GH 3 cells present spontaneous Ca 2ϩ action potentials and oscillations of intracellular Ca 2ϩ , which can be modified by altering the activity of K ϩ or Ca 2ϩ channels. We took advantage of this spontaneous activity to screen for effects of a purified toxin (Tx3-1) from the venom of Phoneutria nigriventer on ion channels. We report that Tx3-1 increases the frequency of Ca 2ϩ oscillations, as do two blockers of potassium channels, 4-aminopyridine and charybdotoxin. Whole-cell patch clamp experiments show that Tx3-1 reversibly inhibits the A-type K ϩ current (I A ) but does not block other K ϩ currents (delayed-rectifying, inward-rectifying, and largeconductance Ca 2ϩ -sensitive) or Ca 2ϩ channels (T and L type) in these cells. In addition, we describe the sequence of a full cDNA clone of Tx3-1, which shows that Tx3-1 has no homology to other known blockers of K ϩ channels and gives insights into the processing of this neurotoxin. We conclude that Tx3-1 is a selective inhibitor of I A , which can be used to probe the role of this channel in the control of cellular function. Based on the effect of Tx3-1, we suggest that I A is an important determinant of the frequency of Ca 2ϩ oscillations in unstimulated GH 3 cells.

The present experiments investigated the effect of some of the toxic components present in the venom of the spider Phoneutria nigriventer on the release of neurotransmitter. The toxic fraction, Phoneutria nigriventer toxin-3 (PhTx3), abolished Ca2+-dependent glutamate release, but did not alter Ca2+-independent secretion of glutamate when rat brain cortical synaptosomes were depolarized with 33 mM KCl. This effect was most likely due to interference with the entry of calcium through voltage-gated calcium channels, because PhTx3 reduced by 50% the increase in intrasynaptosomal free calcium induced by membrane depolarization, and did not affect the release of glutamate evoked by a calcium ionophore (ionomycin). A polypeptide (Tx3-3) present in the PhTx3 fraction reproduced the effects of the PhTx3 fraction on transmitter release and intrasynaptosomal free calcium in the low nanomolar range. We compared the alterations produced by the Tx3-3 with the actions of toxins known to block calcium channels coupled to exocytosis: the results indicated that the Tx3-3 inhibition of glutamate release and intrasynaptosomal calcium resemble that observed with omega-conotoxin MVIIC. We suggest that the Tx3-3 is a calcium-channel antagonist that blocked glutamate exocytosis.

PhTX2, one of the components of the venom of the South American spider Phoneutria nigriventer, inhibits the closure of voltage-sensitive Na+ channels. Incubation of cerebral-cortical synaptosomes with PhTX2 causes a rapid increase in the intrasynaptosomal free Ca2+ concentration and a dose-dependent release of glutamate. This release is made up of a slow component, which appears to be due to reversal of Na(+)-dependent glutamate uptake, and more rapid component that is dependent on the entry of extrasynaptosomal Ca2+. It has previously been shown that membrane depolarization using KCl can cause rapid Ca(2+)-dependent release of glutamate from synaptosomes. This requires Ca2+ entry through a specific type of Ca2+ channel that is sensitive to Aga-GI, a toxic component of the venom of the spider Agelenopsis aperta. We have compared the effects of PhTX2 and KCl on elevation of intrasynaptosomal free Ca2+ and glutamate release, and a number of differences have emerged. Firstly, PhTX2-mediated Ca2+ influx and glutamate release, but not those caused by KCl, are inhibited by tetrodotoxin. Secondly, KCl produces a clear additional increase in Ca2+ and glutamate release following those elicited by PhTX2. Finally, 500 microM MnCl2 abolishes PhTX2-mediated, but not KCl-mediated, glutamate release. These findings suggest that more than one mechanism of Ca2+ entry may be coupled to glutamate release from nerve endings.

Optical tracers in conjunction with fluorescence microscopy have become widely used to follow the movement of synaptic vesicles in nerve terminals. The present review discusses the use of these optical methods to understand the regulation of exocytosis and endocytosis of synaptic vesicles. The maintenance of neurotransmission depends on the constant recycling of synaptic vesicles and important insights have been gained by visualization of vesicles with the vital dye FM1-43. A number of questions related to the control of recycling of synaptic vesicles by prolonged stimulation and the role of calcium to control membrane internalization are now being addressed. It is expected that optical monitoring of presynaptic activity coupled to appropriate genetic models will contribute to the understanding of membrane traffic in synaptic terminals.

Tityustoxin (TsTX), a toxin obtained from the venom of the Brazilian scorpion Tityus serrulatus, stimulates Na+ influx through tetrodotoxin (TTX)-sensitive Na+ channels which, in turn, promotes both Ca(2+)-dependent and Ca(2+)-independent release of glutamate from rat cerebrocortical synaptosomes. The level of Ca(2+)-dependent glutamate release after addition of 0.5 microM TsTX is greater than that produced by a maximally depolarizing concentration of KCl. This effect of TsTX, which is entirely dependent on Na+ entry, suggests that Na+ has a role in modulating Ca2+ entry and glutamate release that is not simply related to membrane depolarization. In order to investigate possible modulatory role(s) of Na+ on Ca(2+)-dependent glutamate release, we compared the effects of TsTX with those of KCl and the Na+ ionophore gramicidin D. When used alone, 100 nM gramicidin D produced a larger increase in intrasynaptosomal free Na+ than did 0.5 microM TsTX, and a similar rise in intrasynaptosomal free Ca2+, but was much less effective in promoting glutamate release. Even the combination of membrane depolarization (by 33 mM KCl) and elevation of intrasynaptosomal free Na+ (by 100 nM gramicidin) was still less effective than TsTX at causing Ca(2+)-dependent glutamate release. These data suggest that localized Na+ entry, through TTX-sensitive Na+ channels, exerts a modulatory role on Ca(2+)-dependent glutamate release from nerve endings in the cerebral cortex.

O edema cerebral, devido a suas repercussões sobre a morbidade e mortalidade de milhões de pacientes em todo o mundo, ainda constitui um desafio para a medicina. A última década trouxe novos conhecimentos sobre como a água transita pelas diversas interfaces de membrana no cérebro. Hoje sabemos que várias proteínas que formam canais estão envolvidas na redistribuição de volumes de água pelo tecido cerebral. Essas proteínas, chamadas aquaporinas, descobertas em 1992, estão elucidando diversos mecanismos da distribuição de água no cérebro e, possivelmente, serão alvos para novos fármacos com ação potencial sobre o edema cerebral. Nossa expectativa sobre essas possibilidades é reforçada pelo conhecimento de que, há muitos anos, já manipulamos proteínas similares usando fármacos hoje bem conhecidos.