Calcium currents from jellyfish striated muscle cells: preservation of phenotype, characterisation of currents and channel localisation.

When striated muscle cells of the jellyfish Polyorchis penicillatus were dissociated at 30 degrees C they retained their in vivo morphology and the integrity of ionic currents. This contrasted with cells dissociated at room temperature that rarely expressed any inward currents. Whole-cell, patch-clamp recordings from dissociated muscle cells revealed that the inward component of the total ionic current consisted of only one calcium current. This calcium current activated at -70 mV, peaked at -30 mV, and inactivated within 5 ms. In comparison with barium and strontium ions, calcium ions were the preferred current carriers. Calcium channels can be blocked by dihydropyridines and nickel ions at micromolar levels. Several properties of this current are reminiscent of T-type calcium currents. Localisation of this channel using the fluorescent channel blocker fDHP and the fluorescent dye RH414 indicated that myofibres had a higher density of these channels than the somata.

Localisation of intracellular calcium stores in the striated muscles of the jellyfish Polyorchis penicillatus: possible involvement in excitation-contraction coupling.

When jellyfish striated muscles were stimulated directly, the amplitude of contractile tension increased as the stimulation frequency increased. Application of 10 mmol l(-1) caffeine reduced the amplitude of contractile tension and abolished this facilitatory relationship, indicating that calcium stores participate in excitation-contraction coupling. Calcium stores were identified ultrastructurally using enzymatic histochemistry to localize CaATPases, and potassium dichromate to precipitate calcium. Electron energy-loss spectroscopy was used to verify the presence of calcium in precipitates. Both CaATPase and calcium were localised in membrane-bound vesicles beneath the sarcolemma. We concluded that sub-sarcolemmal vesicles could act as calcium stores and participate in excitation-contraction coupling.

The anatomy of the nervous system of the hydrozoan jellyfish, Polyorchis penicillatus, as revealed by a monoclonal antibody.

Dissociated cells from the margin and tentacles of the hydromedusa Polyorchis penicillatus were centrifuged in a Percoll gradient to remove cnidocytes. The resulting formaldehyde-fixed cells were used to inoculate mice to produce monoclonal antibodies. One of the hybridomas, which secreted antibodies against all neurons, was cloned and designated as mAb 5C6. Immunohistochemical labelling with mAb 5C6 of whole-mount preparations and paraffin sections provided a far more complete picture of the organisation of the hydromedusan nervous system than was previously available when using neuronal labelling techniques that restrict labelling to certain neuronal types. Besides confirming anatomical features described in earlier studies these techniques allowed us to discover a number of new structures and to determine connections that were only suspected. Such findings included:1. The discovery of an arch-like connection between the swimming motor neuron network at the apices of the subumbrellar muscle sheets 2. An orthogonal network connecting each pair of radial nerves in each radius 3. Continuity of a central branch of the radial nerve with the radial innervation of the manubrium 4. Details of the sensory neuronal contribution to the microanatomy of the ocelli and cnidocyte batteries 5. Presence of specialised receptor cells in the margin at the bases of tentacles 6. Neurons apparently innervating the radial muscles of the velum 7. Isolated neurons in the peduncle and gonads

K(+) currents in cultured neurones from a polyclad flatworm.

Cells from the brain of the polyclad flatworm Notoplana atomata were dispersed and maintained in primary culture for up to 3 weeks. Whole-cell patch-clamp of presumed neurones revealed outwardly directed K(+) currents that comprised, in varying proportions, a rapidly activating (time constant tau =0.94+/-0.79 ms; N=15) and inactivating ( tau =26.1+/-1.9 ms; N=22) current and a second current that also activated rapidly ( tau =1.1+/-0.2 ms; N=9) (means +/- s.e.m.) but did not inactivate within 100 ms. Both current types activated over similar voltage ranges. Activation and steady-state inactivation overlap and are markedly rightward-shifted compared with most Shaker-like currents (half-activation of 16.9+/-1. 9 mV, N=7, half-inactivation of -35.4+/-3.0 mV, N=5). Recovery from inactivation was rapid (50+/-2.5 ms at -90 mV). Both currents were unaffected by tetraethylammonium (25 mmol l(-1)), whereas 4-aminopyridine (10 mmol l(-1)) selectively blocked the inactivating current. The rapidly inactivating current, like cloned K(+) channels from cnidarians and certain cloned K(+) channels from molluscs and the Kv3 family of vertebrate channels, differed from most A-type K(+) currents reported to date. These findings suggest that K(+) currents in Notoplana atomata play novel roles in shaping excitability properties.

Wound healing in jellyfish striated muscle involves rapid switching between two modes of cell motility and a change in the source of regulatory calcium.

Small wounds (1.2 mm in diameter) made in the sheet of myoepithelial cells forming the "swimming" muscle of the jellyfish, Polyorchis penicillatus, were closed within 10 h by epithelial cells migrating centripetally to the wound center. Some 24 to 48 h later these cells redifferentiated into fully contractile muscle cells. Labeling with bromodeoxyuridine failed to reveal any cell proliferation during this process. Phenotype switching (within 1 h) from contractile muscle cells to migratory cells did not require synthesis of new protein as shown by treatment with 40 microM cycloheximide. Excitation-contraction coupling in undamaged muscle depended on entry of Ca(2+) through voltage-gated ion channels, as shown by a block of contractility by 40 microM nitrendipine and also on calcium released from intracellular stores since caffeine (10 mM) caused a 25% reduction in contractile force. In contrast, migratory cells did not require a source of extracellular calcium since migration was unimpeded by low (1 microM) free Ca(2+) or nitrendipine. Instead, modulatory calcium was derived from intracellular stores since caffeine (10 mM) and thapsigargin (10 microM) slowed migration. This lack of dependence on calcium influx in migratory cells was further confirmed by a dramatic down-regulation in voltage-gated inward current as shown by whole-cell patch recordings.

Modulation of jellyfish potassium channels by external potassium ions.

The amplitude of an A-like potassium current (I(Kfast)) in identified cultured motor neurons isolated from the jellyfish Polyorchis penicillatus was found to be strongly modulated by extracellular potassium ([K(+)](out)). When expressed in Xenopus oocytes, two jellyfish Shaker-like genes, jShak1 and jShak2, coding for potassium channels, exhibited similar modulation by [K(+)](out) over a range of concentrations from 0 to 100 mM. jShak2-encoded channels also showed a decreased rate of inactivation and an increased rate of recovery from inactivation at high [K(+)](out). Using site-directed mutagenesis we show that inactivation of jShak2 can be ascribed to an unusual combination of a weak "implicit" N-type inactivation mechanism and a strong, fast, potassium-sensitive C-type mechanism. Interaction between the two forms of inactivation is responsible for the potassium dependence of cumulative inactivation. Inactivation of jShak1 was determined primarily by a strong "ball and chain" mechanism similar to fruit fly Shaker channels. Experiments using fast perfusion of outside-out patches with jShak2 channels were used to establish that the effects of [K(+)](out) on the peak current amplitude and inactivation were due to processes occurring at either different sites located at the external channel mouth with different retention times for potassium ions, or at the same site(s) where retention time is determined by state-dependent conformations of the channel protein. The possible physiological implications of potassium sensitivity of high-threshold potassium A-like currents is discussed.

Residues in a jellyfish shaker-like channel involved in modulation by external potassium.

The jellyfish gene, jShak2, coded for a potassium channel that showed increased conductance and a decreased inactivation rate as [K(+)](out) was increased. The relative modulatory effectiveness of K(+), Rb(+), Cs(+), and Na(+) indicated that a weak-field-strength site is present. Cysteine substituted mutants (L369C and F370C) of an N-terminal truncated construct, (jShak2Delta2-38) which only showed C-type inactivation, were used to establish the position and nature of this site(s). In comparison with jShak2Delta2-38 and F370C, L369C showed a greater relative increase in peak current when [K(+)](out) was increased from 1 to 100 mM because the affinity of this site was reduced at low [K(+)](out). Increasing [K(+)](out) had little effect on the rate of inactivation of L369C; however, the appearance of a second, hyperbolic component to the inactivation curve for F370C indicated that this mutation had increased the affinity of the low-affinity site by bringing the backbone oxygens closer together. Methanethiosulphonate reagents were used to form positively (MTSET), negatively (MTSES), and neutrally (MTSM) charged side groups on the cysteine-substituted residues at the purported K(+) binding site(s) in the channel mouth and conductance and inactivation kinetic measurements made. The reduced affinity of the site produced by the mutation L369C was probably due to the increased hydrophobicity of cysteine, which changed the relative positions of carbonyl oxygens since MTSES modification did not form a high-field-strength site as might be expected if the cysteine residues project into the pore. Addition of the side chain -CH(2)-S-S-CH(3), which is similar to the side chain of methionine, a conserved residue in many potassium channels, resulted in an increased peak current and reduced inactivation rate, hence a higher affinity binding site. Modification of cysteine substituted mutants occurred more readily from the inactivated state confirming that side chains probably rotate into the pore from a buried position when no K ions are in the pore. In conclusion we were able to show that, as for certain potassium channels in higher taxonomic groups, the site(s) responsible for modulation by [K(+)](out) is situated just outside the selectivity filter and is represented by the residues L(369) and F(370) in the jellyfish Shaker channel, jShak2.

The effects of level of expression of a jellyfish Shaker potassium channel: a positive potassium feedback mechanism.

1. When jellyfish Shaker potassium channels (jShak2) are heterologously expressed in Xenopus oocytes at different levels they demonstrate density-dependent changes in electrical and kinetic properties of macroscopic currents. 2. The activation and inactivation properties of jShak2 channels depend on the extracellular potassium concentration. In this study we present experimental data which show that expression-dependent changes in kinetic and electrical properties of jShak2 macroscopic currents can be explained by the positive feedback effect of dynamic accumulation of K+ in the perimembranal space.

Genomic organization of a voltage-gated Na+ channel in a hydrozoan jellyfish: insights into the evolution of voltage-gated Na+ channel genes.

Voltage-gated Na+ channels are responsible for fast propagating action potentials. The structurally simplest animals known to contain rapid, transient, voltage-gated currents carried exclusively by Na+ ions are the Cnidaria. The Cnidaria are thought to be close to the origin of the metazoan radiation and thus are pivotal organisms for studying the evolution of the Na+ channel gene. Here we describe the genomic organization of the Na+ channel alpha subunit, PpSCN1, from the hydrozoan jellyfish, Polyorchis penicillatus. We show that most of the 20 intron sites in this diploblast are conserved in mammalian Na+ channel genes, with some even shared by Ca2+ channels. One of these conserved introns is spliced by a rare U 12-type spliceosome. Such conservation places the origin of the primary exon arrangement of Na+ channels and different intron splicing mechanisms to at least the common ancestors of diploblasts and triploblasts, approximately 600 million-1 billion years ago.

A putative voltage-gated sodium channel alpha subunit (PpSCN1) from the hydrozoan jellyfish, Polyorchis penicillatus: structural comparisons and evolutionary considerations.

Extant cnidarians are probably the simplest metazoans with discrete nervous systems and rapid, transient voltage-gated currents carried exclusively by Na+ ions. Thus cnidarians are pivotal organisms for studying the evolution of voltage-gated Na+ channels. We have isolated a full-length Na+ channel alpha subunit cDNA (PpSCN1) from the hydrozoan jellyfish, Polyorchis penicillatus, that has one of the smallest known coding regions of a four domain Na+ channel (1695 amino acids). Homologous residues that have a critical bearing on the selectivity filter, voltage-sensor and binding sites for tetrodotoxin and lidocaine in vertebrates and most invertebrates differ in cnidarians. PpSCN1 is not alternatively-spliced and may be the only pore-forming alpha subunit available to account for at least three electrophysiologically distinct Na+ currents that have been studied in P. penicillatus.

Voltage sensing in jellyfish Shaker K+ channels.

The S4 segment of the jellyfish (Polyorchis penicillatus) Shaker channel jShak1 contains only six positively charged motifs. All other Shaker channels, including the jellyfish Shaker channel jShak2, have seven charges in this segment. Despite their charge differences, both these jellyfish channels produce currents with activation and inactivation curves shifted by approximately +40 mV relative to other Shaker currents. Adding charge without changing segment length by mutating the N-terminal side of jShak1 S4 does not have a pronounced effect on channel activation properties. Adding the positively charged motif RIF on the N-terminal side of K294 (the homologue of K374 in Drosophila Shaker, which is a structurally critical residue) produced a large positive shift in both activation and inactivation without altering the slope of the activation curve of the channel. When IFR was added to the other side of K294, there was a small negative shift in activation and fast inactivation of the channel was prevented. Our results demonstrate that K294 divides the S4 segment into functionally different regions and that the voltage threshold for activation and inactivation of the channel is not determined by the total charge on S4.

A cardiac-like sodium current in motor neurons of a jellyfish.

1. Whole cell voltage-clamp recordings from isolated swimming motor neurons (SMNs) reveal a rapidly activating and inactivating sodium current. 2. Permeability ratios of PLi/PNa = 0.941 and P(guanidinium)/PNa = 0.124 were measured for the mediating channel, which was impermeable to rubidium. 3. The conductance/voltage and steady state inactivation curves are shifted in a depolarizing direction by approximately 45 mV relative to most neuronal sodium currents in higher animals. 4. Activation could be fitted with two exponents and maximal current peaked at 0.74 +/- 0.06 ms (mean +/- SD). 5. Inactivation could be fitted with fast (Tau 1 = 1.91 +/- 0.07 ms at +10 mV) and slow (Tau 2 = 11.65 +/- 0.55 ms at +10 mV) exponents. 6. Half-recovery from inactivation occurred slowly (52.6 +/- 2.9 ms). 7. A second class of identifiable neurons, "B" neurons, possesses a distinctly different population of sodium channels. they showed different inactivation kinetics and far more rapid recovery from inactivation (half-recovery < 5 ms). 8. We conclude that there was physiological diversification of sodium channels early in metazoan evolution and that there has been considerable cell-specific selection of channel properties.

Multiple Shaker potassium channels in a primitive metazoan.

Voltage-gated potassium channels are critical elements in providing functional diversity in nervous systems. The diversity of voltage-gated K+ channels in modern triploblastic metazoans (such as mollusks, arthropods and vertebrates) is provided primarily by four gene subfamilies (Shaker, Shal, Shab, and Shaw), but there has been no data from the ancient diploblastic metazoans until now. Diploblasts, represented by jellyfish and other coelenterates, arose during the first major metazoan radiation and are the most structurally primitive animals to have true nervous systems. By comparing the K+ channels of diploblasts and triploblasts, we may determine the fundamental set of K+ channels present in the first nervous systems. We now report the isolation of two Shaker subfamily cDNA clones, jShak1 and jShak2, from the hydrozoan jellyfish Polyorchis penicillatus (Phylum Cnidaria). JShak1 and jShak2 express transient outward currents in Xenopus oocytes most similar to Shaker currents from Drosophila in their rates of inactivation and recovery from inactivation. The finding of multiple Shaker subfamily genes is significant in that multiple Shaker genes also exist in mammals. In Drosophila, multiple Shaker channels are also produced, but by a mechanism of alternative splicing. Thus, the Shaker K+ channel subfamily had an established functional identity prior to the first major radiation of metazoans, and multiple forms of Shaker channels have been independently selected for in a wide range of metazoans.

Voltage-activated potassium currents in isolated motor neurons from the jellyfish Polyorchis penicillatus.

1. We describe two voltage-activated potassium currents in the swim motor neurons (SMNs) of the hydrozoan jellyfish, Polyorchis penicillatus. Recordings from neurons isolated in primary cultures were made using the tight-seal, whole-cell technique. 2. One current, IK-fast, turned on rapidly (time to peak = 6-15 ms), was half-activated at -10 to 0 mV, decayed with two exponential phases (tau were approximately 70 ms and approximately 1 s), and was half-inactivated by prepulses around -53 mV. It likely plays an important role in regulating the duration of SMN action potentials. IK-fast has features shared by delayed rectifiers and A-like currents in other invertebrates and vertebrates. 3. Another current, IK-slow, elicited from a holding potential of -30 mV, exhibited a slow onset (tau = 65-250 ms), was half-activated approximately +24 mV, exhibited a shallower voltage dependence than IK-fast, and did not inactivate. It was slower than most known delayed rectifiers.

Voltage-activated calcium currents in identified neurons from a hydrozoan jellyfish, Polyorchis penicillatus.

Calcium currents were studied in isolated "swim motor neurons" from the jellyfish Polyorchis penicillatus, using the tight-seal, whole-cell, voltage-clamp technique. Two high-voltage-activated (HVA) currents were observed. The transient current, HVA-t, activated rapidly (time to peak, 4 msec), inactivated with two time constants (26 msec, 187 msec) in a positive voltage range (Vi = -23 mV), and was larger when carried by calcium than by barium ions. The sustained current, HVA-s, inactivated slowly or not at all, even at very positive voltages and had the same amplitude whether carried by Ca2+ or Ba2+. It is likely that the two HVA current components arise from distinct channel populations, because the ionic selectivity of calcium channels is not known to depend on their inactivation kinetics. A third current appeared to activate at very positive voltages, and at a slower rate than did HVA-t. It is likely to be an artifact of inhomogeneous space clamping. A low-voltage-activated, cadmium-insensitive calcium current may also be present. Calcium currents in this primitive, multicellular animal have properties similar to calcium currents in other phyla; however, they do not fit neatly into the "T, N, L" classification scheme of vertebrate calcium currents.

Isolation of the neuropeptide less than Glu-Trp-Leu-Lys-Gly-Arg-Phe-NH2 (Pol-RFamide II) from the hydromedusa Polyorchis penicillatus.

Using a radioimmunoassay for the sequence Arg-Phe-NH2 (RFamide), we have isolated the peptide less than Glu-Trp-Leu-Lys-Gly-Arg-Phe-NH2 (Pol-RFamide II) from acetic acid extracts of the hydromedusa Polyorchis penicillatus. This peptide is a neuropeptide and constitutes a peptide family together with less than Glu-Leu-Leu-Gly-Gly-Arg-Phe-NH2 (Pol-RFamide I), the first neuropeptide isolated from Polyorchis, and less than Glu-Gly-Arg-Phe-NH2 (Antho-RFamide), a neuropeptide isolated from sea anemones and sea pansies.

Presynaptic spike broadening reduces junctional potential amplitude.

Presynaptic modulation of action potential duration may regulate synaptic transmission in both vertebrates and invertebrates. Such synaptic plasticity is brought about by modifications to membrane currents at presynaptic release sites, which, in turn, lead to changes in the concentration of cytosolic calcium available for mediating transmitter release. The 'primitive' neuromuscular junction of the jellyfish Polyorchis penicillatus is a useful model of presynaptic modulation. In this study, we show that the durations of action potentials in the motor neurons of this jellyfish are negatively correlated with the amplitude of excitatory junctional potentials. We present data from in vitro voltage-clamp experiments showing that short duration voltage spikes, which elicit large excitatory junctional potentials in vivo, produce larger and briefer calcium currents than do long duration action potentials, which elicit small excitatory junctional potentials.

The importance of cnidarian synapses for neurobiology.

Despite being the most primitive organisms to possess a nervous system, cnidarians afford rare opportunities for studying various, general aspects of chemical synaptic transmission. This is made possible by the unique organization of their nervous systems and by the fact that in certain species the neurons and synapses are readily accessible for intracellular recordings and voltage clamp. The results obtained from such studies are summarized here, with particular emphasis on work with two species, Cyanea capillata (Scyphozoa) and Polyorchis pennicilatus (Hydrozoa). The potential of these preparations for providing additional data is also discussed.

Isolation of pyroGlu-Leu-Leu-Gly-Gly-Arg-Phe-NH2 (Pol-RFamide), a novel neuropeptide from hydromedusae.

The hydromedusa Polyorchis penicillatus is a good model system to study neurotransmission in coelenterates. Using a radioimmunoassay for the peptide sequence Arg-Phe-NH2 (RFamide), two peptides have now been purified from acetic acid extracts of this medusa. The structure of one of these peptides was established as pyroGlu-Leu-Leu-Gly-Gly-Arg-Phe-NH2, and was named Pol-RFamide. This peptide belongs to the same peptide family as a recently isolated neuropeptide from sea anemones (pyroGlu-Gly-Arg-Phe-NH2). Using antisera to Pol-RFamide, the peptide was found to be exclusively localized in neurones of Polyorchis, among them neurones associated with smooth-muscle fibres. This suggests that Pol-RFamide might be a transmitter or modulator at neuromuscular junctions.

FMRFamide immunoreactivity in the nervous system of the medusa Polyorchis penicillatus.

Three different antisera to the molluscan neuropeptide Phe-Met-Arg-Phe-amide (FMRFamide) and two different antisera to the fragment RFamide were used to stain sections or whole mounts of the hydrozoan medusa Polyorchis penicillatus. All antisera stained the same neuronal structures. Strong immunoreactivity was found in neurons of the ectodermal nerve nets of the manubrium and tentacles, in neurons of the sensory epithelium, and in neurons at the periphery of the sphincter muscle. Strong immunoreactivity was also present in processes and perikarya of the whole outer nerve ring, in the ocellar nerves, and in nerve cells lying at the periphery of the ocellus. The inner nerve ring contained a moderate number of immunoreactive processes and perikarya, which were distinct from the swimming motor neurons. In contrast to the situation in the hydrozoan polyp Hydra attenuata, no immunoreactivity was found with several antisera to oxytocin/vasopressin and bombesin/gastrin-releasing peptide. The morphology and location of most FMRFamide-immunoreactive neurons in Polyorchis coincides with two identified neuronal systems, which have been recently discovered from neurophysiological studies.