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Publications and source records attributed to L K Kaczmarek.
The protein kinase inhibitor H-7 has been shown to prevent the potentiation of action potentials that normally accompanies an afterdischarge in the bag cell neurons of Aplysia. We have now shown that H-7 attenuates the release of ELH from these neurons during an afterdischarge without influencing the firing frequency or length of the afterdischarge.
The bag cell neurons of Aplysia provide a model system in which to investigate the effects of hyperosmolality on the electrical and secretory properties of neurons. Brief stimulation of these neurons triggers an afterdischarge of action potentials that lasts approximately 20-30 min, during which time they release several neuroactive peptides. We have found that pre-incubation of intact clusters of bag cell neurons in hyperosmotic media prior to stimulation prevents the initiation of afterdischarges. Furthermore, an increase in osmolality of the external medium during an ongoing afterdischarge causes its premature termination. Hyperosmotic media attenuate the release of peptide evoked by both electrically stimulated afterdischarges and potassium-induced depolarization. The ability of high potassium to depolarize the bag cell neurons is, however, not impaired. Exposure of isolated bag cell neurons to hyperosmotic media also inhibits the amplitude of action potentials evoked by depolarizing current injection and attenuates the voltage-dependent calcium current. In isolated bag cell neurons loaded with the calcium indicator dye, fura-2, hyperosmotic media reduced the rise in intracellular calcium levels that normally occurs in response to depolarization. Our results suggest that the effects of hyperosmotic media on peptide secretion in bag cell neurons can largely be attributed to their effects on calcium entry.
MinK is a novel protein which induces an extremely slowly activating potassium channel when expressed in Xenopus oocytes. We discuss the properties and regulation of the current and localization and possible physiological roles of the MinK protein.
Activation of protein kinase C (PKC) in Aplysia bag cell neurons causes the recruitment of voltage-dependent calcium channels. Using imaging techniques on isolated cells, we have now found that an activator of PKC, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), promotes the rapid appearance of new sites of calcium influx associated with a change in the morphology of neurite endings. In untreated cells, calcium influx triggered by action potentials occurs along neurites and in the central region of growth cones, but does not usually occur at the leading edge of lamellipodia. TPA produces extension of the lamellipodium, and action potentials now trigger calcium influx at the distal edge of the newly extended endings. Cotreatment with TPA and a cyclic AMP analog promotes movement of secretory organelles toward the new sites of calcium influx. Our results suggest that these second messenger systems promote the rapid formation of morphological structures that contribute to the potentiation of peptide release.
1. The gene for a mammalian Shaw K+ channel has recently been cloned and has been shown, by alternative splicing, to give rise to two different transcripts, Kv3.1 alpha and Kv3.1 beta. To determine whether these channels are associated with specific types of neurons and to determine whether or not the alternately spliced K+ channel variants are differentially expressed, we used ribonuclease (RNase) protection assays and in situ hybridization histochemistry to localize the specific subsets of neurons containing Kv3.1 alpha and Kv3.1 beta mRNAs in the adult and developing rat brain. 2. In situ hybridization histochemistry revealed a heterogeneous expression pattern of Kv3.1 alpha mRNA in the adult rat brain. Highest Kv3.1 alpha mRNA levels were expressed in the cerebellum. High levels of hybridization were also detected in the globus pallidus, subthalamus, and substantia nigra reticulata. Many thalamic nuclei, but in particular the reticular thalamic nucleus, hybridized well to Kv3.1 alpha-specific probes. A subpopulation of cells in the cortex and hippocampus, which by their distribution and number may represent interneurons, were also found to contain high levels of Kv3.1 alpha mRNA. In the brain stem, many nuclei, including the inferior colliculus and the cochlear and vestibular nuclei, also express Kv3.1 alpha mRNA. Low or undetectable levels of Kv3.1 alpha mRNA were found in the caudate-putamen, olfactory tubercle, amygdala, and hypothalamus. 3. Kv3.1 beta mRNA was also detected in the adult rat brain by both RNase protection assays and by in situ hybridization experiments. Although the beta splice variant is expressed at lower levels than the alpha species, the overall expression pattern for both mRNAs is similar, indicating that both splice variants co-expressed in the same neurons. 4. The expression of Kv3.1 alpha and Kv3.1 beta transcripts was examined throughout development. Kv3.1 alpha mRNA is detected as early as embryonic day 17 and then increases gradually until approximately postnatal day 10, when there is a large increase in the amount of Kv3.1 alpha mRNA. Interestingly, the expression of Kv3.1 beta mRNA only increases gradually during the developmental time frame examined. Densitometric measurements indicated that Kv3.1 alpha is the predominant splice variant found in neurons of the adult brain, whereas Kv3.1 beta appears to be the predominant species in embryonic and perinatal neurons. 5. Most of the neurons that express the Kv3.1 transcripts have been characterized electrophysiologically to have narrow action potentials and display high-frequency firing rates with little or no spike adaptation.(ABSTRACT TRUNCATED AT 400 WORDS)
When expressed in the Xenopus oocyte, the minK protein induces a slowly activating voltage-dependent potassium current (Isk). We studied the modulation of this current by altering intracellular cAMP levels and found that the amplitude of Isk is dramatically increased by treatments that raise cAMP levels and decreased by agents that lower cAMP levels. Preinjection of a protein inhibitor of the cAMP-dependent protein kinase blocked the effects of increased cAMP levels. There were no changes in the voltage dependence or kinetics of Isk. Mutations that eliminate a potential phosphorylation site on the minK protein did not block the effects of activating the kinase. In addition, the membrane capacitance of the oocyte increased and decreased in parallel with Isk. Our results fit a mechanism in which channel proteins are selectively inserted into and removed from the plasma membrane in response to changes in kinase activity.
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Expression of the EJ-ras oncogene in the AtT20 cell line results in several changes in their properties that correspond to a switch of these anterior pituitary-derived cells to a more neuronlike phenotype. The width of action potentials following transfection with ras is reduced 20-fold from over 200 msec in control AtT20 cells to less than 10 msec in ras-transfected cells. This is associated with a two- to threefold increase in the density of voltage-dependent potassium currents. In addition, the rate of inactivation of these currents is decreased approximately twofold in ras-transfected cells. At least part of the change in potassium current may be due to differential expression of potassium channel mRNAs. In the ras-transfected cells, mRNA species were detected using a probe for the voltage-dependent potassium channels, Kv4, a species that appears to be uniquely expressed in the nervous system, and NGK2, an alternatively spliced product transcribed from the same gene. These mRNAs are not detected in control AtT20 cells. The results suggest that the ras protein modulates the phenotype of excitable cells by influencing the expression of specific potassium channels and thereby altering the density and types of channels in the plasma membrane.
Chloride channels in the apical plasma membrane of cells in the dogfish rectal gland have served as a model system for the study of regulation of chloride flux by changes in intracellular cyclic AMP levels. Similar regulation by cyclic AMP has been described for channels in cells of human secretory epithelia where defective regulation by cyclic AMP-dependent protein phosphorylation is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). We have isolated a cDNA clone from the rectal gland encoding a protein that is 72% identical to the human CFTR. One of the major phosphorylation sites in CFTR is absent in the dogfish protein. The dogfish protein has, however, four additional putative substrate sites for the cyclic AMP-dependent protein kinase. A peptide antibody, which was raised against an amino acid sequence common to both the human and dogfish CFTR sequences, recognizes proteins with similar molecular masses (160 kDa) in the dogfish gland and in mammalian lung. Immunolocalization studies with this antibody show that the putative dogfish CFTR is localized to the apical membrane of cells lining the lumen of the rectal gland.
Following brief synaptic stimulation, the bag cell neurons in the abdominal ganglion of Aplysia undergo a series of changes in electrophysiological and secretory properties that triggers egg laying behavior. Activation of protein kinase C appears to play an important role in these changes and, in particular, causes the unmasking of a new species of voltage-dependent calcium channel. We have now used isolated bag cell neurons maintained in cell culture to study changes in protein phosphorylation that are induced by exposure to an activator of protein kinase C. Primary cultures of bag cell neurons were labeled with 32P orthophosphate and then incubated with either tetradecanoyl phorbol 13-acetate (TPA), a potent activator of protein kinase C, or with an inactive phorbol ester. When protein extracts were separated with 2D electrophoresis approximately 100 phosphoproteins could be distinguished. Only four of these proteins, with molecular weights of 20, 32, 200, and 250 kD, underwent a reproducible increase in the extent of phosphorylation of at least twofold in response to TPA. TPA-induced changes in phosphate incorporation were blocked by pretreatment with the protein kinase C inhibitor H7. One of the TPA-regulated phosphoproteins was localized in a plasma membrane-containing fraction and was sensitive to trypsin treatment of intact cells, suggesting that it is a membrane protein with sites exposed to the extracellular medium. Two of the other TPA-regulated phosphoproteins may be associated with the inner face of the plasma membrane. Our results indicate that only a small number of proteins undergo a major change in phosphorylation state following the activation of protein kinase C in isolated bag cell neurons. One or more of these proteins may contribute to the unmasking of the calcium channels.
Many neurotransmitters and hormones regulate secretion from endocrine cells and neurons by modulating voltage-gated Ca2+ channels. One proposed mechanism of neurotransmitter inhibition involves protein kinase C, activated by diacylglycerol, a product of phosphatidyl-inositol inositol hydrolysis. Here we show that thyrotropin-releasing hormone (TRH), a neuropeptide that modulates hormone secretion from pituitary tumor cells, inhibits Ca2+ channels via the other limb of the phosphatidylinositol signaling system: TRH causes inositol trisphosphate-triggered Ca2+ release from intracellular organelles, thus causing Ca2(+)-dependent inactivation of Ca2+ channels. Elevation of intracellular Ca2+ concentration is coincident with the onset of TRH-induced inhibition and is necessary and sufficient for its occurrence. The inhibition is blocked by introducing Ca2+ buffers into cells and mimicked by a variety of agents that mobilize Ca2+. Treatments that suppress protein kinase C have no effect on the inhibition. Hence inactivation of Ca2+ channels occurs not only as a result of Ca2+ influx through plasma membrane channels, but also via neurotransmitter-induced Ca2+ mobilization. This phenomenon may be common but overlooked because of the routine use of Ca2+ buffers in patch-clamp electrodes.
It is now clear that voltage-gated K+ channels are encoded by a set of multigene subfamilies. Expression of different members of these subfamilies, coupled with mutational analysis, has advanced our knowledge of the structure and function of voltage-dependent K+ channels.
Glucocorticoid hormones are released as part of the stress response and regulate secretion by the pituitary. Since the activity of ion channels also influences secretion, we examined the effect of the glucocorticoid agonist dexamethasone on ion channel expression. K+ channel mRNA was detected in rat hypothalamus and anterior pituitary, with probes derived from the rat Kv1 gene, a member of the mammalian voltage-gated K+ channel superfamily. High levels were also detected in PRL-secreting clonal (GH3 and GH4C1) rat pituitary cells. Dexamethasone rapidly increased the steady state concentration of Kv1 mRNA in GH3 cells in a dose-dependent manner. This change in gene expression was accompanied by an increase in whole cell voltage-gated K+ current [lk(i)] with similar pharmacology to the Kv1 gene product. Our findings indicate that hormones may act directly on excitable cells to produce long term effects on electrical activity and secretion by regulating K+ channel expression.
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.
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.
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.
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.
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.