Bag Cell Neurons Research Articles

Identification of a Vesicular Pool of Calcium Channels in the Bag Cell Neurons ofAplysia 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.

Gene expression and regulation of Aplysia californica prohormone convertases aPC1B and aPC2.

Peptide diversity can be regulated in many ways. One of the possible mechanisms of such a regulation may lie in the differential processing of a precursor in a tissue-specific manner or in a modulated processing within a single cell. To address these questions the enzymes responsible for the release of active neuropeptides first need to be well characterized. In this study, we have established the cellular distributions of the recently cloned prohormone convertases aPC2 and aPC1B in Aplysia californica and found the distribution of these enzymes to be mainly neuronal and endocrine. We then investigated in two sets of neurons (bag cell neurons and sensory neurons) the possible regulation of these two prohormone convertases by a neurotransmitter known to modulate behavior in Aplysia. In these neurons, we found that only aPC2 is regulated by serotonin possibly via the cyclic AMP cascade. Finally, a study of the biosynthesis of the bag cell egg-laying hormone precursor after treatment with serotonin suggests that such a treatment may increase its processing.

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.

Ionic Currents Underlying Developmental Regulation of Repetitive Firing inAplysiaBag 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.

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.

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.

Stimulation of an insulin receptor activates and down-regulates the Ca2+-independent protein kinase C, Apl II, through a Wortmannin-sensitive signaling pathway in Aplysia.

Activation of tyrosine kinase-linked receptors has been shown to stimulate Ca2+-independent protein kinase C isoforms in nonneuronal cells. We have examined this signaling pathway in the nervous system. Incubating bag cell neurons from the marine mollusk Aplysia californica with concentrations of insulin known to stimulate a tyrosine kinase-linked receptor in these cells persistently activated and down-regulated the Ca2+-independent protein kinase C (Apl II), whereas insulin only transiently activated and did not down-regulate the Ca2+-activated protein kinase C (Apl I). The effects of insulin may be mediated by activation of phosphoinositide 3-kinase because (a) diC16phosphatidylinositol 3,4,5-trisphosphate, a synthetic phosphoinositide 3-kinase product, stimulated autophosphorylation of baculovirus-expressed Apl II, but not of Apl I, and (b) wortmannin, an inhibitor of phosphoinositide 3-kinase, blocked the activation and down-regulation of Apl II by insulin but not the transient activation of Apl I. These results suggest that activators of tyrosine kinase-linked receptors may mediate some of their effects in neurons through activation of Ca2+-independent protein kinase C isoforms.

Sign stimulus activates a peptidergic neural system controlling reproductive behavior in Aplysia.

1. In the marine mollusk Aplysia, egg laying is a complex behavior that lasts for up to several hours. We used behavioral and electrophysiological methods to determine how egg laying occurs in groups of animals and how it is related to other aspects of reproductive behavior. 2. Prolonged contact with an existing egg mass by the lips and tentacles of an animal is a sign stimulus for release of egg-laying behavior and two other fixed action patterns in the same individual, mating as a female during egg laying and mating as a male after egg laying. 3. Prolonged contact with the egg mass initiated repetitive spike activity in bag cell neurons, which are part of a peptidergic neural system that modulates neuronal activity in the CNS for up to several hours. The sign stimulus thus activates the neuromodulatory system, which may serve as an innate releasing mechanism, and an associated internal drive, for control of the behavioral sequence.

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.

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.

The function and differential sorting of a family of aplysia prohormone processing enzymes

We have cloned four members of the family of subtilisin-like endoproteases expressed in the bag cell neurons of Aplysia and have demonstrated that two of these enzymes are capable of correctly cleaving the egg-laying hormone precursor. The egg-laying hormone precursor undergoes an ordered series of cleavages, such that different peptides are differentially sorted into distinct secretory vesicles. We have used electron microscopic chemistry to demonstrate that at least one processing enzyme is differentially segregated into a class of secretory vesicles containing the bag cell peptides. The segregation of specific endoproteases, along with specific neuropeptides within a given cell type, may ensure appropriate cleavage and prevent inappropriate cleavage of the polyprotein precursors.

Transient changes in intracellular calcium associated with a prolonged increase in excitability in neurons of Aplysia californica

1. Transient stimulation of an afferent input to the bag cell neurons of Aplysia californica triggers a 30-min period of spontaneous firing termed the afterdischarge. Measurement of free calcium ion concentrations using calcium-sensitive electrodes revealed a biphasic pattern of elevation of intracellular calcium levels during the afterdischarge. Basal calcium levels at the soma were found to rise rapidly during afferent stimulation and then to decline before the onset of spontaneous firing. This early peak in intracellular calcium was followed by a slower, transient elevation of calcium levels during the period of rapid firing that occurs in the first few minutes of afterdischarge. Stimulation of clusters of bag cell neurons in a calcium-free external medium failed to trigger afterdischarge and produced no changes in basal intracellular calcium levels. 2. When calcium ions in the external medium were replaced by barium ions, stimulation of clusters of bag cell neurons triggered afterdischarges that were characterized by long-duration action potentials. Intracellular calcium levels during these afterdischarges rose slowly over the first few minutes of spontaneous firing. Because calcium-sensitive microelectrodes do not respond to barium ions, these data suggest that stimulation of afterdischarge triggers calcium release from an intracellular compartment. 3. During afterdischarges in barium-containing external media, each broadened action potential produced a discrete transient elevation of intracellular calcium levels. A similar effect was observed in isolated bag cell neurons in primary culture when action potentials were stimulated by depolarizing current pulses in a barium-containing medium. These data suggest that, under these conditions, individual action potentials trigger the release of intracellular calcium from some intracellular pool.

Persistence of hormone secretion from neuroendocrine cells of aplysia after termination of electrical afterdischarge

The bag cell neurons of the marine mollusk Aplysia have been used extensively for investigating the electrophysiology and molecular biology of neurosecretory cells. However, there has been little attempt to carefully describe the pattern of secretion of the 36-amino acid peptide hormone that controls egg-laying behavior. This egg-laying hormone (ELH) is secreted in response to a highly characteristic pattern of action potential firing called the afterdischarge. The purpose of this study was to document the pattern of ELH secretion in response to the bag cell afterdischarge. To accomplish this goal, we developed the first RIA for measurement of ELH. The study was conducted in vitro in excised neural preparations that included the bilateral bag cell clusters; preparations were maintained in static culture of filtered artificial sea water (ASW) at 21-22 C. Afterdischarges were electrically stimulated, and action potentials were monitored via extracellular electrodes. The entire volume of ASW was collected every 5 min and immediately replaced with fresh ASW. In all 20 preparations, ELH levels were low to undetectable before stimulation of the afterdischarge; ELH rose above baseline within 5-10 min of the start of the afterdischarge, reached peak levels near the end or after the end of the afterdischarge, and then remained elevated long after action potentials had ceased firing. These results indicate a temporal dissociation between action potential firing and secretion. We suggest that the afterdischarge is important for triggering intracellular events that culminate in peptide release. Importantly, once these secretion-inducing events have been initiated, they can continue in the absence of further action potential stimulation.

Persistence of hormone secretion from neuroendocrine cells of aplysia after termination of electrical afterdischarge.

The bag cell neurons of the marine mollusk Aplysia have been used extensively for investigating the electrophysiology and molecular biology of neurosecretory cells. However, there has been little attempt to carefully describe the pattern of secretion of the 36-amino acid peptide hormone that controls egg-laying behavior. This egg-laying hormone (ELH) is secreted in response to a highly characteristic pattern of action potential firing called the afterdischarge. The purpose of this study was to document the pattern of ELH secretion in response to the bag cell afterdischarge. To accomplish this goal, we developed the first RIA for measurement of ELH. The study was conducted in vitro in excised neural preparations that included the bilateral bag cell clusters; preparations were maintained in static culture of filtered artificial sea water (ASW) at 21-22 C. Afterdischarges were electrically stimulated, and action potentials were monitored via extracellular electrodes. The entire volume of ASW was collected every 5 min and immediately replaced with fresh ASW. In all 20 preparations, ELH levels were low to undetectable before stimulation of the afterdischarge; ELH rose above baseline within 5-10 min of the start of the afterdischarge, reached peak levels near the end or after the end of the afterdischarge, and then remained elevated long after action potentials had ceased firing. These results indicate a temporal dissociation between action potential firing and secretion. We suggest that the afterdischarge is important for triggering intracellular events that culminate in peptide release. Importantly, once these secretion-inducing events have been initiated, they can continue in the absence of further action potential stimulation.

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)

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.

Mode-switching of a voltage-gated cation channel is mediated by a protein kinase A-regulated tyrosine phosphatase.

Tyrosine kinases and tyrosine phosphatases are abundant in central nervous system tissue, yet the role of these enzymes in the modulation of neuronal excitability is unknown. Patch-clamp studies of an Aplysia voltage-gated cation channel now demonstrate that a tyrosine phosphatase endogenous to excised patches determines both the gating mode of the channel and the response of the channel to protein kinase A. Moreover, a switch in gating modes similar to that triggered by the phosphatase occurs at the onset of a prolonged change in the excitability of Aplysia bag cell neurons.

Cytoskeletal remodeling during growth cone-target interactions.

Reorganization of the cytoskeleton of neuronal growth cones in response to environmental cues underlies the process of axonal guidance. Most previous studies addressing cytoskeletal changes during growth cone pathfinding have focused on the dynamics of a single cytoskeletal component. We report here an investigation of homophilic growth cone-target interactions between Aplysia bag cell neurons using digitally enhanced video microscopy, which addresses dynamic interactions between actin filaments and microtubules. After physical contact of a growth cone with a physiological target, mechanical coupling occurred after a delay; and then the growth cone exerted forces on and displaced the target object. Subsequent to coupling, F-actin accumulation was observed at the target contact zone, followed by preferential microtubule extension to the same site. After successful target interactions, growth cones typically moved off highly adhesive poly-L-lysine substrates into native target cell surfaces. These events were associated with modulation of both the direction and rate of neurite outgrowth: growth cone migration was typically reoriented to a trajectory along the target interaction axis and rates of advance increased by about one order of magnitude. Directed microtubule movements toward the contact site appeared to be F-actin dependent as target site-specific microtubule extension and bundling could be reversibly randomized by micromolar levels of cytochalasin B in a characteristic manner. Our results suggest that target contacts can induce focal F-actin assembly and reorganization which, in turn, guides target site-directed microtubule redistribution.

The peptide FMRFa terminates a discharge in Aplysia bag cell neurons by modulating calcium, potassium, and chloride conductances

1. Electrical stimulation of an afferent nerve triggers a 30-min period of firing of action potentials in the bag cell neurons of Aplysia californica. This afterdischarge causes the animal to undergo a long-lasting sequence of stereotyped reproductive behaviors culminating in laying of eggs. The connective sheath surrounding the clusters of bag cell neurons is interspersed with a network of particles that are immunoreactive to an antiserum raised against the tetrapeptide neurotransmitter Phe-Met-Arg-Phe-amide (FMRFa). Because the sheath is known to be rich in processes from the bag cell neurons, these data suggest that an FMRFa-like peptide may be located in neuronal processes that are in close contact with those of the bag cell neurons. 2. Application of FMRFa to bag cell neurons in intact abdominal ganglia effectively suppresses the onset of the afterdischarge in response to electrical stimulation and terminates an ongoing afterdischarge in a reversible manner. 3. Application of FMRFa to isolated bag cell neurons in primary cell culture causes an attenuation of the amplitude of evoked action potentials. This could be attributed in part to an attenuation of the voltage-activated calcium current, which in voltage-clamp experiments was found to be reduced by 10-40%. 4. Application of FMRFa to bag cell neuron in primary culture also causes a hyperpolarization of the membrane potential by activating an outward current with a reversal potential of approximately -67 mV. Ion substitution experiments, together with application of channel blockers, indicate that this current is carried by both potassium and chloride ions. Activation of this current is suppressed by treatment of the cells with either a cyclic AMP analogue or a phorbol ester activator of protein kinase C. 5. FMRFa exerts a powerful inhibitory influence on the bag cell neurons by altering the properties of ion currents involved in both the generation of action potentials and control of the resting potential. This suggests that this neuropeptide plays a role in the regulation of the onset of afterdischarge in vivo.

Expression of mutant ELH prohormones in AtT-20 cells: the relationship between prohormone processing and sorting.

Posttranslational processing of many proteins is essential to the synthesis of fully functional molecules. The ELH (egg-laying hormone) prohormone is cleaved by endoproteases in a specific order at a variety of basic residue processing sites to produce mature peptides. The prohormone is first cleaved at a unique tetrabasic site liberating two intermediates (amino and carboxy) which are sorted to different classes of dense core vesicles in the bag cell neurons of Aplysia. When expressed in AtT-20 cells, the ELH prohormone is also first cleaved at the tetrabasic site. The amino-terminal intermediate is then sorted to the constitutive pathway, and a portion of the carboxy-terminal intermediate is sorted to the regulated pathway. Here, we use mutant constructs of the ELH prohormone expressed in AtT-20 cells to examine the relationship between prohormone processing and consequent sorting. Prohormone which has a dibasic site in place of the tetrabasic site is processed and sorted similarly to wild type. Furthermore, mutant prohormone which lacks the tetrabasic site is processed at an alternative site comprising three basic residues. In these mutant prohormones, mature ELH is still produced and stored in dense core vesicles while amino-terminal products are constitutively secreted. However, deletion of the tetrabasic and tribasic sites results in the rerouting of the amino-terminal intermediate products from the constitutive pathway to the regulated secretory pathway. Thus, in the ELH prohormone, the location of the proteolytic processing events within the secretory pathway and the order of cleavages regulate the sorting of peptide products.