Ionic current of an identifiable giant neurone, d-RPLN, of an African giant snail (Achatina fulica Férussac), measured under voltage clamping--II. Outward currents

1. The outward currents of d-RPLN (dorsal-right parietal large neurone), one of the largest identifiable neurones of an African giant snail, were studied. 2. At the holding potential (-90 mV) and at the command voltage (Vc20 mV), the current values were 3.05 +/- 0.13 microA (M +/- SE) for the peak (n = 38), and 1.96 +/- 0.10 microA for the plateau (n = 37). 3. The peak time constant (Vc = 0 mV) was 2.05 +/- 0.08 msec. 4. Tetraethylammonium at 50 mM reduced the plateau value up to 50-55% of the normal, but had little effect on the peak. 5. 5-Aminopyridine at 5.0 mM diminished the peak value to about 50-55%, and delayed the peak time. 6. Quinine at 0.25 mM decreased both the peak and the plateau approximately to 55-65% of their controls, but shortened the peak time when Vc was beyond 0 mV, in contrast to the case of 4-AP. 7. The calcium-free state (replaced with cobalt) reduced these currents to about 75% of the normal, and evidently delayed the peak time.     

Comparison of antagonistic effects of several drugs on calcium current in a giant neuron of an African snail (Achatina fulica Férussac)

Antagonistic effects of several drugs on the calcium current (ICa) of a giant neurone, PON (periodically oscillating neurone), of an African snail (Achatina fulica Ferussac) were compared under the voltage clamp. According to their IC50 values and the confidence limit at 95%, the order of effectiveness of the drugs was: brovincamine, verapamil greater than or equal to isoperisone, eperisone, d-diltiazem greater than or equal to vincamine, l-diltiazem, tolperisone. Their IC50 values were assumed to be the dissociation constants of Ca2+ channels in the resting state (Kr). Voltage dependence of these drugs was examined by measuring the steady-state inactivation curves. From the results obtained, the dissociation constants of the channels in the inactivated state (Ki) and the Kr/Ki ratio were calculated for each drug. The order of Ki values of these drugs was: isoperisone, brovincamine, verapamil less than or equal to eperisone, l-diltiazem, d-diltiazem, tolperisone. The use dependence of the drugs was also examined by measuring the further decrease of ICa amplitude caused by high frequent stimulations. We observed that verapamil, brovincamine, d-diltiazem and l-diltiazem were use dependent, whereas eperisone, tolperisone and isoperisone were not.     

Neuronal calcium channel inhibitors. Synthesis of omega-conotoxin GVIA and effects on 45Ca uptake by synaptosomes

We previously described a 27-amino acid peptide neurotoxin from the venom of Conus geographus, omega-conotoxin GVIA, which inhibits neuronal voltage-activated calcium channels. In this paper we describe the total synthesis of omega-conotoxin GVIA and demonstrate that it efficiently blocks voltage-activated uptake of 45Ca by standard synaptosomal preparations from chick brain. Dihydropyridines do not block 45Ca uptake under these conditions. Thus, the omega-conotoxin-sensitive, but dihydropyridine-insensitive uptake of 45Ca2+ by chick brain synaptosomes serves as a functional assay for a Ca channel target of omega-conotoxin. The use of synthetic GVIA should rapidly accelerate our understanding of the molecular biology of Ca2+ channels and their role in neuronal function.     

A five-conductance model of the action potential in the rat sympathetic neurone

The origin of the action potential in neurones has yet to be answered satisfactorily for most cells. We present here a five-conductance model of the somatic membrane of the mature and intact sympathetic neurone studied in situ in the isolated rat superior cervical ganglion under two-electrode voltage-clamp conditions. The neural membrane hosts five separate types of voltage-dependent ionic conductances, which have been isolated at 37 degrees C by using simple manipulations such as conditioning-test protocols and external ionic pharmacological treatments. The total current could be separated into two distinct inward components: (1) the sodium current, INa, and (2) the calcium current, ICa; and three outward components: (1) the delayed rectifier, IKV, (2) the transient IA, and (3) the calcium-dependent IKCa. Each current has been kinetically characterized in the framework of the Hodgkin-Huxley scheme used for the squid giant axon. Continuous mathematical functions are now available for the activation and inactivation (where present) gating mechanisms of each current which, together with the maximum conductance values measured in the experiments, allow for a satisfactory reconstruction of the individual current tracings over a wide range of membrane voltage. The results obtained are integrated in a full mathematical model which, by describing the electrical behaviour of the neurone under current-clamp conditions, leads to a quantitative understanding of the physiological firing pattern. While, as expected, the fast inward current carried by Na+ contributes to the depolarizing phase of the action potential, the spike falling phase is more complex than previous explanations. IKCa, with a minor contribution from IKV, repolarizes the neurone only under conditions of low cell internal negativity. Their role becomes less pronounced in the voltage range negative to -60 mV, where membrane repolarization allows IA to deinactivate. In the spike arising from these voltage levels the membrane repolarization is mainly sustained by IA, which proves to be the only current sufficiently fast and large enough to recharge the membrane capacitor at the speed observed during activity. Different modes of firing coexist in the same neurone and the switching from one to another is fast and governed by the membrane potential level, which makes the selection between the different voltage-dependent channel systems. The neurone thus seems to be prepared to operate within a wide voltage range; the results presented indicate the basic factors underlying the different discrete behaviours.     

Effects of synthetic peptides on giant neurones identified in the ganglia of an African giant snail (Achatina fulica Férussac). IV

1. The previous papers (Ku et al., 1986; Kim et al., 1987; Yongsiri et al., 1987) reported the effects of the synthetic peptides, i.e. Met-enkephalin, substance P, neurotensin, oxytocin, Arg-vasopressin, proctolin, FMRFamide, ranatensin C etc., on about 20 identifiable giant neurones of an African giant snail (Achatina fulica Férussac). 2. In the present study, the effects of the same peptides on the following Achatina neurones, other than those of the previous papers, were investigated: v-RPLN, v-LPSN, v-VNAN, v-VLN, r-VMN, l-VMN, v-l-VOrN and d-RCDN. 3. Of the neurones tested here, v-RPLN (ventral-right parietal large neurone) was excited slightly by Met-enkephalin, excited markedly by oxytocin, and inhibited by FMRFamide, at 10(-4) M. 4. Of these effects, those of oxytocin and FMRFamide were undoubtedly the direct effects on the neurone tested, whereas those of Met-enkephalin were probably due to the synaptic activations. 5. Another neurone, v-LPSN (ventral-left parietal large neurone), was affected by oxytocin and ranatensin C at 10(-4) M. The two substances sometimes showed similar simple excitatory effects, in other cases biphasic (excitation followed by inhibition) effects, and in a few cases almost no effect. 6. The rest of the neurones tested were not sensitive at all to any of the peptides examined.     

APGW-amide as an inhibitory neurotransmitter of Achatina fulica Ferussac

APGWamide (L-Ala-L-Pro-Gly-L-Trp-NH2) was purified from the ganglia of an African giant snail (Achatina fulica Ferussac). This peptide inhibited (hyperpolarized) more than half of the Achatina neurone types tested. This produced an outward current with the membrane conductance increase of RAPN (right anterior pallial neurone) under voltage clamp. The ED50 of the peptide was 6.2 x 10(-6) M (95% confidence limit: 5.0-7.8 x 10(-6) M) and the Emax was 3.9 +/- 0.2 nA. The effects were due to a membrane permeability increase to K+. The peptide is proposed as an inhibitory neurotransmitter of the Achatina neurones.     

Effects of clavines, ergot alkaloids, on the membrane potential of an identifiable giant neuron of the African giant snail Achatina fulica Ferussac

The effects of seven clavines, alkaloids of ergot, on the electrical activity of an identifiable giant neurone (TAN, tonically autoactive neurone) of the African giant snail were examined. All the substances examined, lysergine, agroclavine, elymoclavine, festuclavine, chanoclavine, rugulovasine A and rugulovasine B, at 2 X 10(-4) kg/l have no constant effect on TAN, indicating that they have no direct effect on this neurone. However, the substances examined, except for chanoclavine, in the same concentration occasionally caused the transient depression with an augmentation of trans-synaptic influences. This depression may be due to the trans-synaptic influences. The four substances examined, lysergine, agroclavine, elymoclavine and festuclavine, in the same concentration produced TAN abnormal spike discharges, doublet or triplet spikes.     

Isolation of achatin-I, a neuroactive tetrapeptide having a D-phenylalanine residue, from Achatina ganglia, and its effects on Achatina giant neurones.

1. We have isolated a neuroexcitatory tetrapeptide having a D-phenylalanine (Gly-D-Phe-L-Ala-L-Asp) from the ganglia of Achatina fulica Férussac. This peptide was termed achatin-I (Kamatani et al., 1989). In the present report, we shall present highlights from the original paper concerning the process of peptide isolation and the examination of its effects. 2. From the ganglia of about 30,000 animals, we obtained 50 micrograms of achatin-I and 17 micrograms of its stereoisomer consisting of only L-amino acid residues (Gly-L-Phe-L-Ala-L-Asp) which was termed achatin-II. The data of instrumental analyses (1H-NMR, SIMS, CD and HPLC) of isolated achatin-I and achatin-II were identical to those of synthetic ones. 3. Achatin-I showed marked excitatory effects on the three Achatina giant neurones, PON (periodically oscillating neurone), TAN (tonically autoactive neurone) and v-RCDN (ventral-right cerebral distinct neurone), whereas achatin-II had no effect. Among their stereoisomers, [D-Ala3]-achatin-I (Gly-D-Phe-D-Ala-L-Asp) had slight excitatory effects on the Achatina neurones tested. Amide derivatives of achatin-I and achatin-II were ineffective. 4. Dose-response curves of achatin-I and [D-Ala3]-achatin-I for producing the inward current of PON were measured under voltage clamp at a holding membrane voltage (Vh) of -50 mV.(ABSTRACT TRUNCATED AT 250 WORDS)     

Decreases of action potential amplitudes, in sodium-free and calcium-free conditions, of identifiable giant neurons of an African giant snail (Achatina fulica Férussac)--I. The right parietal ganglion

Decreases of the action potential amplitude in sodium- and calcium-free states were observed with respect to the four giant neurons, PON (periodically oscillating neuron), Tan (tonically autoactive neuron), RAPN (right anterior pallial neuron) and d-RPLN (dorsal-right parietal large neuron), identified in the right parietal ganglion of the suboesophageal ganglia of an African giant snail (Achatina fulica Férussac). The decrease of the PON action potential amplitude, caused in the sodium-free state, was observed to be 25.4 +/- 2.1% (23.0 +/- 2.0 mV), expressed by M +/- SE, while that of the calcium-free state was 35.0 +/- 2.1% (30.9 +/- 1.7 mV). Then, the two ionic dependencies of the PON action potential were estimated to be about 40-50% on sodium and 50-60% on calcium. The decrease of the TAN action potential in the sodium-free state, was observed to be 20.7 +/- 1.2% (18.8 +/- 1.3 mV), whereas that of the calcium-free state was 42.2 +/- 2.7% (39.0 +/- 2.2 mV), indicating that the two ionic dependencies were 30-40% on sodium and 60-70% on calcium. The decrease of the RAPN action potential in the sodium-free state, was 45.8 +/- 3.7% (40.3 +/- 3.1 mV), whereas that of the calcium-free state was 21.7 +/- 2.5% (17.8 +/- 2.0 mV), indicating that the two ionic dependencies were about 70% on sodium and about 30% on calcium. The decrease of the d-RPLN action potential in the sodium-free state was found to be 17.6 +/- 2.4% (15.2 +/- 1.8 mV), whereas that of the calcium-free state was 23.1 +/- 1.4% (20.8 +/- 1.4 mV), indicating that the two ionic dependencies were 40-50% on sodium and 50-60% on calcium. The action potential amplitudes of all the neurons tested were decreased in both sodium-free and calcium-free states. However, their ionic dependencies were estimated to vary from 70% on sodium (30% on calcium) to 30% on the sodium (70% on the calcium), according to the neurons tested.     

Inhibitory effects of dipeptides containing tryptophan on the excitability on the giant neuron of the giant African snail (Achatina fulica, Ferussac)]

Besides an inhibitory effect of L-Lys-L-Phe-L-Tyr and L-Phe-L-Tyr, L-Phe-L-Trp showed the same effect on the excitability of a giant neurone (the TAN, tonically autoactive neurone) identified in suboesophageal ganglia of Achatina futica Férussac. The critical concentration of L-Phe-L-Trp to produce the effect was 10(-5)--3 X 10(-5) kg/1. The inhibitory effect of this substance was relatively slow and independent no chloride ions, as well as in the case of L-Phe-L-Tyr. L-Trp-L-Phe, Gly-L-Trp and L-Trp-Gly did not show any effect on the same neurone. The I-V curve of the TAN's neuromembrane, measured by the injection of a transmembrane triangular current, were not modified markedly by L-Phe-L-Trp at 4 X 10(-4) kg/1. The pattern of the TAN's spike firing produced by the depolarizing current injection were not influenced by this substance.     

Different effects of the replacement of extracellular sodium with lithium on the abnormal spike discharges caused by 2 convulsants, Metrazol and strychnine, on an identifiable giant neuron of the mollusc Achatina fulica Ferussac

The influences of the replacement of sodium with lithium in the extracellular medium on the abnormal spike discharges, caused by two convulsants, metrazol and strychnine, of a giant neurone (TAN, tonically autoactive neurone) identified in the suboesophageal ganglia of the African giant snail (Achatina fulica Férussac) were examined. The slow oscillations of potential caused by metrazol disappeared after this replacement. On the other hand, the abnormal action potentials caused by strychnine, as well as the normal action potentials, still remained after the removal of sodium in the medium.     

Purification and sequence of a presynaptic peptide toxin from Conus geographus venom

A novel toxin, omega-conotoxin (omega-CgTX), from the venom of the fish-eating marine mollusc Conus geographus has been purified and biochemically characterized. Recently, this omega-conotoxin has been shown to inhibit the voltage-activated entry of Ca2+, thus providing a potentially powerful probe for exploring the vertebrate presynaptic terminal [Kerr, L. M., & Yoshikami, D. (1984) Nature (London) 308, 282-284]. The toxin is a basic 27 amino acid peptide amide with three disulfide bridges. An unusual feature is a remarkable preponderance of hydroxylated amino acids. The sequence of omega-CgTx GVIA is Cys-Lys-Ser- Hyp-Gly5-Ser-Ser-Cys-Ser-Hyp10-Thr-Ser-Tyr-Asn-Cys15-C ys-Arg-Ser- Cys-Asn20-Hyp-Tyr-Thr-Lys-Arg25-Cys-Tyr-NH2.     

Further study of effects of synthetic peptides on identifiable giant neurones of an African giant snail (Achatina fulica Férussac)

1. Effects of the following peptides at 10(-4) M on identifiable giant neurones of Achatina fulica Férussac were examined: physalaemin, eledoisin, bradykinin, neurokinin A, neurokinin B, neuromedin B, gastrin releasing peptide decapeptide (neuromedin C), gastrin releasing peptide (14-27), cholecystokinin tetrapeptide, cholecystokinin octapeptide, thyrotropin releasing hormone, Arg-vasotocin, gamma-melanocyte stimulating hormone. 2. The six neurones tested were as follows: PON (periodically oscillating neurone), TAN (tonically autoactive neurone), RAPN (right anterior pallial neurone), d-RPLN (dorsal-right parietal large neurone), VIN (visceral intermittently firing neurone) and d-VLN (dorsal-visceral large neurone). 3. Of the peptides examined, only Arg-vasotocin at 10(-4) M produced the excitatory effects on PON, VIN and d-VLN. Physalaemin showed slight inhibitory effects on TAN; this substance was sometimes almost ineffective on the neurone. 4. The other peptides examined were completely ineffective on all of the neurones tested.     

Refined solution structure of omega-conotoxin GVIA: implications for calcium channel binding.

The polypeptide omega-conotoxin GVIA (GVIA) is an N-type calcium channel blocker from the venom of Conus geographus, a fish-hunting cone shell. Here we describe a high-resolution solution structure of this member of the 'inhibitor cystine knot' protein family. The structure, based on NMR data acquired at 600 MHz, has mean pairwise RMS differences of 0.25 +/- 0.06 and 1.07 +/- 0.14 A over the backbone heavy atoms and all heavy atoms, respectively. The solvent-accessible side chains are better defined than in previously published structures and provide an improved basis for docking GVIA with models of the calcium channel. Moreover, some side chain interactions important in GVIA folding in vitro and in stabilizing the native structure are defined clearly in the refined structure. Two qualitatively different backbone conformations in the segment from Thr11 to Asn14 persisted in the restrained simulated annealing calculations until a small number of lower bound constraints was included to prevent close contacts from occurring that did not correspond with peaks in the NOESY spectrum. It is possible that GVIA is genuinely flexible at this segment, spending a finite time in the alternative conformation, and this may influence its interaction with the calcium channel.     

Effects of synthetic biologically active peptides on giant neurones identified in the left buccal ganglion of an African giant snail (Achatina fulica Férussac)

1. The effects of synthetic biologically active peptides, including Met-enkephalin, substance P, oxytocin, Arg-vasopressin, proctolin and FMRFamide, on the following four buccal neurones were examined: d-LBAN (dorsal-left buccal anterior neurone), d-LBMN (dorsal-left buccal medial neurone), d-LBCN (dorsal-left buccal central neurone) and d-LBPN (dorsal-left buccal posterior neurone). These peptides were examined at 10(-4) M. 2. Oxytocin excited d-LBAN and slightly excited d-LBCN, while this inhibited d-LBMN. Arg-vasopressin excited slightly d-LBAN and d-LBCN, but this had some times no effect. FMRFamide inhibited d-LBAN, and slightly inhibited d-LBCN. 3. No direct synaptic connection from the two ventral cerebral giant neurones, v-LCDN and v-RCDN, to the four buccal giant neurones was found, though the two cerebral neurones innervate the cerebro-buccal connectives.     

Neuroeffector connections of multimodal neurons in the African snail (Achatina fulica)

Using a new method of animal preparation, the efferent connections of giant paired neurons on the dorsal surface of visceral and right parietal ganglia of snail, Achatina fulica, were examined. It was found that spikes in giant neurons d-VLN and d-RPLN evoke postjunctional potentials in different points of the snail body and viscerae (in the heart, in pericardium, in lung cavity and kidney walls, in mantle and body wall muscles, in tentacle retractors and in cephalic artery). The preliminary analysis of synaptic latency and facilitation suggests a direct connections between giant neurons and investigated efferents.     

Sodium and calcium currents in neuroblastoma x glioma hybrid cells before and after morphological differentiation by dibutyryl cyclic AMP

Sodium and calcium inward currents (INa and ICa) were measured in neuroblastoma X glioma hybrid cells of clones 108CC5 and 108CC15 by a single suction pipette method for internal perfusion and voltage clamp. Morphologically undifferentiated, exponentially growing cells were compared with cells differentiated by cultivation with 1 mmol/l dibutyryl cyclic AMP. Outward currents were eliminated by perfusing the cells with a K+-free solution. Voltage dependence and ion selectivity as well as steady state inactivation characteristics of INa and ICa resembled those of differentiated mouse neuroblastoma cells, clone N1E-115 (Moolenaar and Spector 1978, 1979). These parameters were identical in undifferentiated and differentiated cells of both clones. After differentiation the average density of the peak sodium and calcium currents was increased two and four-fold, respectively, in both cell lines. Our data indicate that exponentially growing, morphologically undifferentiated 108CC5 and 108CC15 neuroblastoma X glioma hybrid cells possess functional Na+ and Ca2+ channels undistinguishable from those of non-proliferating cells of these clones differentiated morphologically by treatment with dibutyryl cyclic AMP. That Na+ and Ca2+ spikes were not detected by other authors in these cells prior to morphological differentiation by dibutyryl cyclic AMP may be attributed to the fact that at the low resting membrane potential measured the Na+ and Ca2+ channels are inactivated.     

The potential-dependent incoming ionic currents of myoblasts from the frog Rana temporaria developing in culture]

Voltage-dependent inward ionic currents in 1-6-day cultured skeletal myoblasts have been studied using whole-cell patch clamp technique. Sodium (INa) and two types of calcium (ICa) currents were recorded at all stages. INa did not differ from that described in frog striated muscle fibres. Both types of ICa were found to be DHP-sensitive. They differ in their activation time. It is suggested that two types of ICa correspond to ICa of twitch and tonic muscle fibres.     

Structural organization of an identified giant neuron of Helix lucorum

Method of intracellular staining with cobalt was used for detailed study of processes branching of the giant cell in the left parietal ganglion of the snail Helix lucorum L. Dendritic and axonal branches are described and quantitatively characterized. Terminals of axonal collaterals of this neurone innervating presumed neurosecretory bodies are described in the tissue surrounding the ganglion.     

Solution structure of the calcium channel antagonist omega-conotoxin GVIA.

The three-dimensional solution structure is reported for omega-conotoxin GVIA, which is a potent inhibitor of presynaptic calcium channels in vertebrate neuromuscular junctions. Structures were generated by a hybrid distance geometry and restrained molecular dynamics approach using interproton distance, torsion angle, and hydrogen-bonding constraints derived from 1H NMR data. Conformations of GVIA with low constraint violations converged to a common peptide fold. The secondary structure in the peptide is an antiparallel triple-stranded beta-sheet containing a beta-hairpin and three tight turns. The NMR data are consistent with the region of the peptide from residues S9 to C16 being more dynamic than the rest of the peptide. The peptide has an amphiphilic structure with a positively charged hydrophilic side and an opposite side that contains a small hydrophobic region. Residues that are thought to be important in binding and function are located on the hydrophilic face of the peptide.