge of spontaneous action potentials. During the discharge, the action potentials of the bag cell neurons become enhanced in height and width. The afterdischarge triggers release of neuroactive peptides that initiate egg-laying behavior in this animal. Evidence suggests that changes in excitability of the bag cell neurons may be mediated by activation of protein kinase C (PKC) and cAMP-dependent protein kinase (cAMP-PK). PKC activators, such as the phorbol ester TPA (12-O-tetradecanoyl-13-phorbol acetate), enhance the amplitude of action potentials in isolated bag cell neurons in cell culture. These agents act by unmasking a previously covert species of voltage-dependent calcium channel resulting in an increase in calcium current. In the accompanying paper (Conn et al., 1989), we showed that H-7, a protein kinase inhibitor, inhibits the effect of TPA, and is a selective inhibitor of PKC relative to cAMP-PK in these cells. We now report that another PKC inhibitor, sphinganine, also inhibits the effect of TPA on action potential height and calcium current in cultured bag cell neurons, and that N-acetylsphinganine, an inactive sphinganine analog, fails to inhibit the effects of PKC activators. Although both H-7 and sphinganine prevent the effects of TPA when added prior to TPA addition, neither compound reverses the effects of TPA when added after the action potentials have already become enhanced by TPA.(ABSTRACT TRUNCATED AT 250 WORDS)

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1988

Inositol trisphosphate releases intracellularly stored calcium and modulates ion channels in molluscan neurons.

Stimulation of the bag cell neurons of Aplysia triggers a long-lasting afterdischarge in these cells. In vivo, such a discharge causes the onset of a sequence of reproductive behaviors. We have found that treatments that trigger discharges in vitro stimulate the hydrolysis of phosphoinositides in the bag cell neurons, as measured by increased incorporation of 3H-inositol into fractions containing membrane lipids and water-soluble inositol phosphates. The electrophysiological effects of inositol trisphosphate, one of the products of phosphoinositide turnover that has been shown to mobilize intracellular calcium in non-neuronal cells, were investigated using isolated bag cell neurons in cell culture. Microinjection of inositol trisphosphate into cultured bag cell neurons caused a transient hyperpolarization of the membrane (approximately 35 sec), together with an increase in conductance. This effect of inositol trisphosphate was abolished by 50 mM tetraethylammonium ions. Inositol trisphosphate also reduced the amplitude of action potentials. Injection of calcium ions directly into bag cell neurons mimicked these responses seen after inositol trisphosphate injection. Using the cell-attached patch-clamp technique in conjunction with inositol trisphosphate microinjection, we observed that inositol trisphosphate evoked increases in the activity of a channel carrying outward current at the resting potential and more positive potentials. The estimated slope conductance of the channel modulated by inositol trisphosphate was approximately 40 pS, and its reversal potential was close to that predicted for potassium ions. The increased opening of this channel in response to inositol trisphosphate injection appeared to result from a transient shift of its voltage-dependence to more negative potentials. In a few cases, inositol trisphosphate injection also elicited an increase in the activity of a channel passing inward current at rest. Direct measurements of changes in intracellular calcium in response to inositol trisphosphate were made using digital imaging of isolated neurons loaded with the fluorescent calcium indicator fura-2. These revealed that injection of inositol trisphosphate significantly elevated intracellular calcium levels, and that this inositol trisphosphate-induced rise in cytosolic calcium was not affected by removal of extracellular calcium. In contrast to the effects of trains of action potentials in calcium-containing media, which produced increases in calcium primarily in neurites, the inositol trisphosphate-induced elevation of calcium appeared more localized to the somata of these neurons.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1987

Xenopus oocytes injected with rat uterine RNA express very slowly activating potassium currents.

Under the influence of estrogen, uterine smooth muscle becomes highly excitable, generating spontaneous and prolonged bursts of action potentials. In a study of the mechanisms by which this transition in excitability occurs, polyadenylated RNA from the uteri of estrogen-treated rats was injected into Xenopus oocytes. The injected oocytes expressed a novel voltage-dependent potassium current. This current was not observed in oocytes injected with RNA from several other excitable tissues, including rat brain and uterine smooth muscle from ovariectomized rats not treated with estrogen. The activation of this current on depolarization was exceptionally slow, particularly for depolarizations from relatively negative membrane potentials. Such a slowly activating channel may play an important role in the slow, repetitive bursts of action potentials in the myometrium.

Animals↗
PubMed1987

Cyclic AMP induces changes in distribution and transport of organelles within growth cones of Aplysia bag cell neurons.

This report examines cAMP-induced regulation of directed organelle transport in bag cell neuron growth cones using video-enhanced differential interference contrast (DIC) microscopy (Allen et al., 1981; Inoue, 1981) and digital image analysis techniques. Under control conditions, organelle transport is evident in the central cytoplasmic regions of bag cell neuron growth cones, but not in lamellae. Motility of lamellae takes the form of slow (less than 0.01 micron/sec) extension of margins and ruffling motions that propagate as waves (velocity, approximately 0.07 micron/sec) in a retrograde direction. Application of forskolin and a phosphodiesterase (PDE) inhibitor at concentrations known to induce changes in bag cell protein phosphorylation resulted in (1) rapid extension of directed organelle transport into lamellae, and (2) inhibition of the retrograde ruffling waves. These changes effected transformation of lamellae into neurite endings packed with microtubules and organelles, a large proportion of which appeared to be neurosecretory granules. The effects were reversible, dose-dependent, potentiated by a variety of PDE inhibitors, and mimicked by 6-N-butyl-8-benzyl-thio-cAMP (BT-cAMP). Though forskolin may normally promote depolarization and Ca entry, these changes in growth cone structure are not secondary to influx of external Ca, as they persist in Ca-free/EGTA solutions; furthermore, they do not resemble the effects of depolarization induced by perfusion with elevated K solutions. The cAMP-induced changes in growth cone morphology that we report here suggest a possible role for protein phosphorylation in promoting growth cone differentiation and structural changes accompanying secretion.

Animals↗
PubMed1987

Alpha bag cell peptide directly modulates the excitability of the neurons that release it.

Brief electrical or hormonal stimulation of the bag cell neurons of Aplysia triggers a long-lasting discharge during which alpha bag cell peptide (alpha-BCP) and other neuropeptides are released from the cells. We have carried out experiments, using both intact abdominal ganglia and isolated neurons, demonstrating that alpha-BCP acts directly on the bag cell neurons to influence cAMP levels and voltage-dependent potassium currents. Exposure of the bag cell neurons within intact ganglia to alpha-BCP, at concentrations greater than 1 nM, inhibited an ongoing discharge. alpha-BCP also significantly reduced both basal and forskolin-stimulated levels of cAMP in bag cell clusters. The inhibition of the discharge by alpha-BCP could be prevented and reversed by pharmacological elevation of intracellular cAMP levels. Immunohistochemical staining of neurons maintained in cell culture showed that all isolated bag cell neurons exhibit immunoreactivity with antisera against alpha-BCP. Application of the adenylate cyclase activator forskolin to such isolated cells, in the presence of a phosphodiesterase inhibitor, attenuates the amplitude of the delayed voltage-dependent outward currents measured in voltage-clamp experiments. Pretreatment of the cells with alpha-BCP significantly reduced the ability of forskolin to attenuate these currents, demonstrating that alpha-BCP acts directly at autoreceptors on bag cell neurons. Experiments with the isolated cells showed that a second autoreceptor-mediated effect of alpha-BCP was the enhancement of an inwardly rectifying potassium current that was activated at potentials more negative than -40 mV. The reversal potential and conductance of the current induced by alpha-BCP were dependent on the external K+ concentration. This response to alpha-BCP could be blocked by rubidium, cesium, and barium ions. Our data demonstrate that alpha-BCP can exert inhibitory biochemical and electrophysiological actions on the bag cell neurons that release it and suggest that autoreceptors for alpha-BCP play an important role in the termination of a discharge in the bag cell neurons.

Adenylate Cyclase Toxin↗