We describe the first potent and selective blocker of the class E Ca2+channel. SNX-482, a novel 41 amino acid peptide present in the venom of the African tarantula, Hysterocrates gigas, was identified through its ability to inhibit human class E Ca2+ channels stably expressed in a mammalian cell line. An IC50 of 15-30 nM was obtained for block of the class E Ca2+ channel, using either patch clamp electrophysiology or K+-evoked Ca2+ flux. At low nanomolar concentrations, SNX-482 also blocked a native resistant or R-type Ca2+ current in rat neurohypophyseal nerve terminals, but concentrations of 200-500 nM had no effect on R-type Ca2+ currents in several types of rat central neurons. The peptide has the sequence GVDKAGCRYMFGGCSVNDDCCPRLGCHSLFSYCAWDLTFSD-OH and is homologous to the spider peptides grammatoxin S1A and hanatoxin, both peptides with very different ion channel blocking selectivities. No effect of SNX-482 was observed on the following ion channel activities: Na+ or K+ currents in several cultured cell types (up to 500 nM); K+ current through cloned potassium channels Kv1.1 and Kv1. 4 expressed in Xenopus oocytes (up to 140 nM); Ca2+ flux through L- and T-type Ca2+ channels in an anterior pituitary cell line (GH3, up to 500 nM); and Ba2+ current through class A Ca2+ channels expressed in Xenopus oocytes (up to 280 nM). A weak effect was noted on Ca2+ current through cloned and stably expressed class B Ca2+ channels (IC50 > 500 nM). The unique selectivity of SNX-482 suggests its usefulness in studying the diversity, function, and pharmacology of class E and/or R-type Ca2+ channels.

The sperm acrosome reaction is a Ca 2؉ -dependent exocytotic event that is triggered by adhesion to the mammalian egg's zona pellucida. Previous studies using ionselective f luorescent probes suggested a role of voltagesensitive Ca 2؉ channels in acrosome reactions. Here, wholecell patch clamp techniques are used to demonstrate the expression of functional T-type Ca 2؉ channels during mouse spermatogenesis. The germ cell T current is inhibited by antagonists of T-type channels (pimozide and amiloride) as well as by antagonists whose major site of action is the somatic cell L-type Ca 2؉ channel (1,4-dihydropyridines, arylalkylamines, benzothiazapines), as has also been reported for certain somatic cell T currents. In sperm, inhibition of T channels during gamete interaction inhibits zona pellucidadependent Ca 2؉ elevations, as demonstrated by ion-selective f luorescent probes, and also inhibits acrosome reactions. These studies directly link sperm T-type Ca 2؉ channels to fertilization. In addition, the kinetics of channel inhibition by 1,4-dihydropyridines suggests a mechanism for the reported contraceptive effects of those compounds in human males.

In many cells, receptor activation initiates sustained Ca2+ entry which is critical in signal transduction. Mammalian transient receptor potential (Trp) proteins, which are homologous to the Drosophila photoreceptor-cell Trp protein, have emerged as candidate subunits of the ion channels that mediate this influx. As a consequence of overexpression, these proteins produce cation currents that open either after depletion of internal Ca2+ stores or through receptor activation. However, determining the role of endogenous Trp proteins in signal transduction is complicated by the absence of selective antagonists. Here we examine Trp function during sperm-egg interaction. The sperm acrosome reaction is a Ca2+-dependent secretory event that must be completed before fertilization. In mammals, exocytosis is triggered during gamete contact by ZP3, a glycoprotein constituent of the egg's extracellular matrix, or zona pellucida (ZP). ZP3 activates trimeric G proteins and phospholipase C and causes a transient Ca2+ influx into sperm through T-type Ca2+ channels. These early responses promote a second Ca2+-entry pathway, thereby producing sustained increases in intracellular Ca2+ concentration ([Ca2+]i) that drive acrosome reactions. Our results show that Trp2 is essential for the activation of sustained Ca2+ influx into sperm by ZP3.

Multiple types of voltage-dependent Ca(2+) channels are involved in the regulation of neurotransmitter release (Tsien et al., 1991; Dunlap et al., 1995). In the nerve terminals of the neurohypophysis, the roles of L-, N-, and P/Q-type Ca(2+) channels in neuropeptide release have been identified previously (Wang et al., 1997a). Although the L- and N-type Ca(2+) currents play equivalent roles in both vasopressin and oxytocin release, the P/Q-type Ca(2+) current only regulates vasopressin release. An oxytocin-release and Ca(2+) current component is resistant to the L-, N-, and P/Q-type Ca(2+) channel blockers but is inhibited by Ni(2+). A new polypeptide toxin, SNX-482, which is a specific alpha(1E)-type Ca(2+) channel blocker (Newcomb et al., 1998), was used to characterize the biophysical properties of this resistant Ca(2+) current component and its role in neuropeptide release. This resistant component was dose dependently inhibited by SNX-482, with an IC(50) of 4.1 nM. Furthermore, SNX-482 did not affect the other Ca(2+) current types in these CNS terminals. Like the N- and P/Q-type Ca(2+) currents, this SNX-482-sensitive transient Ca(2+) current is high-threshold activated and shows moderate steady-state inactivation. At the same concentrations, SNX-482 blocked the component of oxytocin, but not of vasopressin, release that was resistant to the other channel blockers, indicating a preferential role for this type of Ca(2+) current in oxytocin release from neurohypophysial terminals. Our results suggest that an alpha(1E) or "R"-type Ca(2+) channel exists in oxytocinergic nerve terminals and, thus, functions in controlling only oxytocin release from the rat neurohypophysis.

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The nerve endings of rat neurohypophyses were acutely dissociated and a combination of pharmacological, biophysical and biochemical techniques was used to determine which classes of Ca2+ channels on these central nervous system (CNS) terminals contribute functionally to arginine vasopressin (AVP) and oxytocin (OT) secretion. Purified neurohypophysial plasma membranes not only had a single high‐affinity binding site for the N‐channel‐specific ω‐conopeptide MVIIA, but also a distinct high‐affinity site for another ω‐conopeptide (MVIIC), which affects both N‐ and P/Q‐channels. Neurohypophysial terminals exhibited, besides L‐ and N‐type currents, another component of the Ca2+ current that was only blocked by low concentrations of MVIIC or by high concentrations of ω‐AgaIVA, a P/Q‐channel‐selective spider toxin. This Ca2+ current component had pharmacological and biophysical properties similar to those described for the fast‐inactivating form of the P/Q‐channel class, suggesting that in the neurohypophysial terminals this current is mediated by a ‘Q’‐type channel. Pharmacological additivity studies showed that this Q‐component contributed to rises in intraterminal Ca2+ concentration ([Ca2+]i) in only half of the terminals tested. Furthermore, the non‐L‐ and non‐N‐component of Ca2+‐dependent AVP release, but not OT release, was effectively abolished by the same blockers of Q‐type current. Thus Q‐channels are present on a subset of the neurohypophysial terminals where, in combination with N‐ and L‐channels, they control AVP but not OT peptide neurosecretion.

We have investigated the action of SNX482, a toxin isolated from the venom of the tarantula Hysterocrates gigas, on voltage-dependent calcium channels expressed in tsa-201 cells. Upon application of 200 nM SNX482, R-type alpha(1E) calcium channels underwent rapid and complete inhibition, which was only poorly reversible upon washout. However, upon application of strong membrane depolarizations, rapid and complete recovery from inhibition was obtained. Tail current analysis revealed that SNX482 mediated an approximately 70 mV depolarizing shift in half-activation potential, suggesting that the toxin inhibits alpha(1E) calcium channels by preventing their activation. Experiments involving chimeric channels combining structural features of alpha(1E) and alpha(1C) subunits indicated that the presence of the domain III and IV of alpha(1E) is a prerequisite for a strong gating inhibition. In contrast, L-type alpha(1C) channels underwent incomplete inhibition at saturating concentrations of SNX482 that was paralleled by a small shift in half-activation potential and which could be rapidly reversed, suggesting a less pronounced effect of the toxin on L-type calcium channel gating. We conclude that SNX482 does not exhibit unequivocal specificity for R-type channels, but highly effectively antagonizes their activation.

Rat magnocellular neurones with cell bodies in the supraoptic and paraventricular nuclei of the hypothalamus send their axonal nerve endings into the neurohypophysis, where they release oxytocin or vasopressin into the bloodstream during highly specific firing patterns. Peptide release from the neurohypophysis is closely controlled by an interplay between the duration and frequency of the action potential burst, and the silence that separates the bursts generated by these neurones (Cazalis et al. 1985). Thus, understanding of peptide release from the hypothalamicneurohypophysial system requires characterization of the conductances that shape action potentials in both the cell bodies and their axonal endings. The physical separation of cell bodies and nerve endings in this system allows a unique opportunity to elucidate the differences in neuronal excitability, and underlying conductances in different cellular domains. Large conductance Ca¥-activated K¤ (BK) channels are ubiquitous in neurones. They typically exhibit high permeability and selectivity for K¤, and activation upon 1. Large conductance, Ca¥-activated K¤ (BK) channels were identified in freshly dissociated rat supraoptic neurones using patch clamp techniques. 2. The single channel conductance of cell body BK channels, recorded from inside-out patches in symmetric 145 mÒ K¤, was 246·1 pS, compared with 213 pS in nerve ending BK channels (P < 0·01). 3. At low open probability (Pï), the reciprocal of the slope in the ln(NPï)-voltage relationship (N, number of available channels in the patch) for cell body and nerve ending channels were similar: 11 vs. 14 mVper e-fold change in NPï, respectively. 4. At 40 mV, the [Ca¥]é producing half-maximal activation was 273 nÒ, as opposed to > 1·53 ìÒ for the neurohypophysial channel, indicating the higher Ca¥ sensitivity of the cell body isochannel. 5. Cell body BK channels showed fast kinetics (open time constant, 8·5 ms; fast closed time constant, 1·6 and slow closed time constant, 12·7 ms), identifying them as 'type I' isochannels, as opposed to the slow gating (type II) of neurohypophysial BK channels. 6. Cell body BK activity was reduced by 10 nÒ charybdotoxin (NPï, 37% of control), or 10 nÒ iberiotoxin (NPï, 5% of control), whereas neurohypophysial BK channels are insensitive to charybdotoxin at concentrations as high as 360 nÒ. 7. Whilst blockade of nerve ending BK channels markedly slowed the repolarization of evoked single spikes, blockade of cell body channels was without effect on repolarization of evoked single spikes. 8. Ethanol reversibly increased neurohypophysial BK channel activity (EC50, 22 mÒ; maximal effect, 100 mÒ). In contrast, ethanol (up to 100 mÒ) failed to increase cell body BK channel activity. 9. In conclusion, we have characterized BK channels in supraoptic neuronal cell bodies, and demonstrated that they display different electrophysiological and pharmacological properties from their counterparts in the nerve endings. Keywords: