PubMed Health⌕ Search

Biomedical subjects

R Rahamimoff

Publications and source records attributed to R Rahamimoff.

At least 37 records · Page 2Linked to original sources

Ion channel activity in lobster skeletal muscle membrane.

Ion channel activity in the sarcolemmal membrane of muscle fibers is critical for regulating the excitability, and therefore the contractility, of muscle. To begin the characterization of the biophysical properties of the sarcolemmal membrane of lobster exoskeletal muscle fibers, recordings were made from excised patches of membrane from enzymatically induced muscle fiber blebs. Blebs formed as evaginations of the muscle sarcolemmal membrane and were sufficiently free of extracellular debris to allow the formation of gigaohm seals. Under simple experimental conditions using bi-ionic symmetrical recording solutions and maintained holding potentials, a variety of single channel types with conductances in the range 32-380 pS were detected. Two of these ion channel species are described in detail, both are cation channels selective for potassium. They can be distinguished from each other on the basis of their single-channel conductance and gating properties. The results suggest that current flows through a large number of ion channels that open spontaneously in bleb membranes in the absence of exogenous metabolites or hormones.

Animals↗

Currents and channels in the presynaptic nerve terminal.

Synaptic transmission is a key element in neuronal communication in the nervous system. It is determined by two basic sets of processes: release of transmitter from the presynaptic nerve ending and the detection and response by the postsynaptic cell. The release of transmitter is crucially dependent on the interplay among the ion channels in the surface membrane of the presynaptic terminal. In this article we describe the methods used in the study of the ion channels in secretory nerve endings and present the 16 calcium, sodium, chloride, cationic and potassium ion channels discovered in various preparations. We speculate about the physiological and pathophysiological roles of some of these ion channels.

Animals↗

Confocal microscopy reveals coordinated calcium fluctuations and oscillations in synaptic boutons.

Calcium ions are one of the main factors regulating quantal transmitter release and thus synaptic transmission in the nervous system. Using confocal microscopy, fluorescent imaging with the calcium indicator Rhod-2, and time series analysis, we show that the levels of calcium ions inside single synaptic boutons of the lizard neuromuscular junction are not constant at rest, but undergo coordinated fluctuations in the space domain, which cover a large fraction of the synaptic bouton. Furthermore, oscillations in intracellular calcium were frequently observed in the time domain. Control experiments showed no coordinated fluctuations or oscillations at locations outside the synaptic boutons. Edge detection analysis showed that the coordinated fluctuations and oscillations were not due to movement artifacts. No coordinated fluctuations and oscillations were seen when similar measurements and analyses were performed on artificial fluorescent beads. A variance analysis was performed on artificial fluorescent beads and on synaptic boutons. The variance of the fluorescent signal at the synaptic boutons was larger than the variance in artificial beads with the same mean fluorescence. This extra variance was greatly reduced when the extracellular calcium concentration was decreased from 2.0 mM to 0.4 mM. We conclude that the coordinated fluctuations and oscillations in the calcium-induced fluorescence at the synaptic boutons are genuine biological phenomena and may be of significance in the regulation of transmitter release.

Animals↗

A long closed state of the synaptosomal bursting potassium channel confers a statistical memory.

1. The statistical properties of the bursting potassium channel from fused Torpedo synaptosomes were studied by using patch-clamp recording and time series analysis. 2. Voltage steps produce channel openings; the number of channels opening fluctuates from trial to trial. The maximal current observed in each trial is strongly dependent on the previous history of the membrane patch. Trials with no activity are frequently clumped together and so are trials with intense activity. 3. Autocorrelation analysis reveals a strong interdependence of successive responses, which is voltage dependent. 4. We propose that the strong statistical interdependence of responses to successive depolarizing pulses (the statistical "memory") is a manifestation of a long lived closed state. We speculate that this statistical memory may be of significance in frequency modulation of transmitter release.

Animals↗

Protein phosphatase inhibitor okadaic acid enhances transmitter release at neuromuscular junctions.

To test the hypothesis that continual phosphorylation and dephosphorylation of protein components of nerve terminals might be important determinants of synaptic efficacy, the effect of okadaic acid, a potent natural inhibitor of two serine threonine protein phosphatases (phosphatase 1 and phosphatase 2A), was examined on synaptic transmission at frog (cholinergic) and lobster (glutamatergic and GABAergic) neuromuscular junctions. At frog junctions, the addition of 1 microM okadaic acid to the extracellular fluid caused almost a doubling of the amplitude of the end-plate potential. The effect of okadaic acid was reversible. Quantal analysis showed that the augmenting effect was presynaptic, resulting from an increase in the number of quanta of transmitter released by a nerve impulse. Where was no significant change in the amplitude of spontaneously liberated miniature end-plate potentials, but their frequency of release increased in parallel with the increase in amplitude of the nerve-evoked synaptic potential. Similar studies with lobster neuromuscular junctions showed increases in the size of both excitatory and inhibitory synaptic responses that were similar in magnitude to the effects seen in the frog junctions. No significant changes in membrane potential or in input resistance accompanied the increased response size. These results suggest that transmitter release at a variety of junctions using different transmitters is constantly modulated by phosphorylation and dephosphorylation of important protein components within nerve terminals.

Animals↗

A bursting potassium channel in isolated cholinergic synaptosomes of Torpedo electric organ.

1. Pinched-off cholinergic nerve terminals (synaptosomes) prepared from the electric organ of Torpedo ocelata were fused into large structures (greater than 20 microns) using dimethyl sulphoxide and polyethylene glycol 1500, as previously described for synaptic vesicles from the same organ. 2. The giant fused synaptosomes were easily amenable to the patch clamp technique and 293 seals with a resistance greater than 4 G omega were obtained in the 'cell-attached' configuration. In a large fraction of the experiments, an 'inside-out' patch configuration was achieved. 3. Several types of unitary ionic currents were observed. This study describes the most frequently observed single-channel activity which was found in 247 out of the 293 membrane patches (84.3%). 4. The single-channel current-voltage relation was linear between -60 and 20 mV and showed a slope conductance of 23.8 +/- 1.3 pS when the pipette contained 350-390 mM-Na+ and the bath facing the inside of the synaptosomal membrane contained 390 mM-K+. 5. From extrapolated reversal potential measurements, it was concluded that this channel has a large selectivity for K+ over Na+ (70.4 +/- 11.5, mean +/- S.E.M.). Chloride ions are not transported significantly through this potassium channel. 6. This potassium channel has a low probability of opening. The probability of being in the open state increases upon depolarization and reaches about 1% when the inside of the patch is 20 mV positive compared to the pipette side. 7. The mean channel open time increases with depolarization; thus the product current x time (= charge) also increases upon depolarization, showing properties of an outward rectifier. 8. The potassium channel in the giant synaptosome membrane has a bursting behaviour. Open-time distribution, closed-time distribution and a Poisson analysis indicate that the minimal kinetic scheme requires one open state and three closed states.

Animals↗

Vasopressin produces long-lasting increase in transmitter release.

Arginine-vasopressin has a widespread distribution in the nervous system, and has been implicated in cellular and behavioral functions. Its effect on the neuromuscular synapse reveals that it produces long-lasting augmentation in synaptic transmission due to an increase in spontaneous and nerve stimulation evoked quantal transmitter release. No significant postsynaptic effect was detected.

Action Potentials↗

Ionic channels in synaptic vesicles: are they involved in transmitter release?

Synaptic vesicles were isolated from the nerve terminals of Torpedo electric organ. After fusion, 'giant' vesicles were formed which could be examined by the patch clamp technique. One of the cationic channels, the P channel, shows a small preference for K+ compared to Na+ and has multiple conductance levels. Its rate of opening is voltage and calcium dependent. Fractal analysis of the P channels reveals that its behaviour does not seem to be fractal in nature. At voltages where only one conductance level is observed, fractal analysis shows at least one discrete open state and at least two discrete closed states. There are considerable similarities between the P channel and channels found in granules from the hypophysis. These channels resemble, in turn, the channels found in gap junctions. Therefore, it is not unwarranted to speculate that a gap-junction-like communication between the secretory vesicle and the extracellular space may occur during exocytosis.

Animals↗

Neuromuscular transmission in diabetes: response to high-frequency activation.

In searching for the cellular correlates of diabetic neuropathy, we examined the response to tetanic stimulation of diabetic neuromuscular junctions and of age-matched controls. The experiments were performed on the soleus nerve muscle preparation of the rat in which diabetes was induced by streptozotocin. Tetanic potentiation was substantially lower in diabetic rats. In addition, it was found that the diabetic neuromuscular junction is more resistant to high-frequency stimulation than normal age-matched controls, in which such stimulation causes a progressively increasing number of failures in synaptic transmission. Tetanic failures cannot be predicted from the stochastic properties of transmitter release and are due to propagation block of action potentials into the nerve terminals. The resistance of diabetic nerves to tetanic stimulation is a function of the duration of diabetes; the earliest significant difference between the number of tetanic failures in diabetic and normal age-matched controls was observed after 19 d of diabetes, and this difference grew with increased duration of diabetes. The resistance to tetanic stimulation in diabetic rats is reversed by insulin in vivo but not in vitro. Elevation in extracellular [K+] increases the number of tetanic failures in both diabetic and normal preparations. Furthermore, elevating extracellular [K+] to 8.5 mM brings the number of tetanic failures into the range of tetanic failures in normal nerves. This finding is consistent with the hypothesis that differences in extracellular [K+] accumulation during high-frequency stimulation are responsible for the diabetic nerve's relative resistance to high-frequency stimulation. The lower number of failures corrects only partially the impaired neuromuscular transmission in the diabetic state, and there is an overall reduction in tetanic potentiation in diabetes.

Aging↗

Ion channels in synaptic vesicles from Torpedo electric organ.

A simple method has been developed for fusing synaptic vesicles into spherical structures 20-50 micron in diameter. The method has been applied to purified cholinergic synaptic vesicles from Torpedo electric organ, and the membrane properties of these fused structures have been studied by the "cell"-attached version of the patch clamp technique. A large conductance potassium-preferring channel, termed the P channel, was consistently observed in preparations of fused synaptic vesicles. The selectivity of the channel for potassium over sodium was approximately equal to 2.8-fold. Two major conductance levels were observed during P-channel activity, and their relative proportion was dependent on the voltage applied to the membrane through the patch pipette. P channels were not seen in fused preparations of purified Torpedo lipids, nor was the frequency of their occurrence increased in preparations enriched with plasma membrane or nonvesicular membranes. We suggest, therefore, that the P channels are components of the synaptic vesicle membrane. Their function in synaptic transmission physiology is still unknown.

Animals↗

Ionic basis of tetanic and post-tetanic potentiation at a mammalian neuromuscular junction.

1. The ionic basis of tetanic and post-tetanic potentiation (TP and PTP) was studied at the rat soleus neuromuscular junction (NMJ), using the miniature endplate potential (MEPP) frequency as an index for transmitter release. Conventional intracellular recording and computer-assisted data analysis were employed. 2. The experimental results in this study indicate that contrary to previous suggestions, there is a substantial similarity in the ionic basis of TP and PTP at the mammalian and amphibian motor nerve terminals which can be subdivided into [Ca2+]o-dependent and [Ca2+]o-independent parts. 3. Tetanic and post-tetanic increase in MEPP frequency at the rat soleus NMJ is similar to that at the frog NMJ in the following aspects: (i) Tetanic potentiation is substantially larger in calcium-containing solutions than in calcium-deficient solutions. About 90% of tetanic potentiation is contributed by extracellular calcium. (ii) Increase in [Mg2+]o reduces tetanic potentiation in calcium-containing solutions and enhances TP in calcium-defient solutions. Elevated [Mg2+]o prolongs the post-tetanic potentiation both in calcium-containing and in calcium-deficient solutions. (iii) A post-tetanic jump in MEPP frequency was observed in 44% of the experiments performed in calcium-deficient solutions. (iv) The augmentation phase of post-tetanic potentiation, evident in calcium-containing solutions, is completely abolished by removal of [Ca2+]o. (v) Tetanic and post-tetanic potentiations are enhanced by increasing the rate and duration of tetanic stimulation in calcium-containing solutions. 4. The [Ca2+]o-independent part of tetanic potentiation is presumably due to entry of sodium ions and their accumulation in the nerve terminal, since it is increased by measures known to inhibit the sodium pump: reduction in [K+]o and partial substitution of sodium by lithium. 5. Sodium ions contribute substantially to the [Ca2+]o-independent part of posttetanic potentiation, since its duration is markedly prolonged by ouabain, reduction in [K+]o and partial substitution of sodium by lithium. 6. Tetanic potentiation is manifested earlier in calcium-containing media than in calcium-deficient media. This difference may indicate that sodium entry into the terminal during tetanic stimulation is at locations remote from the releasing sites. Alternatively, this time difference may be due to the delay between intracellular sodium accumulation and the increase in transmitter release.

Action Potentials↗

Adrenocorticotropic hormone causes long-lasting potentiation of transmitter release from frog motor nerve terminals.

Exposure of frog neuromuscular preparations to adrenocorticotropic hormone for several minutes increased both nerve-evoked and spontaneous transmitter release for several hours. No changes in postsynaptic sensitivity to transmitter were detected. The long-lasting potentiation shows little sensitivity to changes in extracellular calcium concentration and seems to be entirely presynaptic in origin.

Action Potentials↗

Stereospecific glucose transport across motor nerve terminal membrane: an electrophysiological study.

Spontaneous transmitter release at the neuromuscular junction of the frog and rat was monitored during exposures to hyperosmotic solutions containing different sugars. Raising the osmolarity of the medium with D-glucose causes a marked, but transient, increase in the frequency of miniature end-plate potentials (MEPPs): after the initial elevation in frequency there is a subsequent decline towards the control levels, in spite of a continuous perfusion with the hyperosmotic solution. This decline occurs more rapidly in the frog. Two nonmetabolized analogues of glucose, 2-deoxy-D-glucose and 3-O-methylglucose, cause a transient hyperosmotic increase in MEPP frequency, which is very similar to the effect of D-glucose. The elevation of MEPP frequency with hyperosmotic glucose is stereospecific. Hyperosmotic solutions of L-glucose cause a sustained increase in transmitter release in the rat and frog. Insulin dramatically reduces the response of the frog nerve terminal to hyperosmotic D-glucose. Phenolphthalein, a glucose transport blocker, reduces or eliminates the secondary decline in MEPP frequency. It is suggested that the transient nature of the response to hyperosmotic solutions reflects the penetration of the hyperosmotic agent into the nerve terminal. The rate of decline of the MEPP frequency presumably indicates the rate of transport, which determines the rate of osmotic equilibration. This rate can then serve as an index of the relative permeability of the functioning presynaptic membrane to different sugars.

Animals↗

Regulatory role of intracellular sodium ions in neurotransmitter secretion.

Calcium ions are the main inducer of quantal transmitter release of the frog neuromuscular junction; but even in their virtual absence from the extracellular medium, nerve stimulation causes a prolonged augmentation of transmitter release. These facts led to the hypothesis that an accumulation of intracellular sodium can serve as a slow secondary regulator of neurosecretion. Three lines of evidence presented in this article substantiate this hypothesis: firstly, veratridine, which is known to increase sodium fluxes through the voltage-dependent sodium channels, increases transmitter release after nerve stimulation. Secondly, monensin, which was shown to induce sodium transport through nerve membranes, increases evoked transmitter release, tetanic potentiation and posttetanic potentiation. Thirdly, sodium-filled phosphatidylcholine liposomes increase transmitter release. These effects of sodium are probably not due to a direct effect on the transmitter release mechanism, but are caused by sodium-induced calcium translocation from intracellular stores.

Animals↗

Noradrenaline augments tetanic potentiation of transmitter release by a calcium dependent process.

Noradrenaline (25 microM-50 microM) causes an increase in tetanic potentiation and in the augmentation phase of posttetanic potentiation of miniature and plate potential frequency. These effects were observed at both the frog and the rat neuromuscular junctions. The action of noradrenaline on quantal transmitter release depends on the presence of calcium ions in the extracellular medium.

Animals↗

The action of the sodium ionophore, monensin, or transmitter release at the frog neuromuscular junction.

The action of the sodium ionophore, monensin, on spontaneous and evoked transmitter release at the frog neuromuscular junction was studied. Ringer's solutions with low calcium concentrations (0.4 mM or 0 Ca 1 mM EGTA) were used to bathe the preparation. Following addition of monensin in the bathing solution: (1) substantial increases in miniature end-plate potential frequency occurred; (2) tetanic nerve stimulation caused increases in potentiation of approximately 10 times over control values; (3) there was a dual action on the amplitude of the end-plate potential. We conclude that sodium ions take part in the regulation of transmitter release at the neuromuscular junction.

Animals↗

A study of tetanic and post-tetanic potentiation of miniature end-plate potentials at the frog neuromuscular junction.

1. The involvement of calcium sodium, potassium and magnesium in tetanic and post-tetanic potentiation of miniature end-plate potential frequency was examined at the frog neuromuscular junction using conventional electrophysiological techniques. 2. Tetanic potentiation is larger in calcium containing solutions, than in solutions which generate reversed electrochemical gradient for calcium during nerve activity. 3. Tetanic potentiation increases with stimulation frequency and duration, under both inward and reversed electrochemical gradient for calcium conditions. This indicates that factors, other than calcium entry, participate in tetanic potentiation. 4. Addition of the potassium conductance blocking agent, 3-aminopyridine (5 mM), increases tetanic potentiation in calcium containing media, while depressing it under reversed calcium gradient. 5. Electronic depolarization of the nerve terminal in tetrodotoxin-containing Ringer solution, produces tetanic potentiation under inward gradient, but fails to do so under reversed gradient. This indicates that the entry of sodium ions participates in the generation of tetanic potentiation. 6. Addition of magnesium ions suppresses tetanic potentiation in calcium containing solution, but increases tetanic potentiation under reversed gradient. 7. The results are explained by the hypothesis that calcium entry and intracellular calcium translocation participate in the generation of tetanic potentiation. 8. Both the fast and the slow components (augmentation and potentiation respectively) of post-tetanic potentiation increase in duration, with increase in the tetanic stimulation rate. 9. The decay of post-tetanic potentiation increases: when [Ca]o is elevated by ionophoretic application during the decay phase only, when ouabain is present in the medium or when [Mg]o is elevated. These finding suggest that calcium, sodium and possibly magnesium take part in post-tetanic potentiation.

Animals↗