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.

A short-chain peptide toxin isolated from Centruroides sculpturatus scorpion venom inhibits ether-à-go-go-related gene K(+) channels.

From the venom of the American scorpion Centruroides sculpturatus Ewing we have isolated a minute peptide fraction (named CsEKerg1) which reversibly inhibits the current through ERG (ether-à-go-go-related gene) K(+) channels. Isolation was done by CM-cellulose column chromatography and reversed phase high-performance liquid chromatography. To test for an effect on ERG channels we used NG108-15 neuroblastomaxglioma hybrid cells voltage-clamped in the whole-cell mode. CsEKerg1 contains 43 amino acids and has a molecular weight of 4833. Its amino acid sequence is similar but not identical to that of ergtoxin, a peptide isolated recently from the venom of the Mexican scorpion Centruroides noxius [FASEB J. 13 (1999) 953]. Half inhibition of ERG current occurs at a peptide concentration of 1.12microg/ml.

Slowing of ERG current deactivation in NG108-15 cells by the histidine-specific reagent diethylpyrocarbonate.

The aim of this study was to explore and characterize the effect of the histidine-specific reagent diethylpyrocarbonate (DEPC) on the ERG (ether-à-go-go related gene) channels of whole-cell voltage-clamped NG108-15 neuroblastoma x glioma hybrid cells. The channels were fully activated by long depolarizing prepulses. Hyperpolarizing pulses elicited K+ inward currents which deactivated after reaching a peak. DEPC (0.26-2.1 mM, externally applied for 5-12 min) irreversibly decreased tau(-1), the rate constant of deactivation. At a pulse potential of -120 mV x tau(-1) decreased on average by 60%. The effect can be described as a -25 mV shift of the tau(-1)(V) curve. The activation curve and the curve relating steady-state current to pulse potential were shifted by similar amounts. The decrease of tau(-1) was the same at 0.26 and at 2.1 mM, but developed faster at the higher concentration. The slowing of deactivation was only seen when the cells were held at a potential of -20 mV. At this potential it developed with a time constant of 47 s. At more negative holding potentials (-40 or -70 mV) only a slight reduction of the peak occurred. The observations suggest preferential binding of DEPC to the open and inactivated channel states. The DEPC effect can possibly be explained by the reaction of DEPC with histidine residues or other amino acids in the external loops of the channel. However, dichloro-(2,2':6',2"-terpyridine)-platinum (II) dihydrate (DTPD), another histidine-specific reagent, markedly decreased the peak current without affecting tau(-1). Therefore, the possibility that (some of) the effects of DEPC and DTPD are unrelated to their property as histidine-specific reagents cannot be excluded.

Effect of low external calcium on the ERG current of NG108-15 cells.

K(+) currents through ERG (ether-à-go-go related gene) channels were recorded in whole-cell voltage clamped NG108-15 neuroblastomaxglioma hybrid cells. The channels were fully activated by low holding potential (V(H)=-20 mV) and long depolarizing prepulses. Hyperpolarizing pulses elicited inward currents which deactivated after reaching a peak. Lowering [Ca(2+)](o) from 5 to 1. 5 or 0.5 mM decreased tau(-1), the rate constant of deactivation. The effect can be explained by a shift of the tau(-1)(V) curve to more negative potentials caused by an increase in surface charge density. Plotting tau(-1) against [Ca(2+)](o) for different potentials yielded straight lines; their slope was independent of potential at -140 to -120 mV and decreased at more positive potentials. The time to peak curve and the maximum of the steady-state inward current were also shifted to more negative potentials. In addition, peak ERG inward current increased. Raising [Ca(2+)](o) from 5 to 10 mM accelerated deactivation and decreased the peak current. 5 mM Ba(2+) affected tau(-1) similarly and inhibited peak current more strongly whereas 5 mM Mg(2+) was less potent. As found by Faravelli et al. (J. Physiol. 496 (1996) 13), bath solutions devoid of divalent cations (0 Ca(2+), 0 Mg(2+), 0.1 or 1.1 mM EGTA) abolished deactivation almost completely. The phenomenon was seen with bath containing either 40 or 6.5 mM K(+). Its occurrence was favored by raising the temperature to 34 degrees C. It suggests a particular requirement of channel closing for Ca(2+).

Inactivation of the ERG current in NG108-15 cells.

Differentiated NG108-15 neuroblastoma x glioma hybrid cells were whole-cell voltage clamped. The rate of inactivation of ERG (ether-à-go-go related gene) potassium channels was measured with a three-pulse protocol. Contamination with delayed rectifier current at positive potentials was avoided by using the selective ERG channel blocker E-4031. The curve relating time constant of inactivation tau to membrane potential V could be fitted by a Gauss curve. In a bath with 40 mM K(+), the curve peaked at V = -36 mV. Lowering [K(+)](o) decreased tau. At V = -20 mV, the average tau was 25.4 ms in 40 mM K(+), 20.6 ms in 6.5 mM K(+), and 15.0 ms in 0 mM K(+). This resembles the relation between tau and [K(+)](o) in ERG channels expressed in Xenopus oocytes.

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

Differentiated NG108-15 neuroblastoma x 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 microM selectively inhibited the fast component (IC50 = 14.7 microM). The slow component was less sensitive to linopirdine (IC50>20 microM). The class III antiarrhythmics [(4-methylsulphonyl)amido]benzenesulphonamide (WAY-123.398) and 1-[2-(6-methyl-2-pyrydinil)ethyl]-4-(4-methylsulphonylaminobenz oyl) 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 microM). 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, EAGI, 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.

1. Bradykinin has multiple effects on differentiated NG108-15 neuroblastoma x 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. 2. Preincubation with 1 or 5 microM U73122 for 15 min partly suppressed Ins(1,4,5)P3 generation and the increase in [Ca2+]i induced by 1 microM bradykinin. U73122 10 microM caused total and irreversible inhibition. The inactive analogue U73343 was without effect. 3. Resting levels of Ins(1,4,5)P3 were not affected. However, resting [Ca2+]i was increased by 10 microM U73122, but not by U73343. Individual cells responded to 10 microM U73122 with a small increase in [Ca2+]i, followed in some cells by a large further rise. 4. Pretreatment of whole-cell clamped cells with 1 microM 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 microM U73122 was required. Again, U73343 was without effect. 5. U73122 and U73343 at concentrations of 5-10 microM 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. 6. M current (IM) measured with 1 s pulses, was also decreased by 5-10 microM U73122 and U73343, but short applications of U73122 could cause a small increase. The bradykinin-induced inhibition of IM was not affected by U73122. 7. 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. 8. 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.

Modulation of neuronal calcium channels by arachidonic acid and related substances.

Low-voltage-activated (l-v-a) and high-voltage-activated (h-v-a) Ca2+ currents (ICa) were recorded in whole-cell voltage clamped NG108-15 neuroblastoma x glioma hybrid cells. We studied the effects of arachidonic acid (AA), oleic acid, myristic acid and of the positively charged compounds tetradecyltrimethylammonium (C14TMA) and sphingosine. At pulse potentials > -20 mV, AA (25-100 microM) decreased l-v-a and h-v-a ICa equally. The decrease developed slowly and became continually stronger with increasing time of application. It was accompanied by a small negative shift and a slight flattening of the activation and inactivation curves of the l-v-a ICa. The shift of the activation curve manifested itself in a small increase of l-v-a ICa at pulse potentials < -30 mV. The effects were only partly reversible. The AA effect was not prevented by 50 microM 5, 8, 11, 14-eicosatetraynoic acid, an inhibitor of the AA metabolism, and not mimicked by 0.1-1 microM phorbol 12, 13-dibutyrate, an activator of protein kinase C. Probably, AA directly affects the channel protein or its lipid environment. Oleic and myristic acid acted similarly to AA but were much less effective. The positively charged compounds C14TMA and sphingosine had a different effect: They shifted the activation curve of l-v-a ICa in the positive direction and suppressed l-v-a more than h-v-a ICa; their effect reached a steady-state within 5-10 min and was readily reversible. C14TMA blocked l-v-a ICa with an IC50 of 4.2 microM while sphingosine was less potent.

Model experiments on squid axons and NG108-15 mouse neuroblastoma x rat glioma hybrid cells.

Three types of ionic current essentially determine the firing pattern of nerve cells: the persistent Na+ current, the M current and the low-voltage-activated Ca(2)+ current. The present article summarizes recent experiments concerned with the basic properties of these currents. Keynes and Meves (Proc R Soc Lond B (1993) 253, 61-68) studied the persistent or steady-state Na+ current on dialysed squid axons and measured the probability of channel opening both for the peak and the steady-state Na+ current (PF(peak) and PF(ss)) as a function of voltage. Whereas PF(peak) starts to rise at -50 mV and reaches a maximum at +40 to +50 mV, PF(ss) only begins to rise appreciably at around 0 mV and is still increasing at +100 mV. This differs from observations on vertebrate excitable tissues where the persistent Na+ current tums on in the threshold region and saturates at around 0 mV. Schmitt and Meves (Pflugers Arch (1993) 425, 134-139) recorded M current, a non-inactivating K+ current, from NGI08-15 neuroblastoma x glioma hybrid cells, voltage-clamped in the whole-cell mode, and studied the effects of phorbol 12,13-dibutyrate (PDB), an activator of protein kinase C (PKC), and arachidonic acid (AA). PDB and AA both decreased I(M), the effective concentrations being 0.1-1 mu M and 5-25 mu M, respectively; while the PDB effect was regularly observed, the M current depression by AA was highly variable from cell to cell. The PKC 19-31 peptide, an effective inhibitor of PKC, in a concentration of 1 muM almost totally prevented the effects of PDB and AA on M current, suggesting that both are mediated by PKC. Schmitt and Meves (Pflugers Arch (1994a) 426, Suppl R 59) measured low-voltage-activated (l-v-a) and high-voltage-activated (h-v-a) Ca2+ currents on NG108-15 cells and investigated the effect of AA and PDB on both types of current. At pulse potentials > -20 mV, AA (25-100 mu M) decreased 1-v-a and h-v-a I(Ca). The decrease was accompanied by a small negative shift and a slight flattening of the activation and inactivation curves of the l-v-a I(Ca). The AA effect was not prevented by 50 mu M eicosa-5,8,11,14-tetraynoic acid (ETYA), an inhibitor of AA metabolism, or PKC 19-31 peptide and not mimicked by 0.1-1 mu M PDB. Probably, AA acts directly on the channel protein or its lipid environment. The physiological relevance of these three sets of observations is briefly discussed.

Bovine serum albumin selectively increases the low-voltage-activated calcium current of NG108-15 neuroblastoma x glioma cells.

The effect of bovine serum albumin (BSA) on the neuronal Ca2+ current (ICa) has been studied in NG108-15 cells. BSA selectively increased the low-voltage-activated (l-v-a), transient ICa but did not affect the high-voltage-activated (h-v-a) ICa. The increase of l-v-a ICa was partly due to a small shift of its activation curve to more negative values of membrane potential and partly to an increase of the maximal activatable current. Fatty-acid-free (FA-free) BSA was about equally effective as ordinary BSA; the smallest effective concentration of FA-free BSA was between 0.01 and 0.1 mg/ml.

Protein kinase C as mediator of arachidonic acid-induced decrease of neuronal M current.

The M current, IM, of NG108-15 neuroblastoma x glioma hybrid cells, a non-inactivating K+ current, is decreased by arachidonic acid (5-25 microM), often after an initial transitory increase. To test the possibility that the decrease is caused by activation of protein kinase C (PKC) we used the PKC 19-31 peptide, which is an effective inhibitor of PKC. With 1 microM peptide in the pipette solution the normally observed strong reduction of IM by 1 microM phorbol 12,13-dibutyrate (PDB) was almost totally prevented, indicating that PKC is completely inhibited; also the voltage dependence of the M conductance, gM(V), was shifted to more negative membrane potentials. In the presence of 1 microM peptide the effect of 25 microM arachidonic acid on IM was significantly reduced, suggesting that the effect, or at least a large part of it, is mediated by PKC.

Properties of the voltage sensor for the opening and closing of the sodium channels in the squid giant axon.

A combination of data from standard I-V curves, and from steps applied either at the initial current peak or in the inactivated steady state, yielded values of the total probability of the two open states of the sodium channel, multiplied by a constant scaling factor, as a function of membrane potential. The probability function PFpeak was found to reach a maximum for pulses to 40-50 mV, but for larger test potentials it underwent a slight decline. The curve for its rise was shifted in a positive direction by several millivolts when the temperature was raised. Measurements of the probability function PFss in the final steady condition, when almost the whole population of channels was inactivated, but a small flow of Na+ current persisted, showed that the voltage sensor responsible for the actual opening of the channels carried 0.8 electronic charges, and that its equilibrium potential had been shifted nearly 100 mV by inactivation to lie close to 50 mV. The charge carried by the C<-->O voltage sensor was the same for all the dialysis and bathing solutions that were tested, but when dialysing with 350 mM NaF and bathing with full Na seawater plus 16 nM TTX, the equilibrium potential in the inactivated state was increased by about 25 mV.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of arachidonic acid on the M current of NG108-15 neuroblastoma x glioma hybrid cells.

The M current, IM, a voltage-dependent non-inactivating K+ current, was recorded in NG108-15 neuroblastoma x glioma hybrid cells, using the whole-cell mode of the patch-clamp technique. We studied the effect of arachidonic acid, other fatty acids and inhibitors of the arachidonic acid metabolism. In relatively high concentrations (25-50 microM) arachidonic acid first increased and later decreased the current, Ih, which holds the membrane potential at -30 mV and mainly flows through open M channels. It shifted the midpoint potential, Vo, of the relation between M conductance, gM, and membrane potential, V, to more negative values and decreased the maximum conductance gM and the time constant tau M. In smaller concentrations (5-10 microM) arachidonic acid merely decreased Ih and gM with little effect on Vo and tau M. Eicosatetraynoic acid and docosahexaenoic acid acted similarly to arachidonic acid whereas stearic acid had no effect. Of the three enzyme inhibitors studied, nordihydroguaiaretic acid acted similarly to arachidonic acid. i.e. caused a biphasic change in Ih. Indomethacin and quinacrine caused, respectively, a pure increase and a pure decrease of Ih and gM. Possible explanations are build-up of internally produced arachidonic acid, depletion of eicosanoid products or an inhibitory effect unrelated to arachidonic acid metabolism.

The dual effect of internal tetramethylammonium ions on the open states of the sodium channel in the squid giant axon.

Voltage-clamp recordings of INa in squid axons dialysed with Cs or TMA, and bathed in low Na choline seawater, showed that, except close to threshold, the initial peak of fast-inactivating current was invariably decreased by TMA, whereas the non-inactivating current in the steady state was simultaneously increased. The results suggest that although TMA does not act directly on the movements of the voltage sensors that activate the sodium system, it blocks single-channel conductance in a voltage-dependent fashion in both the open states of the Na channel, while it has an entirely different type of action by increasing the probability of late openings in the steady state. Another difference between the two open states was that the sodium permeability coefficient had a Q10 of 1.8 in the initial open state, whereas in the steady state the effect of temperature was much smaller or even negative.

The effect of phloretin on single potassium channels in myelinated nerve.

The effect of phloretin, a dipolar organic compound, on single potassium channel currents of myelinated nerve fibres of Xenopus laevis has been investigated, using inside-out patches prepared by the method of Jonas et al. (1989). The I channel, a potential dependent K channel with intermediate deactivation kinetics, was reversibly blocked by 20 microM phloretin applied on the inside; the block was strongest at negative membrane potentials and less pronounced at positive potentials. Phloretin shifted the curve relating open probability to membrane potential towards more positive potentials and reduced its slope and maximum. This confirms previous findings on the effect of phloretin on the voltage dependence of the fast macroscopic K conductance. Single channel conductance and deactivation kinetics were not altered by phloretin.

Inhibition of the M current in NG 108-15 neuroblastoma x glioma hybrid cells.

The M current, IM, a voltage-dependent non-inactivating K current, was recorded in NG108-15 neuroblastoma x 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 microM bradykinin inhibited IM by about 60%; 5 nM bradykinin inhibited by about 40%. Bath application of 0.1 microM and 1 microM 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 microM significantly reduced the effect of 1 microM PDBu. M current blockage by PDBu could be partly reversed by bath application of H-7 (51-64 microM), 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 microM PDBu was fully developed, 0.1 microM bradykinin produced a further inhibition of IM. Down-regulation of PKC by long-term treatment with PDBu reduced the effect of 0.1 microM bradykinin significantly but did not abolish it. Staurosporine (0.3 microM, 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, isobutyl-methylxanthine, theophylline) in the millimolar range, probably because of a direct action on the M channels.

Phloretin affects the fast potassium channels in frog nerve fibres.

The effect of phloretin (20-100 microM), a dipolar organic compound, on the voltage clamp currents of the frog node of Ranvier has been investigated. The Na currents are simply reduced in size but not otherwise affected. Phloretin has no effect on the slow 4-aminopyridine-resistant K channels. However, the voltage dependence and time course of the fast K conductance (gK) is markedly altered. The gK (E) curve, determined by measuring fast tail currents at different pulse potentials, normally exhibits a bend at -50 mV, indicating the existence of two types of fast K channels. Phloretin shifts the gK (E) curve to more positive potentials, reduces its slope and its maximum and abolishes the distinction between the two types of fast K channels. The effect becomes more pronounced with time. Phloretin also markedly slows the opening of the fast K channels, but has much less effect on the closing. Opening can be accelerated again by a long depolarizing prepulse which presumably removes part of the phloretin block. It is concluded that phloretin selectively affects the fast K channels of the nodal membrane. The results are compared with similar observations on the squid giant axon.

A slow component in the gating current of the frog node of Ranvier.

The slow component of the gating current on-response has been studied on voltage-clamped nodes of Ranvier of the frog Rana esculenta. At 0 mV and 20 degrees C the charge and the time constant of the slow component were on average 37.1 fC and 0.39 ms, respectively. The slow component could be abolished by shifting the holding potential from its usual value (-100 mV) to -60 mV. Repolarization to -100 mV for 2 ms was sufficient for complete recovery of the slow component. The local anaesthetic benzocaine (1 mM) reduced the fast component (measured at 0 mV) on average to 54% and the slow component (also measured at 0 mV) to 70% of the control value. In the potential range from -60 to 60 mV the charge of the slow component increased slightly with voltage. No significant voltage dependence of its time constant was observed. The slow component most likely reflects charge movement between different open states; it does not seem to be related to inactivation of the sodium channels or activation of the potassium channels.