Karen N. Bradley

What can toxins tell us for drug discovery

Toxins are of interest in drug design because the toxins provide three-dimensional templates for creating small molecular mimics with interesting pharmacological properties. Toxins are also useful in drug discovery because they can be used as pharmacological tools to uncover potential therapeutic targets. With their high potency and selectivity, toxins are often more useful in functional experiments than standard pharmacological agents. We have used two groups of neurotoxins, the dendrotoxins and the muscarinic toxins (MTs), to explore the involvement of subtypes of potassium ion channels and muscarinic receptors, respectively, in processes involved in cognition and the changes in neuronal properties with aging. From our current work, quantitative autoradiographic studies with radiolabelled dendrotoxins reveal widespread distribution of binding sites throughout rat brain sections, but few differences exist between young adult and aged rats. However, displacement studies with toxin K, which preferentially binds to the Kv1.1 subtype of cloned potassium channel, show the selective loss of such sites in regions of the hippocampus and septohippocampal pathway with aging. MTs have been tested for effects on performance of rats in memory paradigms. MT2, which activates m1 receptors, improves performance of rats in a step-down inhibitory avoidance test, whereas MT3, which blocks m4 receptors, decreases performance when given into the hippocampus. This is the first clear demonstration of a role for m4 muscarinic receptors in cognition.

Origin and Mechanisms of Ca2+ Waves in Smooth Muscle as Revealed by Localized Photolysis of Caged Inositol 1,4,5-Trisphosphate

The cytosolic Ca2+ concentration ([Ca2+]c) controls diverse cellular events via various Ca2+ signaling patterns; the latter are influenced by the method of cell activation. Here, in single-voltage clamped smooth muscle cells, sarcolemma depolarization generated uniform increases in [Ca2+]c throughout the cell entirely by Ca2+ influx. On the other hand, the Ca2+ signal produced by InsP3-generating agonists was a propagated wave. Using localized uncaged InsP3, the forward movement of the Ca2+ wave arose from Ca2+-induced Ca2+ release at the InsP3 receptor (InsP3R) without ryanodine receptor involvement. The decline in [Ca2+]c (the back of the wave) occurred from a functional compartmentalization of the store, which rendered the site of InsP3-mediated Ca2+ release, and only this site, refractory to the phosphoinositide. The functional compartmentalization arose by a localized feedback deactivation of InsP3 receptors produced by an increased [Ca2+]c rather than a reduced luminal [Ca2+] or an increased cytoplasmic [InsP3]. The deactivation of the InsP3 receptor was delayed in onset, compared with the time of the rise in [Ca2+]c, persisted (>30 s) even when [Ca2+]c had regained resting levels, and was not prevented by kinase or phosphatase inhibitors. Thus different forms of cell activation generate distinct Ca2+ signaling patterns in smooth muscle. Sarcolemma Ca2+ entry increases [Ca2+]c uniformly; agonists activate InsP3R and produce Ca2+ waves. Waves progress by Ca2+-induced Ca2+ release at InsP3R, and persistent Ca2+-dependent inhibition of InsP3R accounts for the decline in [Ca2+]c at the back of the wave.

Muscarinic toxins from the green mamba

Muscarinic acetylcholine receptors are involved in many important physiological processes. Discovery of different subtypes of muscarinic receptors that are responsible for modulating specific physiological events was a key development in muscarinic receptor research. However, the lack of highly selective muscarinic agonists and antagonists has made the classification of a muscarinic receptor subtype responsible for the mediation or modulation of a particular response very difficult. Toxins have previously proved to be highly useful pharmacological tools, due to their high potency and selectivity. This review looks at a new class of muscarinic ligand isolated from the venom of the Eastern green mamba (Dendroaspis angusticeps). Just over a decade ago, it was found that two toxins from the green mamba venom appeared to distinguish between different muscarinic receptor subtypes. Since then, at least 10 more muscarinic toxins (MTs) have been isolated from mamba venom. In recent years, some of the MTs have been used as pharmacological tools; for example, to determine the muscarinic receptor subtype involved in inhibition of adenylyl cyclase in rat striatum. This review looks at the progress that has been made over the past 10 years in the area of MT research and examines whether or not these new peptides are a new way forward in the field of muscarinic receptor research.

In smooth muscle, FK506-binding protein modulates IP3 receptor-evoked Ca2+ release by mTOR and calcineurin.

Ca2+ release from the sarcoplasmic reticulum (SR) by the IP3 receptors (IP3Rs) crucially regulates diverse cell signalling processes from reproduction to apoptosis. Release from the IP3R may be modulated by endogenous proteins associated with the receptor, such as the 12 kDa FK506-binding protein (FKBP12), either directly or indirectly by inhibition of the phosphatase calcineurin. Here, we report that, in addition to calcineurin, FKPBs modulate release through the mammalian target of rapamycin (mTOR), a kinase that potentiates Ca2+ release from the IP3R in smooth muscle. The presence of FKBP12 was confirmed in colonic myocytes and co-immunoprecipitated with the IP3R. In aortic smooth muscle, however, although present, FKBP12 did not co-immunoprecipitate with IP3R. In voltage-clamped single colonic myocytes rapamycin, which together with FKBP12 inhibits mTOR (but not calcineurin), decreased the rise in cytosolic Ca2+ concentration ([Ca2+]c) evoked by IP3R activation (by photolysis of caged IP3), without decreasing the SR luminal Ca2+ concentration ([Ca2+]l) as did the mTOR inhibitors RAD001 and LY294002. However, FK506, which with FKBP12 inhibits calcineurin (but not mTOR), potentiated the IP3-evoked [Ca2+]c increase. This potentiation was due to the inhibition of calcineurin; it was mimicked by the phosphatase inhibitors cypermethrin and okadaic acid. The latter two inhibitors also prevented the FK506-evoked increase as did a calcineurin inhibitory peptide (CiP). In aortic smooth muscle, where FKBP12 was not associated with IP3R, the IP3-mediated Ca2+ release was unaffected by FK506 or rapamycin. Together, these results suggest that FKBP12 has little direct effect on IP3-mediated Ca2+ release, even though it is associated with IP3R in colonic myocytes. However, FKBP12 might indirectly modulate Ca2+ release through two effector proteins: (1) mTOR, which potentiates and (2) calcineurin, which inhibits Ca2+ release from IP3R in smooth muscle.

Ca2+ regulation in guinea‐pig colonic smooth muscle: the role of the Na+‐Ca2+ exchanger and the sarcoplasmic reticulum

To study the contribution of the Na+‐Ca2+ exchanger to Ca2+ regulation and its interaction with the sarcoplasmic reticulum (SR), changes in cytoplasmic Ca2+ concentration ([Ca2+]c) were measured in single, voltage clamped, smooth muscle cells. Increases in [Ca2+]c were evoked by either depolarisation (−70 mV to 0 mV) or by release from the SR by caffeine (10 mm) or flash photolysis of caged InsP3 (InsP3). Depletion of the SR of Ca2+ (verified by the absence of a response to caffeine and InsP3) by either ryanodine (50 μm), to open the ryanodine receptors (RyRs), or thapsigargin (500 nm) or cyclopiazonic acid (CPA, 10 μm), to inhibit the SR Ca2+ pumps, reduced neither the magnitude of the Ca2+ transient nor the relationship between the influx of and the rise in [Ca2+]c evoked by depolarisation. This suggested that Ca2+‐induced Ca2+ release (CICR) from the SR did not contribute significantly to the depolarisation‐evoked rise in [Ca2+]c. However, although Ca2+ was not released from it, the SR accumulated the ion following depolarisation since ryanodine and thapsigargin each slowed the rate of decline of the depolarisation‐evoked Ca2+ transient. Indeed, the SR Ca2+ content increased following depolarisation as assessed by the increased magnitude of the [Ca2+]c levels evoked each by InsP3 and caffeine, relative to controls. The increased SR Ca2+ content following depolarisation returned to control values in approximately 12 min via Na+‐Ca2+ exchanger activity. Thus inhibition of the Na+‐Ca2+ exchanger by removal of external Na+ (by either lithium or choline substitution) prevented the increased SR Ca2+ content from returning to control levels. On the other hand, the Na+‐Ca2+ exchanger did not appear to regulate bulk average Ca2+ directly since the rates of decline in [Ca2+]c, following either depolarisation or the release of Ca2+ from the SR (by either InsP3 or caffeine), were neither voltage nor Na+ dependent. Thus, no evidence for short term (seconds) control of [Ca2+]c by the Na+‐Ca2+ exchanger was found. Together, the results suggest that despite the lack of CICR, the SR removes Ca2+ from the cytosol after its elevation by depolarisation. This Ca2+ is then removed from the SR to outside the cell by the Na+‐Ca2+ exchanger. However, the exchanger does not contribute significantly to the decline in bulk average [Ca2+]c following transient elevations in the ion produced either by depolarisation or by release from the store.

Two Ca2+ entry pathways mediate InsP3‐sensitive store refilling in guinea‐pig colonic smooth muscle

1 Sarcolemma Ca2+ influx, necessary for store refilling, was well maintained, over a wide range (‐70 to + 40 mV) of membrane voltages, in guinea‐pig single circular colonic smooth muscle cells, as indicated by the magnitude of InsP3‐evoked Ca2+ transients. 2 This apparent voltage independence of store refilling was achieved by the activity of sarcolemma Ca2+ channels some of which were voltage gated while others were not. At negative membrane potentials (e.g. ‐70 mV), Ca2+ influx through channels which lacked voltage gating provided for store refilling while at positive membrane potentials (e.g. +40 mV) voltage‐gated Ca2+ channels were largely responsible. 3 Sarcolemma voltage‐gated Ca2+ currents were not activated following store depletion. 4 Removal of external Ca2+ or the addition of the Ca2+ channel blocker nimodipine (1 μM) inhibited store refilling, as assessed by the magnitude of InsP3‐evoked Ca2+ transients, with little or no change in bulk average cytoplasmic Ca2+ concentration. One hypothesis for these results is that the store may refill from a high subsarcolemma Ca2+ gradient. 5 Influx via channels, some of which are voltage gated and others which lack voltage gating, may permit the establishment of a subsarcolemma Ca2+ gradient. Store access to the gradient allows InsP3‐evoked Ca2+ signalling to be maintained over a wide voltage range in colonic smooth muscle.

Effects of muscarinic toxins MT1 and MT2 from green mamba on different muscarinic cholinoceptors.

MT1 and MT2, polypeptides from green mamba venom, known to bind to muscarinic cholinoceptors, behave like muscarinic agonists in an inhibitory avoidance task in rats. We have further characterised their functional effects using different preparations. MT1 and MT2 behaved like relatively selective muscarinic M1 receptor agonists in rabbit vas deferens, but their effects were not reversed by washing or prevented by muscarinic antagonists, although allosteric modulators altered responses to MT1. Radioligand binding experiments indicated that both toxins irreversibly inhibited [3H]N-methylscopolamine binding to cloned muscarinic M1 and M4 receptors, and reduced binding to M5 subtype with lower affinity, while they reversibly inhibited the binding of [3H]prazosin to rat cerebral cortex and vas deferens, with 20 fold lower affinity. High concentrations of MT1 reversibly blocked responses of vas deferens to noradrenaline. MT1 and MT2 appear to irreversibly activate muscarinic M1 receptors at a site distinct from the classical one, and to have affinity for some α-adrenoceptors.

Effects of muscarinic toxins MT2 and MT7, from green mamba venom, on m1, m3 and m5 muscarinic receptors expressed in Chinese hamster ovary cells

Several small proteins called muscarinic toxins (MTs) have been isolated from venom of green mamba (Dendroaspis angusticeps). They have previously been shown in radioligand binding studies to have high selectivity and affinity for individual muscarinic receptor subtypes, but less is known of their functional effects. This study has examined the actions of two of these MTs, MT2 and MT7, using changes in cytosolic Ca(2+) ([Ca(2+)](i)) measured using the fluorescent indicator fura-2 in Chinese Hamster Ovary (CHO) cells stably transfected with individual muscarinic receptor subtypes, m1, m3 and m5. MT2 activated the m1 receptor: at concentrations above 100 nM it caused significant and concentration-dependent increases in [Ca(2+)](i). From 25 to 800 nM MT2 also produced increases in [Ca(2+)](i) by activating m3 receptors, although these increases in [Ca(2+)](i) were not strictly concentration-dependent with only intermittent responses being recorded (i.e. it was not always possible to obtain a response to the agonist with each application of the compound). MT2 (800-1600 nM) also caused significant increases in [Ca(2+)](i) in CHO cells expressing the m5 muscarinic receptor subtype. MT7 (1 microM) displayed no agonist activity at any of the muscarinic receptors but was a potent non-competitive antagonist (at 20 nM) at the m1 muscarinic receptor subtype. It had no antagonist activity at the m3 or m5 subtypes. These results indicate that MT7 is a highly specific antagonist at the m1 muscarinic receptor subtype as suggested by results from radioligand binding studies. However, MT2 is less selective for the m1 muscarinic receptor than previously described as it also exhibits agonist activity at the m3 and m5 muscarinic receptors, which was not detected in radioligand binding studies.

Use of muscarinic toxins, mtx1, mtx2 and mtx3, in the study of synaptic transmission in the peripheral nervous system

Abstract of poster presentation on the use of muscarinic toxins in the study of synaptic transmission in the peripheral nervous system. Presented at the Seventh International Symposium of Subtypes of Muscarinic Receptors. Vienna, Virginia, USA, 12 November - 15 November 1996.

Effects of brucine, a plant alkaloid, on M1 muscarinic receptors and α1-adrenoceptors in the rabbit vas deferens preparation

The plant alkaloid brucine is an analogue of strychnine and is known to be an allosteric modulator at cloned M(1) muscarinic receptors. The functional effects of brucine were examined on the M(1) muscarinic receptors in the rabbit isolated vas deferens preparation. Brucine (10-100 microM) enhanced the effects of the muscarinic agonist McN-A-343 at presynaptic M(1) muscarinic receptors in the rabbit isolated vas deferens preparation, but only when brucine was added prior to McN-A-343. This effect is indicative of a positive allosteric action. It was poorly reversed on washing. Brucine did not affect the responses to the mamba venom muscarinic toxins MT2 and MT4, which are also allosteric activators in this preparation. Brucine (10-100 microM) caused a significant decrease in the twitch response to electrical stimulation in the rabbit vas deferens preparation, which was not antagonised by 100 nM pirenzepine (an M(1) muscarinic antagonist). Brucine and MT4, but not MT2, caused significant decreases (p<0.05) in the responses to noradrenaline in the rabbit vas deferens preparation. Responses to ATP and KCl were not affected. In radioligand binding assays, brucine displaced the alpha(1)-adrenoceptor ligand prazosin from its specific binding sites in membranes made from rat cerebral cortex and rat vas deferens. The apparent K(i) values were 150 and 3.4 microM in the cortical and vas deferens membranes, respectively. The positive allosterism found with brucine at cloned M(1) receptors seems to be mirrored at native M(1) receptors. However, the unexpected blocking effects at alpha(1)-adrenoceptors indicates that more selective ligands than brucine are required as starting points for the design of specific enhancers of the activity of M(1) receptors with therapeutic potential.