Modulation of Ca(2+)-dependent currents in metabolically stressed cultured sensory neurones by intracellular photorelease of ATP.

1. The whole cell recording technique was used to study high voltage-activated Ca2+ currents and Ca(2+)-activated Cl- tail currents from cultured neonatal dorsal root ganglion neurones of the rat which were metabolically stressed. The neurones were metabolically stressed with 2-deoxy-D-glucose (5 mM) for 30 min to 3 h. The aim of the project was to examine the actions of intracellular photorelease of ATP on the properties of Ca(2+)-dependent currents and determine if the effects of metabolic stress could be reversed. 2. The mean duration of Ca(2+)-activated Cl- tail currents was significantly increased by metabolic stress and this effect was reversed by intracellular photorelease of approximately 300 microM ATP. Intracellular photolysis of 'caged' photolabile compounds was achieved with a xenon flash lamp. 3. Intracellular photorelease of ATP and adenosine 3':5'-cyclic monophosphate (cyclic AMP) (about 40 microM) also accelerated the inactivation of high voltage-activated Ca2+ currents evoked by 500 ms depolarizing step commands from -90 mV to 0 mV. This effect was prevented by intracellular application of the calcineurin (protein phosphatase-2B) inhibitor cyclosporin A (14 nM) and cyclophilin A (50 nM) either applied together or individually. In contrast the protein phosphatase 1 and 2A inhibitor, calyculin A, increased voltage-activated Ca2+ currents, but failed to prevent enhanced inactivation induced by intracellular photorelease of ATP. Intracellular photorelease of ATP had no effect on Ca2+ currents recorded from control neurones which were not metabolically stressed and supplied with glucose and ATP in the extracellular and patch pipette solutions respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium-activated currents in cultured neurones from rat dorsal root ganglia.

1. Voltage-activated Ca2+ currents and caffeine (1 to 10 mM) were used to increase intracellular Ca2+ in rat cultured dorsal root ganglia (DRG) neurones. Elevation of intracellular Ca2+ resulted in activation of inward currents which were attenuated by increasing the Ca2+ buffering capacity of cells by raising the concentration of EGTA in the patch solution to 10 mM. Low and high voltage-activated Ca2+ currents gave rise to Cl- tail currents in cells loaded with CsCl patch solution. Outward Ca2+ channel currents activated at very depolarized potentials (Vc + 60 mV to + 100 mV) also activated Cl- tail currents, which were enhanced when extracellular Ca2+ was elevated from 2 mM to 4 mM. 2. The Ca(2+)-activated Cl- tail currents were identified by estimation of tail current reversal potential by use of a double pulse protocol and by sensitivity to the Cl- channel blocker 5-nitro 2-(3-phenyl-propylamino) benzoic acid (NPPB) applied at a concentration of 10 microM. 3. Cells loaded with Cs acetate patch solution and bathed in medium containing 4 mM Ca2+ also had prolonged Ca(2+)-dependent tail currents, however these smaller tail currents were insensitive to NPPB. Release of Ca2+ from intracellular stores by caffeine gave rise to sustained inward currents in cells loaded with Cs acetate. Both Ca(2+)-activated tail currents and caffeine-induced inward currents recorded from cells loaded with Cs acetate were attenuated by Tris based recording media, and had reversal potentials positive to 0 mV suggesting activity of Ca(2+)-activated cation channels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of metabolic blockers on Ca(2+)-dependent currents in cultured sensory neurones from neonatal rats.

1. The whole cell variant of the patch clamp technique was used to record high voltage-activated Ca2+ currents and Ca(2+)-activated Cl- tail currents from cultured neonatal rat dorsal root ganglion neurones. The aim of the project was to use these currents as physiological indices of intracellular Ca2+ regulation under control conditions and in the presence of metabolic inhibitors. 2. Carbonyl cyanide p-trifluoromethoxyphenylhydrazone (5 microM) and sodium cyanide (1 microM) inhibited Ca2+ currents within 20 s, even when ATP was present in the patch pipette solution, suggesting a direct action on Ca2+ channels. These metabolic inhibitors did not affect Ca2+ current 'run down' or inactivation kinetics. 3. Cultured neonatal dorsal root ganglion neurones of the rat were relatively insensitive to the removal of glucose and ATP from the recording solutions for up to 3 h. These data suggest that the Ca2+ homeostatic mechanisms in these cells are highly resistant to metabolic insult. 4. However 2-deoxy-D-glucose (5 mM) in the extracellular recording medium with no ATP or glucose present did prolong the deactivation time of Ca(2+)-activated Cl- tail currents and increase the total charge flow following activation of a 500 ms voltage-activated Ca2+ current. This effect was prevented by inclusion of D-fructose 1,6-diphosphate (500 microM) in the patch pipette solution. 5. We conclude that some agents used to induce chemical hypoxia, such as carbonyl cyanide p-trifluoromethoxyphenylhydrazone and sodium cyanide, may interact directly with voltage-activated Ca2+ channels and are therefore not appropriate for use in studying disturbed neuronal Ca2+ homeostasis. However, the use of 2-deoxy-D-glucose in the absence of glucose and ATP does represent a model of disturbed Ca2+ homeostasis in cultured dorsal root ganglion neurones. In this study we have combined the whole cell recording technique with cultured neurones under conditions which produce a degree of metabolic stress as reflected by prolonged Ca(2+)-activated Cl- tail currents. The reduced efficiency of handling of intracellular Ca2+ loads may be an important factor contributing to the onset of neuronal damage during hypoxia and ischaemia.     

Palmitoyl-DL-carnitine has calcium-dependent effects on cultured neurones from rat dorsal root ganglia.

1. The effects of palmitoyl-DL-carnitine (0.01 to 1 mM) on whole cell voltage-activated calcium channel currents carried by calcium or barium and Ca(2+)-activated chloride currents were studied in cultured neurones from rat dorsal root ganglia. 2. Palmitoyl-DL-carnitine applied to the extracellular environment or intracellularly via the patch solution reduced Ca2+ currents activated over a wide voltage range from a holding potential of -90 mV. Inhibition of high voltage activated Ca2+ channel currents was dependent on intracellular Ca2+ buffering and was reduced by increasing the EGTA concentration from 2 to 10 mM in the patch solution. Barium currents were significantly less sensitive to palmitoyl-DL-carnitine than Ca2+ currents. 3. The amplitude of Ca(2+)-activated Cl- tail currents was reduced by palmitoyl-DL-carnitine. However, the duration of these Cl- currents was greatly prolonged by palmitoyl-DL-carnitine, suggesting slower removal of free Ca2+ from the cytoplasm following Ca2+ entry through voltage-activated channels. 4. Palmitoyl-DL-carnitine evoked Ca(2+)-dependent inward currents which could be promoted by activation of the residual voltage-activated Ca2+ currents and attenuated by intracellular application of EGTA. 5. We conclude that palmitoyl-DL-carnitine reduced the efficiency of intracellular Ca2+ handling in cultured dorsal root ganglion neurones and resulted in enhancement of Ca(2+)-dependent events including inactivation of voltage-activated Ca2+ currents. The activation of inward currents by palmitolyl-DL-carnitine may involve Ca(2+)-induced Ca2+ release from intracellular stores, or direct interaction of palmitoyl-DL-carnitine with Ca2+ stores.     

Inhibition of recombinant low-voltage-activated Ca(2+) channels by the neuroprotective agent BW619C89 (Sipatrigine)

T-type Ca(2+) currents were recorded in 2 mM Ca(2+) from HEK 293 cells stably expressing recombinant low-voltage-activated Ca(2+) channel subunits. Current-voltage relationships revealed that these currents were low-voltage activated in nature and could be reversibly antagonised by mibefradil, a known T-type channel blocker. At a test potential of -25 mV alpha(1I)-mediated Ca(2+) currents were rapidly and reversibly inhibited by 1-100 microM BW619C89 (IC(50)=14 microM, Hill coefficient 1.3). In contrast to its actions on N-type Ca(2+) channels, a near IC(50) dose (10 microM) of BW619C89 produced no alterations in either the kinetics or voltage-dependence of T-type currents. In additional single dose experiments, currents mediated by rat alpha(1G), human alpha(1H) or human alpha(1I) channel subunits were also inhibited by BW619C89. Overall our data indicate that T-type Ca(2+) channels are more potently blocked by BW619C89 than either type-II Na(+) channels or N-type Ca(2+) channels. It seems, therefore, that inhibition of low-voltage-activated Ca(2+) channels is likely to contribute to the anticonvulsant and neuroprotective actions of this and related compounds.     

Blockade by ifenprodil of high voltage-activated Ca2+ channels in rat and mouse cultured hippocampal pyramidal neurones: comparison with N-methyl-D-aspartate receptor antagonist actions.

1. The block by ifenprodil of voltage-activated Ca2+ channels was investigated in intracellular free calcium concentration ([Ca2+]i) evoked by 50 mM K+ (high-[K+]o) in Fura-2-loaded rat hippocampal pyramidal neurones in culture and on currents carried by Ba2+ ions (IBa) through Ca2+ channels in mouse cultured hippocampal neurones under whole-cell voltage-clamp. The effects of ifenprodil on voltage-activated Ca2+ channels were compared with its antagonist actions on N-methyl-D-aspartate- (NMDA) evoked responses in the same neuronal preparations. 2. Rises in [Ca2+]i evoked by transient exposure to high-[K+]o in our preparation of rat cultured hippocampal pyramidal neurones are mediated predominantly by Ca2+ flux through nifedipine-sensitive Ca2+ channels, with smaller contributions from nifedipine-resistant, omega-conotoxin GVIA-sensitive Ca2+ channels and Ca2+ channels sensitive to crude funnel-web spider venom (Church et al., 1994). Ifenprodil (0.1-200 microM) reversibly attenuated high-[K+]o-evoked rises in [Ca2+]i with an IC50 value of 17 +/- 3 microM, compared with an IC50 value of 0.7 +/- 0.1 microM for the reduction of rises in [Ca2+]i evoked by 20 microM NMDA. Tested in the presence of nifedipine 10 microM, ifenprodil (1-50 microM) produced a concentration-dependent reduction of the dihydropyridine-resistant high-[K+]o-evoked rise in [Ca2+]i with an IC50 value of 13 +/- 4 microM. The results suggest that ifenprodil blocks Ca2+ flux through multiple subtypes of high voltage-activated Ca2+ channels. 3. Application of the polyamine, spermine (0.25-5 mM), produced a concentration-dependent reduction of rises in [Ca2+]i evoked by high-[K+]o.(ABSTRACT TRUNCATED AT 250 WORDS)     

Potent inhibition of a recombinant low voltage-activated Ca(2+) channel by SB-209712

T-type Ca(2+) currents were recorded in 2 mM Ca(2+) from HEK293 cells stably expressing the low voltage-activated Ca(2+) channel sub-unit alpha(1I). These currents were inhibited by the known Ca(2+) channel antagonist mibefradil with an IC(50) close to 1 microM. SB-209712 (1,6,bis?1-[4-(3-phenylpropyl)piperidinyl]?hexane), a compound originally developed as a high voltage-activated Ca(2+) channel blocker, proved to be a more potent T-type channel antagonist, exhibiting an IC(50) in the region of 500 nM. The antagonism produced by SB-209712 was reversed following drug removal and the observed antagonism exhibited little or no voltage-dependence with respect to either holding or test potential. These data indicate that SB-209712 is amongst the most potent known non-peptide T-type channel antagonists and thus may have some use in understanding the role of these channels in cellular function.     

The actions of ryanodine on Ca2+-activated conductances in rat cultured DRG neurones; evidence for Ca2+-induced Ca2+ release

The whole-cell recording technique was used to investigate the actions of a calcium release channel ligand, ryanodine, on calcium-activated chloride conductances, and to evaluate ryanodine-sensitive Ca²⁺-induced Ca²⁺ release from intracellular stores in cultured neonatal rat DRG neurones. The aim of the project was to use ryanodine as a pharmacological tool to evaluate calcium-induced calcium release in the cell bodies of cultured DRG neurones. Action potential after-depolarizations were attenuated by extracellular application of the chloride channel blocker, niflumic acid (10 μM), and by ryanodine (10 μM); these actions occurred without concurrent changes in evoked action potentials. Ryanodine and caffeine (10 mM) activated calcium-dependent conductances and the responses to ryanodine were attenuated by depletion of caffeine-sensitive Ca²⁺ stores. The current clamp data were complicated by changes in potassium conductances so studies were carried out under voltage clamp and voltage-activated calcium currents and calcium-activated chloride and non-selective cation currents were isolated pharmacologically. Ryanodine (10 μM) evoked delayed, inward, calcium-activated non-selective cation and chloride currents which reversed close to 0 mV and were attenuated by N-methyl-d-glucamine, niflumic acid and dantrolene. Consistent with actions on action potential after-depolarizations, niflumic acid (10 μM) and ryanodine (10 μM) attenuated calcium-activated chloride currents evoked by calcium entry through voltage-activated calcium channels. Niflumic acid and ryanodine had no effects on voltage-activated calcium currents evoked from a holding potential of –90 mV by voltage step commands to 0 mV. In conclusion calcium-activated chloride conductances appear to be activated in part by calcium released from ryanodine-sensitive stores, and significant calcium-induced calcium release may occur locally in cell bodies of DRG neurones as a result of calcium entry through voltage-activated channels during an action potential. Received: 6 July 1998 / Accepted: 30 November 1998     

Blockade by sigma site ligands of high voltage-activated Ca2+ channels in rat and mouse cultured hippocampal pyramidal neurones.

1. The effects of a series of structurally-dissimilar sigma site ligands were examined on high voltage-activated Ca2+ channel activity in two preparations of cultured hippocampal pyramidal neurones. 2. In mouse hippocampal neurones under whole-cell voltage-clamp, voltage-activated Ca2+ channel currents carried by barium ions (IBa) were reduced with the rank order (IC50 values in microM): 1S,2R-(-)-cis-N-methyl-N-[2-(3,4-dichlorophenyl)ethyl]- 2-(1-pyrrolidinyl)cyclohexylamine (7.8) > rimcazole (13) > haloperidol (16) > ifenprodil (18) > opipramol (32) > carbetapentane (40) = 1-benzylspiro[1,2,3,4-tetrahydronaphthalene-1,4-piperidine] (42) > caramiphen (47) > dextromethorphan (73). At the highest concentrations tested, the compounds almost abolished IBa in the absence of any other pharmacological agent. 3. The current-voltage characteristics of the whole-cell IBa were unaffected by the test compounds. The drug-induced block was rapid in onset and offset, with the exceptions of carbetapentane and caramiphen where full block was achieved only after two to three voltage-activated currents and was associated with an apparent increase in the rate of inactivation of IBa. 4. In rat hippocampal neurones loaded with the Ca(2+)-sensitive dye Fura-2, rises in intracellular free Ca2+ concentration evoked by transient exposure to 50 mM K(+)-containing medium, either in the absence or in the presence of 10 microM nifedipine (to block L-type high voltage-activated Ca2+ channels), were also reversibly attenuated by the sigma ligands. The rank order potencies for the compounds in these experimental paradigms were similar to that observed for blockade of IBa in the electrophysiological studies. 5. These results indicate that, at micromolar concentrations, the compounds tested block multiple subtypes of high voltage-activated Ca2+ channels. These actions, which do not appear to be mediated by high-affinity sigma binding sites, may play a role in some of the functional effects previously described for the compounds.     

Gabapentin-mediated inhibition of voltage-activated Ca2+ channel currents in cultured sensory neurones is dependent on culture conditions and channel subunit expression

We have used the whole cell patch clamp method and fura-2 fluorescence imaging to study the actions of gabapentin (1-(aminoethyl) cyclohexane acetic acid) on voltage-activated Ca(2+) entry into neonatal cultured dorsal root ganglion (DRG) neurones and differentiated F-11 (embryonic rat DRG x neuroblastoma hybrid) cells. Gabapentin (2.5 microM) in contrast to GABA (10 microM) did not influence resting membrane potential or input resistance. In current clamp mode gabapentin failed to influence the properties of evoked single action potentials but did reduce the duration of action potentials prolonged by Ba(2+). Gabapentin attenuated high voltage-activated Ca(2+) channel currents in a dose- and voltage- dependent manner in DRG neurones and reduced Ca(2+) influx evoked by K(+) depolarisation in differentiated F-11 cells loaded with fura-2. The sensitivity of DRG neurones to gabapentin was not changed by the GABA(B) receptor antagonist saclofen but pertussis toxin pre-treatment reduced the inhibitory effects of gabapentin. Experiments following pre-treatment of DRG neurones with a PKA-activator and a PKA-inhibitor implicated change in phosphorylation state as a mechanism, which influenced gabapentin action. Sp- and Rp-analogues of cAMP significantly increased or decreased gabapentin-mediated inhibition of voltage-activated Ca(2+) channel currents. Culture conditions used to maintain DRG neurones and passage number of differentiated F-11 cells also influenced the sensitivity of Ca(2+) channels to gabapentin. We analysed the Ca(2+) channel subunits expressed in populations of DRG neurones and F-11 cells that responded to gabapentin had low sensitivity to gabapentin or were insensitive to gabapentin, by Quantitative TaqMan PCR. The data obtained from this analysis suggested that the relative abundance of the Ca(2+) channel beta(2) and alpha(2)delta subunit expressed was a key determinant of gabapentin sensitivity of both cultured DRG neurones and differentiated F-11 cells. In conclusion, gabapentin inhibited part of the high voltage-activated Ca(2+) current in neonatal rat cultured DRG neurones via a mechanism that was independent of GABA receptor activation, but was sensitive to pertussis toxin. Gabapentin responses identified in this study implicated Ca(2+) channel beta(2) subunit type as critically important to drug sensitivity and interactions with alpha(1) and alpha(2)delta subunits may be implicated in antihyperalgesic therapeutic action for this compound.     

Preferential block of T-type calcium channels by neuroleptics in neural crest-derived rat and human C cell lines.

We have used the whole-cell version of the patch-clamp technique to analyze the inhibition of Ca2+ currents by antipsychotic agents in neural crest-derived rat and human thyroid C cell lines. Diphenylbutylpiperidine (DPBP) antipsychotics, including penfluridol and fluspirilene, potently and preferentially block T-type Ca2+ current in the rat medullary thyroid carcinoma 6-23 (clone 6) cell line. When step depolarizations were applied at 0.1 Hz from a holding potential of -80 mV, with 10 mM Ca2+ as the charge carrier, the DPBP penfluridol inhibited T-type current with an IC50 of 224 nM. High voltage-activated L and N currents were less potently blocked. At a concentration of 500 nM, penfluridol inhibited 78.0 +/- 2.3% (n = 29) of inactivating T-type Ca2+ current, whereas the sustained high voltage-activated current was reduced by 25.6 +/- 3.5% (n = 28). Block of T-type current by penfluridol was enhanced by depolarizing test pulses applied at frequencies above 0.03 Hz. The use-dependent component of block was largely reversed by pulse-free periods at -80 mV. T-type Ca2+ channels in the human TT C cell line were blocked by penfluridol, and the potency was enhanced by reduction of extracellular Ca2+. Non-DPBP antipsychotics, including haloperidol, clozapine, and thioridazine, also blocked T-type channels, but these were 20-100 times less potent than the DPBPs. These results identify the DPBPs as a new class of organic Ca2+ channel antagonists, which are distinctive in their ability to preferentially block T-type channels. These agents will be useful in defining the function of T channels in various excitable cells. Their potent block of T-type Ca2+ channels, which would be enhanced in rapidly firing cells, suggests that this action may be relevant to the therapeutic or toxic effects of these drugs when used in clinical pharmacology.     

Modulation of divalent cation-activated chloride ion currents.

1. Voltage-sensitive calcium channel currents carried by Ca2+ (ICa) or Ba2+ (IBa) were followed by tail currents carried by Cl- ions in approximately 45% of cultured dorsal root ganglion neurones. 2. Extracellular application of (-)-baclofen (100 microM) inhibited IBa and ICl(Ba). Bay K 8644 (5 microM) potentiated both currents. 3. Intracellular GTP-gamma-S increased the proportion of neurones in which ICl(Ba) was recorded. In addition, the activation by GTP-gamma-S of a pertussis toxin-sensitive GTP binding (G)-protein resulted in a steady increase in the Cl- tail current with time, despite a concurrent reduction in IBa. 4. Extracellular application of 10mM caffeine selectively reduced ICl(Ba) without significant change in IBa. When Ca2+ was the charge carrier, caffeine had little effect on ICl(Ca), and increased the inactivation of ICa. 5. We conclude that, in addition to being regulated by divalent cation entry through Ca2+ channels, the Cl- current is also regulated by G-protein activation. The mechanism of activation of ICl(Ba) may involve Ca2+ release from intracellular stores.     

Inhibition of neuronal Ca2+ channel currents by the funnel web spider toxin omega-Aga-IA

Ca2+ channel currents were recorded from cultured rat dorsal root ganglion neurons and cerebellar granule cells using the whole-cell recording variant of the patch clamp technique. omega-Aga-IA, a toxin purified from the venom of the American funnel web spider, Agelenopsis aperta, markedly inhibited high threshold barium currents (lBa) when applied at 10 nM concentration. The low threshold T-type current activated at Vc = -30 mV and the outward (Ca2+ channel) current activated at +120 mV were significantly less sensitive to omega-Aga-IA, omega-Conotoxin GVIA (1 microM) inhibited IBa irreversibly. In contrast, the action of omega-Aga-IA was partially reversed 5 min after its removal. The voltage-activated calcium current (ICa) was inhibited by omega-Aga-IA in a manner different from IBa. ICa measured at the end of a 100-msec voltage step command was reduced to a greater extent than the peak current. The residual ICa following application of omega-Aga-IA was a fast transient current. omega-Aga-IA did not inhibit voltage-activated sodium currents from dorsal root ganglion neurons in the absence of tetrodotoxin. omega-Aga-IA abolished the dihydropyridine (+)-202-791-sensitive L-type current component of IBa. We conclude that omega-Aga-IA is a very potent inhibitor of neuronal voltage-activated Ca2+ channel currents and that it may prove to be a useful tool in the characterization and isolation of Ca2+ channels.     

Multiple actions of anandamide on neonatal rat cultured sensory neurones

1. We have investigated the effects of the endocannabinoid anandamide (AEA) on neuronal excitability and vanilloid TRPV1 receptors in neonatal rat cultured dorsal root ganglion neurones. 2. Using whole-cell patch-clamp electrophysiology, we found that AEA inhibits high-voltage-activated Ca(2+) currents by 33+/-9% (five out of eight neurones) in the absence of the CB(1) receptor antagonist SR141716A (100 nM) and by 32+/-6% (seven out of 10 neurones) in the presence of SR141716A. 3. Fura-2 fluorescence Ca(2+) imaging revealed that AEA produced distinct effects on Ca(2+) transients produced by depolarisation evoked by 30 mM KCl. In a population of neurones of larger somal area (372+/-20 microM(2)), it significantly enhanced Ca(2+) transients (80.26+/-13.12% at 1 microM), an effect that persists after pertussis toxin pretreatment. In a population of neurones of smaller somal area (279+/-18 microM(2)), AEA significantly inhibits Ca(2+) transients (30.75+/-3.54% at 1 microM), an effect that is abolished by PTX pretreatment. 4. Extracellular application of 100 nM AEA failed to evoke TRPV1 receptor inward currents in seven out of eight neurones that responded to capsaicin (1 microM), with a mean inward current of -0.94+/-0.21 nA. In contrast, intracellular application of 100 nM AEA elicited robust inward currents in approximately 62% of neurones, the mean population response was -0.85+/-0.21 nA. When AEA was applied to the intracellular environment with capsazepine (1 microM), the mean population inward current was -0.01+/-0.01 nA. Under control conditions, mean population current fluctuations of -0.09+/-0.05 nA were observed.     

Inhibition of neuronal high-voltage activated calcium channels by the omega-phoneutria nigriventer Tx3-3 peptide toxin

We have investigated the effect of omega-PnTx3-3 (referred to in previous papers simply as Tx3-3), a peptide toxin from the venom of the spider Phoneutria nigriventer, on neuronal high-voltage activated (HVA) Ca(2+) channels, using whole-cell patch-clamp. omega-PnTx3-3 (120 nM) blocked 74+/-8% of the total HVA Ca(2+) currents of cerebellar granule neurones, without affecting the low-voltage activated (LVA) current. P/Q/R-type currents in cerebellar granule neurones, isolated using 4 microM nicardipine and 100 nM omega-conotoxin GVIA, were markedly (79+/-6%) inhibited by 60 nM omega-PnTx3-3. R-type currents, isolated either by additional application of 0.5-1 microM of omega-agatoxin IVA or by pre-incubation with 5 microM omega-conotoxin MVIIC were inhibited almost totally by 120 nM of omega-PnTx3-3. omega-PnTx3-3 reversibly altered the kinetics of the P/Q/R current, increasing the degree of inactivation that occurred during a 50 ms pulse from 20% to 40%. N-type currents, recorded from neuroblastoma N18 cells, were partially (34+/-2%) inhibited by 320 nM omega-PnTx3-3. L-type currents, recorded from GH3 cells, were partially (45+/-12%) inhibited by 80 nM omega-PnTx3-3. We conclude that omega-PnTx3-3 inhibits all known HVA Ca(2+) channels, and most effectively the P/Q- and R-type currents.     

Effects on K+ currents in rat cerebellar granule neurones of a membrane-permeable analogue of the calcium chelator BAPTA.

1. Whole cell recordings of voltage-activated K+ currents were made with the amphotericin B perforated patch technique from cerebellar granule (CG) neurones of 6-8 days rats that had been in culture for 1 to 16 days. By use of appropriate voltage protocols, the effects of the membrane-permeant form of BAPTA, 1,2-bis-(2-amino-phenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM), on the transient A current (IKA), the delayed rectifier current (IKV) and a standing outward current (IKSO) were investigated. 2. Bath application of 25 microM BAPTA-AM inhibited both IKV and IKSO in cultured neurones, but did not seem to affect IKA. Neither 25 microM BAPTA (free acid) nor 25 microM ethylenediaminetetraacetic acid acetoxymethyl ester (EDTA-AM) had any significant effect on the magnitude of IKSO. Similarly in short-term (1-2 days) cultured CG neurones IKV, but not IKA, was inhibited by 25 microM BAPTA-AM. 3. BAPTA-AM (2.5 microM) reduced IKV in short-term culture CG neurones, with further inhibition being seen when the perfusate was changed to one containing 25 microM BAPTA-AM. 4. Tetraethylammonium ions (TEA) (10 mM) reversibly inhibited IKV in these cells with a similar rate of block of IKV to that induced by 25 microM BAPTA-AM. 5. The degree of inhibition of IKV by 25 microM BAPTA-AM was both time- and voltage-dependent, in contrast to the inhibition of this current by TEA. 6. These data indicate that BAPTA-AM reduces K+ currents in cerebellar granule neurones and that this inhibition cannot be explained in terms of intracellular Ca2+ chelation, but is a direct effect on the underlying channels.     

Clotrimazole analogues: effective blockers of the slow afterhyperpolarization in cultured rat hippocampal pyramidal neurones

1. The pharmacology of the slow afterhyperpolarization (sAHP) was studied in cultured rat hippocampal pyramidal neurones. 2. Clotrimazole, its in vivo metabolite, 2-chlorophenyl-bisphenyl-methanol (CBM) and the novel analogues, UCL 1880 and UCL 2027, inhibited the sI(AHP) with similar IC50s (1-2 microM). 3. Clotrimazole and CBM also inhibited the high voltage-activated (HVA) Ca2+ current in pyramidal neurones with IC50s of 4.7 microM and 2.2 microM respectively. UCL 1880 was a less effective Ca2+ channel blocker, reducing the HVA Ca2+ current by 50% at 10 microM. At concentrations up to 10 microM, UCL 2027 had no effect on the Ca2+ current, indicating that its effects on the sI(AHP) were independent of Ca2+ channel block. 4. Clotrimazole also inhibited both the outward holding current (IC50=2.8 microM) present at a potential of -50 mV and the apamin-sensitive medium AHP (mAHP; IC50 approximately amp;10 microM). The other clotrimazole analogues tested had smaller effects on these two currents. The present work also shows that 100 nM UCL 1848, an inhibitor of apamin-sensitive conductances, abolishes the mAHP. 5. Currents were recorded from HEK293 cells transfected with hSK1 and rSK2. The SK currents were very sensitive to inhibition by UCL 1848 but were not significantly reduced by the sI(AHP) inhibitor, UCL 2027 (10 microM). 10 microM UCL 1880 reduced the hSK1 current by 40%. 6. UCL 2027 appears to be the first relatively selective blocker of the sAHP to be described. Furthermore, the ability of UCL 2027 to block the sAHP with minimal effect on SK1 channel activity questions the role of this channel in the sAHP.     

Analysis of GABA(A)- and GABA(B)-receptor mediated effects on intracellular Ca(2+) in DRG hybrid neurones

1. Using pharmacological analysis and fura-2 spectrofluorimetry, we examined the effects of gamma-aminobutyric acid (GABA) and related substances on intracellular Ca(2+) concentration ([Ca(2+)]i) of hybrid neurones, called MD3 cells. The cell line was produced by fusion between a mouse neuroblastoma cell and a mouse dorsal root ganglion (DRG) neurone. 2. MD3 cells exhibited DRG neurone-like properties, such as immunoreactivity to microtubule-associated protein-2 and neurofilament proteins. Bath applications of capsaicin and alpha, beta-methylene adenosine triphosphate reversibly increased [Ca(2+)]i. However, repeated applications of capsaicin were much less effective. 3. Pressure applications of GABA (100 microM), (Z)-3-[(aminoiminomethyl) thio] prop-2-enoic acid sulphate (ZAPA; 100 microM), an agonist at low affinity GABA(A)-receptors, or KCl (25 mM), transiently increased [Ca(2+)]i. 4. Bath application of bicuculline (100 nM - 100 microM), but not picrotoxinin (10 - 25 microM), antagonized GABA-induced increases in [Ca(2+)]i in a concentration-dependent manner (IC(50)=9.3 microM). 5. Ca(2+)-free perfusion reversibly abolished GABA-evoked increases in [Ca(2+)]i. Nifedipine and nimodipine eliminated GABA-evoked increases in [Ca(2+)]i. These results imply GABA response dependence on extracellular Ca(2+). 6. Baclofen (500 nM - 100 microM) activation of GABA(B)-receptors reversibly attenuated KCl-induced increases in [Ca(2+)]i in a concentration-dependent manner (EC(50)=1.8 microM). 2-hydroxy-saclofen (1 - 20 microM) antagonized the baclofen-depression of the KCl-induced increase in [Ca(2+)]i. 7. In conclusion, GABA(A)-receptor activation had effects similar to depolarization by high external K(+), initiating Ca(2+) influx through high voltage-activated channels, thereby transiently elevating [Ca(2+)]i. GABA(B)-receptor activation reduced Ca(2+) influx evoked by depolarization, possibly at Ca(2+)-channel sites in MD3 cells.     

Inhibition of calcium currents in cultured rat dorsal root ganglion neurones by (-)-baclofen.

Voltage-dependent inward calcium currents (ICa) activated in cultured rat dorsal root ganglion neurones were reversibly reduced in a dose-dependent manner by (-)-baclofen (10 microM to 100 microM). Baclofen (100 microM) reduced the calcium-dependent slow outward potassium current (IK(Ca)). This current was abolished in calcium-free medium and by 300 microM cadmium chloride. The action of baclofen on IK(Ca) was reduced when the calcium concentration in the medium was increased from 5 mM to 30 mM. The calcium independent fast transient voltage-dependent outward current (IK(Vt] was also reduced by baclofen; this effect remained present when Ca2+-free medium was used to prevent contamination by IK(Ca). 4-Aminopyridine (500 microM) reduced IK(Vt) and induced a small increase in ICa. The action of baclofen on ICa was partially antagonized by 4-aminopyridine. GABAB receptor-mediated inhibition of ICa in cultured rat dorsal root ganglion neurones involves a direct mechanism rather than resulting indirectly from an increase in the residual outward potassium currents activated by depolarization. The reduction in ICa by baclofen was variable and dependent on the amplitude of control ICa, larger currents being more resistant to the baclofen-induced inhibition.     

Extracellular or intracellular application of argiotoxin-636 has inhibitory actions on membrane excitability and voltage-activated currents in cultured rat sensory neurones

The whole cell variant of the patch clamp technique was used to investigate the actions of the polyamine amide spider toxin, argiotoxin-636, on the excitability of cultured dorsal root ganglion neurones. Synthesized argiotoxin-636 (0.1–100 μM) reduced neuronal excitability when applied to the extracellular environment by low pressure ejection or to the intracellular environment via the patch pipette solution. The toxin prolonged the duration of evoked action potentials and reduced the peak amplitude of action potentials. Intracellular and extracellular application of argiotoxin-636 also decreased the number of action potentials evoked in response to 800-ms depolarizing current commands. This action of the toxin was mimicked by 100 μM tetraethylammonium. Extracellular application of argiotoxin-636 inhibited voltage-activated K⁺ currents in a dose-dependent manner over the complete voltage range. This inhibition occurred without any significant changes in the voltage dependence of activation or inactivation. Intracellular application of argiotoxin-636, during 5–10 min of whole cell recording, also inhibited voltage-activated K⁺ currents without changing the voltage dependence of activation or steady-state inactivation. Extracellular or intracellular spermidine (250 μM) reversibly attenuated the inhibitory actions of extracellular argiotoxin-636. Argiotoxin-636 also inhibited voltage-activated Na⁺ currents; this effect was dependent on repeated activation of the currents and the period during which the neurones were in culture. We conclude that application of argiotoxin-636 to either the extracellular or intracellular environment reduced excitability of cultured sensory neurones from neonatal rats and that this involved inhibition of both voltage-activated K⁺ and Na⁺ currents. The data suggest that the toxin was more effective at attenuating action potentials when neurones were repeatedly excited, and that access to inhibitory sites of action on the voltage-activated ion channels can be achieved from the inside of the neurone.