Potent and voltage-dependent block by philanthotoxin-343 of neuronal nicotinic receptor/channels in PC12 cells.
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1. Block by philanthotoxin-343 (PhTX-343), a neurotoxin from wasps, of ionic currents mediated through neuronal nicotinic acetylcholine (ACh) receptor/channels was characterized in rat phaeochromocytoma PC12 cells, by use of whole cell voltage-clamp techniques. 2. In the cells held at -60 mV, PhTX-343 at 0.1 and 1 microM inhibited an inward current activated by 100 microM ACh. The current inhibition was relieved by depolarizing steps, and augmented at negative potentials, suggesting that PhTX-343 blocks the channel in a voltage-dependent manner. Joro spider toxin-3 (JSTX-3) also exerted voltage-dependent inhibition of ACh-activated currents in a similar concentration range, but argiotoxin636 did not affect the currents. 3. Analysis of the current decay during hyperpolarizing steps indicated that the current inhibition by 100 nM PhTX-343 develops in an order of several hundres of milliseconds. On the other hand, the recovery from the current inhibition during depolarizing steps developed in an order of about 100 ms. 4. The results suggest that PhTX-343 blocks neuronal nicotinic receptor channels in PC12 cells at concentrations lower than those required for channel block in non-mammalian cells, and the block exhibits clear voltage-dependence. Estimated from the voltage-dependence, the binding site of PhTX-343 may be located near the outer mouth of the channel. (+info)
Immunologic reactivity on one year follow-up of subjects without allergy to Hymenoptera stings.
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We studied Hymenoptera stings in 72 pest-control operators without any previous systemic reactions to Hymenoptera stings, and investigated their venom-specific IgE levels in serial specimens collected over one year. At the initial evaluation, venom-specific IgE was present in 25 (34.7%) of 72 pest-control operators, and venom-specific IgE titer significantly decreased as the time interval from the last sting increased (p < 0.001). In most cases, venom-specific IgE disappeared less than 3 years after the last sting. On the other hand, the ratio of subjects with positive CAP for venom-specific IgE was significantly increased with an elevation of total serum IgE level (p < 0.001). After the one year follow-up, venom-specific IgE titer in the 25 subjects with positive CAP decreased significantly (p = 0.026). Total serum IgE level modified the decline significantly (p = 0.011), but the time interval from the last sting did not. In elevated total IgE level (>250 IU/ml), the decline of venom-specific IgE tended to be slow. (+info)
An analysis of philanthotoxin block for recombinant rat GluR6(Q) glutamate receptor channels.
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1. The action of philanthotoxin 343 (PhTX) on rat homomeric GluR6(Q) recombinant glutamate receptor channels was analysed using concentration-jump techniques and outside-out patches from HEK 293 cells. Both onset and recovery from block by external PhTX were dependent on the presence of agonist, indicating that channels must open for PhTX to bind and that channel closure can trap PhTX. 2. Block by external PhTX developed with double-exponential kinetics. The rate of onset of the fast component of block showed an exponential increase per 27 mV hyperpolarization over the range -40 to -100 mV. The rate of onset of the slow component of block showed a non-linear concentration dependence indicating a rate-limiting step in the blocking mechanism. 3. The extent of block by 1 microM external PhTX was maximal at -40 mV and did not increase with further hyperpolarization; the rate of recovery from block by external PhTX increased 6-fold on hyperpolarization from -40 to -100 mV suggesting that PhTX permeates at negative membrane potentials. 4. Apparent Kd values for block by external PhTX estimated from dose-inhibition experiments decreased 300-fold on hyperpolarization from +40 mV (Kd, 19.6 microM) to -40 mV (Kd, 69 nM); there was little further increase in affinity with hyperpolarization to -80 mV (Kd, 56 nM), consistent with permeation of PhTX at negative membrane potentials. 5. Block by internal PhTX showed complex kinetics and voltage dependence. Analysis with voltage ramps from -120 to +120 mV indicated a Kd at 0 mV of 20 microM, decreasing e-fold per 16 mV depolarization. However, at +90 mV the extent of block by 1 and 10 microM internal PhTX (73 % and 95 %, respectively) reached a maximum and did not increase with further depolarization. 6. Voltage-jump analysis of block by 100 microM internal PhTX revealed partial trapping. With 100 ms jumps from -100 to -40 mV, onset and recovery from block were complete within 5 ms. With jumps of longer duration the extent of block increased, with a time constant of 8.1 s, reaching 84 % at 30 s. On repolarization to -100 mV, recovery from block showed fast and slow components. 7. The amplitude of the slow component of block by internal PhTX showed a biphasic voltage dependence, first increasing then decreasing with progressive depolarization. Maximum block was obtained at 0 mV. 8. Our results suggest that PhTX acts as an open channel blocker; however, provided that the toxin remains bound to the channel, an allosteric mechanism destabilizes the open state, inducing channel closing and trapping PhTX. Strong depolarization for internal PhTX, or strong hyperpolarization for external PhTX, forces the toxin to permeate before it triggers entry into closed blocked states. (+info)