Roderick H. Scott

Interactions of polyamines with neuronal ion channels

Polyamines, a group of aliphatic amines, exert selective and complex actions on a variety of ion channels. Polyamines are found endogenously, as normal metabolic intermediates, and also in the venoms of several invertebrates, where they act as potent neurotoxins. In addition, evidence suggests that polyamines may mediate or potentiate excitotoxic mechanisms responsible for neuronal damage during ischaemia. Now that the structures and functions of various polyamines are beginning to be deduced, and synthetic analogues become available, these compounds are of importance, not only as pharmacological tools to study specific receptor/ion channel complexes, but also as templates on which to base drugs designed for neuroprotective effects in a number of neurodegenerative disorders.

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

1 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 μM to 100 μM). 2 Baclofen (100 μM) reduced the calcium‐dependent slow outward potassium current (IK(Ca)). This current was abolished in calcium‐free medium and by 300 μM 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. 3 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 4‐Aminopyridine (500 μM) reduced IK(Vt) and induced a small increase in ICa. The action of baclofen on ICa was partially antagonized by 4‐aminopyridine. 5 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.

Aspects of vertebrate neuronal voltage-activated calcium currents and their regulation

Abbreviations

A comparison of the effect of calcium channel ligands and GABAB agonists and antagonists on transmitter release and somatic calcium channel currents in cultured neurons

Glutamate release has been examined from cultured cerebellar granule neurons in the rat using the technique of prelabelling the releasable pool of glutamate with [3H]glutamine. Glutamate release was stimulated in control neurons by 2-min incubation with 50 mM K+, or in neurons continuously depolarized in Ca2(+)-free 50 mM K+ medium, by 2-min incubation with medium containing 5 mM Ca2+. The ability of the Ca2(+)-channel agonist (+)-202-791 to increase the stimulated release of [3H]glutamate was approximately doubled in the depolarized condition. The antagonist enantiomer (-)-202-791 produced a small inhibition of K(+)-stimulated release, whereas (-)-202-791 completely inhibited Ca2(+)-stimulated release from depolarized neurons at concentrations greater than 10 nM. (-)-Baclofen (100 microM) inhibited transmitter release similarly (25-30%) under the two conditions. Calcium-channel currents were recorded from cultured dorsal root ganglion neurons under control conditions at a holding potential of -80 mV, or in neurons depolarized to -30 mV. (-)-202-791 produced a greater effect at -30 than at -80 mV although even at -30 mV the inhibition was slow in onset and incomplete. (-)-Baclofen (100 microM) inhibited the amplitude of the calcium-channel current at both holding potentials by 30-50%, although it did not clearly slow activation of the current at the depolarized holding potential. The GABAB receptors associated with inhibition of glutamate release and of calcium-channel currents were both markedly blocked by phaclofen but not by 2-OH-saclofen. These findings suggest that the GABAB receptor associated with inhibitory modulation of transmitter release, and that associated with inhibition of calcium-channel currents show pharmacological similarities, and are able to exert their action even at levels of steady depolarization at which most N-type channels should be inactivated.

Photoactivation of intracellular guanosine triphosphate analogues reduces the amplitude and slows the kinetics of voltage-activated calcium channel currents in sensory neurones

The influence of guanine nucleotide analogues on calcium channel currents in cultured rat dorsal root ganglion neurones has been studied using a technique in which the rate of diffusion of the analogues to their site of action is by-passed by photochemical release of the analogues within the neurones. The 1(2-nitrophenyl)ethyl P3-ester derivatives of guanosine 5′-0(3-thio)triphosphate (caged GTP-γ-S) and 5′-guanylylimidodiphosphate (caged GMP-PNP) were synthesised and found to be completely photolysable by light, yielding free GTP-γ-S and GMP-PNP. Calcium channel currents were recorded using the whole cell patch technique and either caged GTP-γ-S or caged GMP-PNP (2 mM) were included in the patch pipette. Stable currents were recorded for 5–10 min, and a single pulse of 300–350 nm irradiation was directed using a liquid light guide onto the recording dish. Calcium channel currents were then recorded every 30–120 s following photochemical release of approximately 20μM GTP-gg-S. The peak calcium channel current was reduced by about 70% with a slow time course [t1/2 1.5±0.2 min (mean±SEM);n=5]. The transient component of the peak current was usually completely abolished, whereas the sustained current measured at the end of the 100 ms depolarising pulse was less affected. Qualitatively similar effects were observed on photolysis of caged GMP-PNP. These results suggest that the channels underlying the transient and the sustained components of the whole cell current may be differentially molulated by GTP analogues.

An investigation into the mechanisms of inhibition of calcium channel currents in cultured sensory neurones of the rat by guanine nucleotide analogues and (−)− baclofen

1 The mechanism of inhibition of calcium channel currents by the guanine nucleotide analogue guanosine 5′‐O‐3 thiotriphosphate (GTP‐γ‐S) and by the GABAB agonist (−)‐baclofen has been studied in cultured dorsal root ganglion neurones of the rat. The inhibition by GTP‐γ‐S is particularly characterized by an abolition of the transient component of calcium channel currents carried either by Ba2+ (IBa) or by Ca2+ (ICa). 2 The effect of agents increasing intracellular cyclic AMP levels has been examined. Neither internal cyclic AMP nor forskolin prevented the inhibition of IBa by baclofen. Neither forskolin nor pretreatment of cells with cholera toxin prevented the inhibition of the transient component of IBa by GTP‐γ‐S. However, both these treatments increased the amplitude of the sustained IBa in the presence of GTP‐γ‐S. The ATP analogue adenosine imido‐diphosphate which inhibits many ATP requiring enzymes did not prevent the effect of GTP‐γ‐S although it reduced the amplitude of IBa. 3 Baclofen (100 μm) produced a 22 ± 2% increase in inositol phosphate production in 30 s, whereas the increase produced by bradykinin (1 μm) was 70 + 14%. However, unlike baclofen, bradykinin did not inhibit IBa or ICa in these cells. 4 The effect of protein kinase C inhibitors was examined. Polymixin B (20 μm in patch pipette) had no effect on the inhibition of IBa by baclofen or GTP‐γ‐S. A higher concentration (100 μm) alone inhibited IBa and no further inhibition by baclofen was observed. Neither H7 (50 μm) nor staurosporine (100 nm), applied extracellularly, prevented the response to GTP‐γ‐S. 5 The protein kinase C activator di‐octanoyl glycerol (20 μm) did not inhibit IBa. Arachidonic acid (100 μm) also produced no inhibition of IBa. 6 In conclusion we have obtained no evidence that a second messenger system mediates the inhibition of calcium channel currents by GTP‐γ‐S or baclofen in dorsal root ganglion neurones. These results support the hypothesis that GABAB receptors are directly coupled to calcium channels by G proteins.

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 Ca2+‐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‐phenylpropylamino) benzoic acid (NPPB) applied at a concentration of 10 μm. 3 Cells loaded with Cs acetate patch solution and bathed in medium containing 4 mm Ca2+ also had prolonged Ca2+‐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 Ca2+‐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 Ca2+‐activated cation channels. 4 Our data may reflect (a) different degrees of association between Ca2+‐activated channels with voltage‐gated Ca2+ channels, (b) distinct relationships between channels and intracellular Ca2+ stores and Ca2+ homeostatic mechanisms, (c) regulation of Ca2+‐activated channels by second messengers, and (d) varying channel sensitivity to Ca2+, in the cell body of DRG neurones.

Activation of Ca(2+)-dependent Cl- currents in cultured rat sensory neurones by flash photolysis of DM-nitrophen.

1. Voltage‐gated Ca2+ currents (ICa) and Ca(2+)‐activated Cl‐ currents (ICl(Ca)) were recorded from cultured rat dorsal root ganglion (DRG) neurones using the whole‐cell configuration of the patch clamp technique. Intracellular photorelease of Ca2+ by flash photolysis of DM‐nitrophen elicited transient inward currents only in those cells which possessed Ca(2+)‐activated Cl‐ tail currents following ICa. The reversal potential of the flash responses was hyperpolarized when extracellular Cl‐ was replaced by SCN‐. The flash responses and the Ca(2+)‐activated Cl‐ tail currents were inhibited by the Cl‐ channel blockers niflumic acid (10‐100 microM) and 5‐nitro‐2‐(3‐phenylpropylamino)benzoic acid (NPPB) (10 microM). 2. After activation by ICa, the Ca(2+)‐activated Cl‐ current could be reactivated during its decay by photorelease of caged Ca2+. Experiments carried out on neurones held at 0 mV demonstrated that ICl(Ca) could be chronically activated due to residual Ca2+ influx. These data directly demonstrated that the decay of ICl(Ca) is not due to inactivation but rather to deactivation as a result of removal of the Ca2+ load from the cell cytoplasm. 3. Photorelease of caged inositol 1,4,5‐trisphosphate (IP3) failed to activate any Ca(2+)‐dependent current responses in cultured DRG neurones, although application of caffeine elicited transient inward currents, and responses to photoreleased IP3 could be obtained from freshly dissociated smooth muscle cells. 4. Photorelease of Ca2+ provides a useful method for investigating the properties of ICl(Ca) independently from other physiological parameters. In addition, we have directly demonstrated that ICl(Ca) in DRG neurones does not inactivate, and so may continue to modulate membrane excitability as long as the intracellular Ca2+ concentration ([Ca2+]i) close to the cell membrane is elevated.(ABSTRACT TRUNCATED AT 250 WORDS)

Actions of arginine polyamine on voltage and ligand-activated whole cell currents recorded from cultured neurones.

1 Toxins from invertebrates have proved useful tools for investigation of the properties of ion channels. In this study we describe the actions of arginine polyamine which is believed to be a close analogue of FTX, a polyamine isolated from the American funnel web spider, Agelenopsis aperta. 2 Voltage‐activated Ca2+ currents and Ca2+‐dependent Cl− currents recorded from rat cultured dorsal root ganglion neurones were reversibly inhibited by arginine polyamine (AP; 0.001 to 100 μm). Low voltage‐activated T‐type Ca2+ currents were significantly more sensitive to AP than high voltage‐activated Ca2+ currents. The IC50 values for the actions of AP on low and high voltage‐activated Ca2+ currents were 10 nm and 3 μm respectively. AP was equally effective in inhibiting high voltage‐activated currents carried by Ba2+, Sr2+ or Ca2+. However, AP‐induced inhibition of Ca2+ currents was attenuated by increasing the extracellular Ca2+ concentration from 2 mm to 10 mm. 3 The actions of AP on a Ca2+‐independent K+ current were more complex, 1 μm AP enhanced this current but 10 μm AP had a dual action, initially enhancing but then inhibiting the K+ current. 4 γ‐Aminobutyric acid‐activated Cl− currents were also reversibly inhibited by 1 to 10 μm AP. In contrast N‐methyl‐d‐aspartate currents recorded from rat cultured cerebellar neurones were greatly enhanced by 10 μm AP. 5 We conclude that at a concentration of 10 nm, AP is a selective inhibitor of low threshold T‐type voltage‐activated Ca2+ currents. However, at higher concentrations 1–10 μm AP interacts with ion channels or other membrane constituents to produce a variety of actions on both voltage and ligand gated ion channels.

Voltage-dependent modulation of rat sensory neurone calcium channel currents by G protein activation : effect of a dihydropyridine antagonist

The ability of a depolarizing prepulse to increase the rate of activation of IBa has been examined in cultured sensory neurones of the rat. Both in control neurones and in the presence internally of the guanine nucleotide analogue, guanosine 5′‐O‐3‐thiotriphosphate (GTPγS) which markedly slows the rate of activation of IBa and reduces its amplitude, a depolarizing prepulse increased the rate of activation of IBa, but did not increase its amplitude measured at the end of the 100 ms voltage step. The calcium channel antagonist (−)‐202–791, which we have previously shown to increase the amplitude of IBa in the presence of GTPγS, did not occlude the response to a depolarizing prepulse, suggesting that the mechanism of action of (−)‐202–791 is not to disrupt the interaction of the channels with activated G proteins.