Hans Meves

Arachidonic acid and ion channels: an update

Arachidonic acid (AA), a polyunsaturated fatty acid with four double bonds, has multiple actions on living cells. Many of these effects are mediated by an action of AA or its metabolites on ion channels. During the last 10 years, new types of ion channels, transient receptor potential (TRP) channels, store‐operated calcium entry (SOCE) channels and non‐SOCE channels have been studied. This review summarizes our current knowledge about the effects of AA on TRP and non‐SOCE channels as well as classical ion channels. It aims to distinguish between effects of AA itself and effects of AA metabolites. Lipid mediators are of clinical interest because some of them (for example, leukotrienes) play a role in various diseases, others (such as prostaglandins) are targets for pharmacological therapeutic intervention.

Effect of toxins isolated from the venom of the scorpionCentruroides sculpturatus on the Na currents of the node of ranvier

Abstract1.The effect of various toxin fractions isolated by Watt et al. (1978) from the venom of the scorpionCentruroides sculpturatus Ewing on the Na currents of the node of Ranvier has been studied with the voltage clamp method.2.The toxin fractions were applied externally. The most potent fractions were toxins III, IV and V which were effective in concentrations of 0.33–3.33 μg/ml. The effect of toxins III and IV was quite different from that of toxin V.3.In toxin III or IV — treated nodes a strong depolarizing pulse was followed by a transient shift of the negative resistance branch of theINa (E) curve to more negative potentials. The amount of shift varied between −10 and −60 mV. A 500ms depolarizing pulse of small amplitude produced a slowly developing Na inward current which slowly decayed after the end of the pulse. Inactivation was incomplete, even with 500 ms pulses to 0 mV.4.The transient shift of theINa (E) curve was not seen in nodes treated with toxin V. This toxin merely caused slow and incomplete Na inactivation. The effect of toxin IV was not suppressed by a four times higher concentration of toxin V, suggesting that the two toxins act on different receptors.5.Toxin I acted like toxin IV but was about 10 times less potent. The effect of high concentrations of variants 1, 2, 3, 5, 6 resembled that of toxin V.6.All effects observed with toxin III or IV were also seen with the whole venom (cf. Cahalan 1975).

The ionic requirements for the production of action potentials in helix pomatia neurones.

Summary1.The effect of polarizing currents, ions and drugs on the action potentials of individual neurones in the sub-oesophageal ganglion of the snail Helix pomatia has been examined.2.The current-voltage relation showed a marked decrease of resistance for the outward current (delayed rectification). AtI=0, the average membrane resistance was 8.3 kΩ×cm2.3.Tetrodotoxin in a concentration of 4.6×10−6 g/ml did not affect the overshoot or the maximum rate of rise. 0.4% cocaine inhibited delayed rectification, but did not abolish the action potential.4.Ringer solution with twice the normal Na concentration augmented the overshoot by 5–6 mV and increased the maximum rate of rise to 130% of its normal value. Na-free Ringer decreased the overshoot by only 5–8 mV and reduced the maximum rate of rise to about half its normal value.5.Even after prolonged perfusion with Na-free solution action potentials were still obtained. The overshoot stayed constant during a 2 hours perfusion period whereas the maximum rate of rise declined slowly. Na-free Ringer with Mn depressed the spike.6.Raising the Ca concentration of the Na-free Ringer increased the overshoot (average 15.9 mV for a 10-fold increase of [Ca]0), shifted the threshold potential towards zero and slightly augmented the maximum rate of rise.7.Nominally Ca-free solution markedly reduced the action potential. Excitability was maintained in Na- and Ca-free Ringer with 10 mM Sr or Ba.8.Part of the results could be explained by assuming a reservoir of Na+ ions in or close to the cell membrane. An alternative explanation is that Ca++ ions participate in carrying charge across the membrane during the rising phase of the action potential. The second hypothesis appears more likely although a definite decision could not be reached.

Interactions of Scorpion Toxins with the Sodium Channel

It is evident from the data reviewed that scorpion toxins can be distinguished on the basis of three properties: their effects on Na currents, their specific binding to excitable membranes, and the effects of depolarization and pH on binding and on effect. Additional work with other scorpion toxins is required to establish the degree of correlation between the three properties for each class of toxin. Further investigations with this family of homologous proteins will undoubtedly contribute not only to our understanding of the toxins themselves but also to our understanding of the structure and function of the Na channel.

Separation of M-like current and ERG current in NG108-15 cells

Differentiated NG108‐15 neuroblastoma×glioma hybrid cells were whole‐cell voltage‐clamped. Hyperpolarizing pulses, superimposed on a depolarized holding potential (−30 or −20 mV), elicited deactivation currents which consisted of two components, distinguishable by fitting with two exponential functions. Linopirdine [DuP 996, 3,3‐bis(4‐pyridinylmethyl)‐1‐phenylindolin‐2‐one), a neurotransmitter‐release enhancer known as potent and selective blocker of the M‐current of rat sympathetic neurons, in concentrations of 5 or 10 μM selectively inhibited the fast component (IC50=14.7 μM). The slow component was less sensitive to linopirdine (IC50>20 μM). The class III antiarrhythmics [(4‐methylsulphonyl)amido]benzenesulphonamide (WAY‐123.398) and 1‐[2‐(6‐methyl‐2‐pyrydinil)ethyl]‐4‐(4‐methylsulphonylaminobenzoyl) piperidine (E‐4031), selective inhibitors of the inwardly rectifying ERG (ether‐à‐go‐go‐related gene) potassium channel, inhibited predominantly the slow component (IC50=38 nM for E‐4031). The time constant of the WAY‐123.398‐sensitive current resembled the time constant of the slow component in size and voltage dependence. Inwardly rectifying ERG currents, recorded in K+‐rich bath at strongly negative pulse potentials, resembled the slow component of the deactivation current in their low sensitivity to linopirdine (28% inhibition at 50 μM). The size of the slow component varied greatly between cells. Accordingly, varied the effect of WAY‐123.398 on deactivation current and holding current. RNA transcripts for the following members of the ether‐à‐go‐go gene (EAG) K+ channel family were found in differentiated NG108‐15 cells: ERG1, ERG2, EAG1, EAG‐like (ELK)1, ELK2; ERG3 was only present in non‐differentiated cells. In addition, RNA transcripts for KCNQ2 and KCNQ3 were found in differentiated and non‐differentiated cells. We conclude that the fast component of the deactivation current is M‐like current and the slow component is deactivating ERG current. The molecular correlates are probably KCNQ2/KCNQ3 and ERG1/ERG2, respectively.

The effects of bradykinin on K+ currents in NG108–15 cells treated with U73122, a phospholipase C inhibitor, or neomycin

Bradykinin has multiple effects on differentiated NG108–15 neuroblastoma×glioma cells: it increases Ins(1,4,5)P3 production and intracellular Ca2+ concentration [Ca2+]i, evokes a Ca2+ activated K+ current (IK(Ca)) and inhibits M current (IM). We studied the effect of the aminosteroid U73122 and the antibiotic neomycin, both putative blockers of phospholipase C (PLC), on these four bradykinin effects. Preincubation with 1 or 5 μm U73122 for 15 min partly suppressed Ins(1,4,5)P3 generation and the increase in [Ca2+]i induced by 1 μm bradykinin. U73122 10 μm caused total and irreversible inhibition. The inactive analogue U73343 was without effect. Resting levels of Ins(1,4,5)P3 were not affected. However, resting [Ca2+]i was increased by 10 μm U73122, but not by U73343. Individual cells responded to 10 μm U73122 with a small increase in [Ca2+]i, followed in some cells by a large further rise. Pretreatment of whole‐cell clamped cells with 1 μm U73122 for 30 min reduced the bradykinin‐induced IK(Ca) to a fifth of its normal size. To suppress it totally, a 7–12 min pretreatment with 5 μm U73122 was required. Again, U73343 was without effect. U73122 and U73343 at concentrations of 5–10 μm irreversibly decreased the holding current (Ih) which at a holding potential of −30 or −20 mV mainly flows through open M channels. The decrease was often preceded by a transient increase. M current (IM) measured with 1 s pulses, was also decreased by 5–10 μm U73122 and U73343, but short applications of U73122 could cause a small increase. The bradykinin‐induced inhibition of IM was not affected by U73122. Preincubation with 1 or 3 mm neomycin for 15 min did not affect Ins(1,4,5)P3 generation and the increase in [Ca2+]i induced by bradykinin. Pretreatment with 3 mm neomycin for about 20 min diminished the bradykinin‐induced IK(Ca) to a fifth of its normal size. The four main conclusions drawn from the results are: (a) U73122 suppresses bradykinin‐induced PLC activation and IK(Ca), but not IM inhibition. (b) This indicates that the transient outward current IK(Ca), but not the decrease of IM in response to bradykinin, is mediated by PLC. (c) U73122 itself inhibits IM and mobilizes Ca2+ from intracellular stores. (d) Externally applied neomycin is not an effective inhibitor of PLC‐mediated signalling pathways in NG108–15 cells.

Inhibition of the M current in NG108-15 neuroblastoma × glioma hybrid cells

The M current, IM, a voltage-dependent non-inactivating K current, was recorded in NG108-15 neuroblastoma × glioma hybrid cells, using the whole-cell mode of the patch-clamp technique. We studied inhibition of the M current by bradykinin, phorbol dibutyrate (PDBu), an activator of protein kinase C (PKC), and methylxanthines. Focal application of 0.1–5 μM bradykinin inhibited IM by about 60%; 5 nM bradykinin inhibited by about 40%. Bath application of 0.1 μM and 1 μM PDBu diminished IM to about half of the control value. Staurosporine, a PKC inhibitor, applied for 35–43 min in a concentration of 0.3 μM significantly reduced the effect of 1 μM PDBu. M current blockage by PDBu could be partly reversed by bath application of H-7 (51–64 μM), another PKC inhibitor. These observations suggest that the PDBu effect is really due to activation of PKC. The findings are compatible with the view [Brown DA, Higashida H (1988) J Physiol (Lond) 397:185–207] that the bradykinin effect on IM is mediated by PKC. However, three further observations suggest that this is only true for part of the bradykinin effect. When the suppression of IM by 1 μM PDBu was fully developed, 0.1 μM bradykinin produced a further inhibition of IM. Down-regulation of PKC by long-term treatment with PDBu reduced the effect of 0.1 μM bradykinin significantly but did not abolish it. Staurosporine (0.3 μM, applied for 31–46 min) failed to reduce the effect of 5 nM bradykinin significantly. The M current could be reversibly blocked by methylxanthines (caffeine, isobutylmethylxanthine, theophylline) in the millimolar range, probably because of a direct action on the M channels.

Die Nachpotentiale isolierter markhaltiger Nervenfasern des Frosches bei tetanischer Reizung

Summary1. The behaviour of the after-potentials of single Ranvier nodes during repetitive stimulation has been analyzed with special reference to effects that could be interpreted as being due to a transient augmentation or diminuition of the potassium concentration in the perinodal space.2. The after-depolarizations which are observed at anodal polarized nodes in ordinary or K+-rich Ringers solution do not add during a train of impulses.3. The absolute membrane potential at the crest of the after-hyperpolarizations which are found in K+-free Ringers solution increases progressively during rapid tetanic stimulation and reaches a steady level which depends on the frequency and duration of the tetanus.4. Rapid repetitive stimulation is followed by a posttetanic hyperpolarization which declines with a half-time of 10–40 msec. The size of the posttetanic hyperpolarization is maximal in K+-free Ringers solution; it is not reduced by 2,4-dinitrophenol.5. The amplitude of the posttetanic hyperpolarization increases with increasing frequency and duration of the tetanus, until a maximum is reached. The early phase of the posttetanic hyperpolarization is accompanied by a slight reduction of membrane resistance.6. It is concluded from the observations under 2)–4) that an appreciable accumulation of released potassium in the perinodal space does not occur and that the posttetanic hyperpolarization of isolated nodes is not due to a transient depletion of potassium from the perinodal space. Unlike the situation in the giant axons of squid and cockroach and in C-fibre-bundles, an effective external barrier to diffusion does not exist at the isolated node.7. The increase of the absolute membrane potential at the crest of the after-hyperpolarizations during repetitive stimulation and the posttetanic hyperpolarization are supposed to reflect a cumulative increase of the potassium permeability of the membrane which builds up during the tetanus and outlasts its end.

Voltage-dependent effect of a scorpion toxin on sodium current inactivation

Abstract1.In voltage clamped nodes of Ranvier inactivation of the sodium permeability is slowed by toxin V from the scorpionCentruroides sculpturatus, by sea anemone toxin ATX II or by internally applied KIO3. The slow decay of the Na inward current is markedly accelerated if the test pulse is preceded by a depolarizing conditioning pulse followed by a 10–500 ms pause. This phenomenon was studied in detail, using conditioning pulses of varying amplitude and up to 15 s duration.2.In nodes treated with toxin V a 20 ms conditioning pulse to positive potentials was sufficient to produce a clear acceleration of the decay of the Na current and a reduction of the inward current remaining at the end of a 50 ms test pulse, i.e. a weakening of the toxin effect. In nodes treated with ATX II or internal KIO3 longer conditioning pulses were required. A similar effect of conditioning pulses on the decaying phase of the Na current was also observed in untreated fibres.3.To study the phenomenon quantitatively we fitted the decaying phase of the inward Na current with the equationINa=A exp(-t/τ1)+B exp(-t/τ2)+C The effect of depolarizing conditioning pulses could be described as an increase of A, a decrease of B and C and a reduction of the time constants τ1 and τ1.4.I50/Ipeak, the normalised inward current remaining at the end of a 50 ms test pulse, decreased exponentially with increasing duration of the conditioning pulse to a steady-state value. The time constant τ and the steady-state value depended on the potential during the conditioning pulse. For nodes treated with toxin V, τ was 0.24 s at 0 mV and 12° C and half inhibition occurred at −42 mV. The time constant τ was larger for nodes treated with ATX II or internal KIO3. At positive potentials, I50 was reduced to 20% of the control value in toxin V-treated nodes, but only to 70% in KIO3-treated nodes.5.Recovery from the effect of the conditioning pulse was studied by varying the pause between conditioning pulse and test pulse; recovery was 66–100% complete after 500 ms.6.The results are interpreted by assuming that a sepolarizing conditioning pulse (a) accelerates inactivation of the sodium permeability and (b) causes dissociation of the toxin-receptor complex or transition into an inactive state. The latter effect occurs in toxin V-treated fibres but not in those treated with ATX II or KIO3.

The effect of veratridine on internally perfused giant axons.

SummaryThe effect of adding small amounts of veratridine to the internal fluid of perfused squid giant axons was investigated. 2×10−5 g/ml veratridine applied internally caused a transient depolarization of 50 mV whereas 8×10−6 g/ml had little effect on the resting potential. The spike was followed by a long lasting after-depolarization of up to 19 mV amplitude which decayed in a non-exponential manner. Repetitive activity following a single brief shock was observed. Repetitive stimulation (4/sec) produced non-linear addition of the after-depolarizations. The effects of internally applied veratridine were fully reversible. The findings are compared with the effects of externally applied veratridine on squid axons.