Blockade of neuronal nitric oxide synthase abolishes the toxic effects of Tx2-5, a lethal Phoneutria nigriventer spider toxin.

The primary goal of this study was to determine whether Tx2-5, a sodium channel selective toxin obtained from the venom of the spider Phoneutria nigriventer, produced penile erection by means of nitric oxide mechanism. Toxin identity was analyzed by MALDI-TOF, ES-MS and N-terminal amino acid sequencing. Pretreating mice with the non-selective nitric oxide synthase (NOS) inhibitor N(omega)-Nitro-L-arginine methyl ester hydrochloride (L-NAME) and the selective neuronal-NOS inhibitor 7-Nitroindazole (7-NI) prior to Tx2-5 i.p. (10 microg/25 g mouse) injection challenged the hypothesis above. Controls were injected with the D-isomer or DMSO or saline. Results demonstrated that L-NAME inhibited penile erections in about half the animals treated, while 7-NI completely abolished this effect. Interestingly 7-NI also abolished all the other symptoms of intoxication induced by Tx2-5, including salivation, respiratory distress and death. Tx2-5 killed all the animals of the control group and no one in the 7-NI-treated group. We conclude that (1) intraperitoneal injections of Tx2-5 induce a toxic syndrome that include penile erection, hypersalivation and death by respiratory distress or pulmonary edema; (2) pretreatment with the non-selective NOS inhibitor L-NAME reduces the penile erection and partially protects from the lethal effects of Tx2-5; (3) pretreatment with the nNOS-selective inhibitor 7-NI completely abolishes all the toxic effects of Tx2-5, including penile erection and death suggesting that nNOS is the major player in this intoxication; (4) toxins from other animals that affect sodium channels in the same way as Tx2-5 and induce similar toxic syndromes may have as a major common target, the activation of nitric oxide synthases.     

Expression of a recombinant Phoneutria toxin active in calcium channels

PnTx3-4 is a toxin isolated from the venom of the spider Phoneutria nigriventer that blocks N-, P/Q-, and R-type voltage-gated calcium channels and has great potential for clinical applications. In this report we used the SUMO system to express large amounts of recombinant PnTx3-4 peptide, which was found in both soluble and insoluble fractions of bacterial extracts. We purified the recombinant toxin from both fractions and showed that the recombinant peptide showed biological activity similar to the native PnTx3-4. In silico analysis of the primary sequence of PnTx3-4 indicated that the peptide conforms to all the criteria of a knottin scaffold. Additionally, circular dichroism spectrum analysis of the recombinant PnTx3-4 predicted that the toxin structure is composed of approximately 53% turns/unordered, 31% α-helix and 16% β-strand, which is consistent with predicted model of the PnTx3-4 knottin scaffold available at the knottin database (http://knottin.cbs.cnrs.fr). These studies provide the basis for future large scale production and structure-function investigation of PnTx3-4.     

PnTx4-3, a new insect toxin from Phoneutria nigriventer venom elicits the glutamate uptake inhibition exhibited by PhTx4 toxic fraction.

Several pools of neurotoxic peptides obtained from fractionated Phoneutria nigriventer venom induce different toxicological effects. One of them, PhTx4, is highly toxic towards insects and displays only a slight toxicity when injected in mice. Also, this fraction contains a class of peptides that are able to inhibit glutamate uptake in preparations of mammalian central nervous systems (CNS). In this work a new toxin called PnTx4-3 was isolated from the PhTx4 fraction by reverse phase and anion exchange steps using high performance liquid chromatography (HPLC). Edman sequencing of PnTx4-3 revealed that it was a polypeptide of 48 amino acid residues, containing 10 cysteines cross-linked by five disulfide bridges. The molecular mass measured by ES-Q-TOF mass spectrometry was 5199.49+/-0.64 Da, which is very close to the calculated mass from amino acid sequence (5199.99 Da). This toxin induces immediate excitatory effects when injected intrathoracically in house flies and cockroaches. Intracerebroventricular injections of 30 microg of PnTx4-3 in mice resulted in no apparent signs of intoxication. In order to make an orthologous comparison, pharmacological characterisation were carried out in rat brain synaptosomes by using [3H]-L-glutamate, showed that the whole PhTx4 fraction as well as the pure toxins PnTx4-3, Tx4(6-1) and Tx4(5-5) obtained of this fraction, were able to inhibit the glutamate uptake in the micromolar concentration range. PnTx4-3 inhibits the glutamate uptake in a dose dependent manner, with an IC50 of approximately 1 microM. PnTx4-3 is highly homologous to the Tx4(6-1) and Tx4(5-5) toxins previously described from the same fraction.     

Molecular cloning and characterization of Phoneutria nigriventer toxins active on calcium channels.

The aim of the present study was the molecular cloning of toxins active on calcium channels expressed by the spider Phoneutria nigriventer. Clones encoding the toxins Pn3-3A, Pn3-4A, Tx3-5, Pn3-5A, Tx3-6, Pn3-6A and Pn3-6B were identified from a cDNA library derived from the venom gland of this spider, revealing toxins of 49, 76, 45, 39, 55 and 58 amino acids residues, respectively, with polypeptide precursors being composed of three major portions: a signal peptide, a propeptide and finally, the mature toxin. A high degree of homology with the amino acid sequence was found between Pn3-3A and the neurotoxin Tx3-3 (identity of 79%), and between Pn3-4A and the neurotoxin Tx3-4 (identity of 95%). The deduced amino acid sequence for the mature polypeptides Tx3-5 and Tx3-6 confirms the polypeptide sequence previously published for these neurotoxins. In addition, the toxin Pn3-5A showed 58% identity to the Tx3-5 amino acid sequence, and the toxins Pn3-6A and Pn3-6B showed 85 and 33% identity, respectively, to the Tx3-6 amino acid sequence.     

Tx2-6 toxin of the Phoneutria nigriventer spider potentiates rat erectile function.

The venom of the spider Phoneutria nigriventer contains several toxins that have bioactivity in mammals and insects. Accidents involving humans are characterized by various symptoms including penile erection. Here we investigated the action of Tx2-6, a toxin purified from the P. nigriventer spider venom that causes priapism in rats and mice. Erectile function was evaluated through changes in intracavernosal pressure/mean arterial pressure ratio (ICP/MAP) during electrical stimulation of the major pelvic ganglion (MPG) of normotensive and deoxycorticosterone-acetate (DOCA)-salt hypertensive rats. Nitric oxide (NO) release was detected in cavernosum slices with fluorescent dye (DAF-FM) and confocal microscopy. The effect of Tx2-6 was also characterized after intracavernosal injection of a non-selective nitric oxide synthase (NOS) inhibitor, L-NAME. Subcutaneous or intravenous injection of Tx2-6 potentiated the elevation of ICP/MAP induced by ganglionic stimulation. L-NAME inhibited penile erection and treatment with Tx2-6 was unable to reverse this inhibition. Tx2-6 treatment induced a significant increase of NO release in cavernosum tissue. Attenuated erectile function of DOCA-salt hypertensive rats was fully restored after toxin injection. Tx2-6 enhanced erectile function in normotensive and DOCA-salt hypertensive rats, via the NO pathway. Our studies suggest that Tx2-6 could be important for development of new pharmacological agents for treatment of erectile dysfunction.     

Role of Ca2+ in vascular smooth muscle contractions induced by Phoneutria nigriventer spider venom.

Phoneutria nigriventer venom (PNV) contracts vascular tissues and increases arterial blood pressure. This study aimed to investigate the mechanisms involved on PNV-induced contractions of rabbit mesenteric and celiac arteries. Strips of mesenteric and celiac arteries were suspended in a cascade system and superfused with warmed and oxygenated Krebs solution. PNV was dialyzed in order to exclude the participation of biogenic amines in the contractions elicited by the venom. Noradrenaline (NA, 30-300 pmol), PNV (1-10 microg), Bay K-8644 (0.3-3 nmol) and KCl (10-100 micromol) dose-dependently contracted the preparations. Ca(2+)-free solution reduced by 38 and 83% the PNV-induced contractions of mesenteric and celiac arteries, respectively. Subsequent infusion of EGTA (0.2 mM) suppressed the residual contractions. Nifedipine (1 microM) and verapamil (10 microM) abolished PNV- and Bay K-8644-evoked contractions, whereas those induced by NA were reduced to a lesser extent. Lanthanum chloride (0.2 mM) inhibited by 75-90% the mesenteric and celiac contractions mediated by PNV. Caffeine (2 mM) fully blocked contractions induced by NA (95% mean inhibition), but only partly reduced those induced by PNV (35% mean inhibition). Ryanodine (10 microM) inhibited by 50% the contractions evoked by NA, but had no effect on the PNV-induced contractions in both tissues. Our findings indicate that PNV contracts vascular smooth muscle mainly due to increased influx of Ca(2+) from extracellular sources.     

Ontogenetic changes in Phoneutria nigriventer (Araneae, Ctenidae) spider venom.

Venom-yield and composition of differently sized individuals of the medically most important Brazilian spider Phoneutria nigriventer (Keyserling, 1891) was analysed. During growth the venom-mass increases according to a fourth order function of the prosoma size, which mainly reflects an increase of the venom gland volume. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed increasing percentages of proteins < or = 17 kDa from 4.1% in the smallest analysed spiders (2-3 months-old) to 79.1% in adult female venom. Additionally, high-pressure liquid-chromatography showed an increase of a single ('main') peak from 4.6 to 64.9%, while the overall number of other major-peaks decreased. Venom from young instars completely lacked lethality in mice up to a dose of 3.28 mg/kg i.v. as compared to a LD(50) of 0.63 mg/kg for adult female or 1.57 mg/kg for adult male venom that we reported previously. In conclusion, ontogenetic changes in venom protein-composition of growing P. nigriventer are suggested to produce increasing lethality in vertebrates.     

A common "hot spot" confers hERG blockade activity to alpha-scorpion toxins affecting K+ channels

While alpha-KTx peptides are generally known for their modulation of the Shaker-type and the Ca(2+)-activated potassium channels, gamma-KTxs are associated with hERG channels modulation. An exception to the rule is BmTx3 which belongs to subfamily alpha-KTx15 and can block hERG channels. To explain the peculiar behavior of BmTx3, a tentative "hot spot" formed of 2 basic residues (R18 and K19) was suggested but never further studied [Huys I, et al. BmTx3, a scorpion toxin with two putative functional faces separately active on A-type K(+) and HERG currents. Biochem J 2004;378:745-52]. In this work, we investigated if the "hot spot" is a commonality in subfamily alpha-KTx15 by testing the effect of (AmmTx3, Aa1, discrepin). Furthermore, single mutations altering the "hot spot" in discrepin, have introduced for the very first time a hERG blocking activity to a previously non-active alpha-KTx. Additionally, we could extend our results to other alpha-KTx subfamily members belonging to alpha-KTx1, 4 and 6, therefore, the "hot spot" represents a common pharmacophore serving as a predictive tool for yet to be discovered alpha-KTxs.     

Primary culture of venom glands from the Brazilian armed spider, Phoneutria nigriventer (Araneae, Ctenidae).

Phoneutria spider venoms are a rich source of bioactive components. The limited amounts of crude material available, however, can be considered as a major hindrance for a faster development in the field. In the present study, we attempted to establish primary cultures of venom glands of Phoneutria nigriventer as an alternative, in vitro source of venom. Three different developmental stages were tried as starting materials: whole embryo (inside the cocoon), nymph (early after cocoon hatching) and young adult (1 year after cocoon hatching). The embryonic cells remained in suspension in the primary cultures, with no signs of adhesion or differentiation, for about 6 months. Nevertheless, this culture was useful for the first chromosome C-banding of Phoneutria. An average of 29+/-1 acrocentric chromosomes were found. Striated muscle cells were the only kind of cells in the culture of venom glands from Phoneutria nymphs. The most promising results were achieved with 1-year-old specimens. Besides muscle, adherent epithelial cells were also obtained in culture. Although these cells remained in culture for a short time (up to 48 h) immunochemical analysis of the culture supernatant evidenced the presence of Phoneutria venom components. This can be considered as a first step toward the functional cultures of venom glands of Phoneutria spiders.     

Activation of K+ channels and Na+/K+ ATPase prevents aortic endothelial dysfunction in 7-day lead-treated rats

Seven day exposure to a low concentration of lead acetate increases nitric oxide bioavailability suggesting a putative role of K⁺ channels affecting vascular reactivity. This could be an adaptive mechanism at the initial stages of toxicity from lead exposure due to oxidative stress. We evaluated whether lead alters the participation of K⁺ channels and Na⁺/K⁺-ATPase (NKA) on vascular function. Wistar rats were treated with lead (1st dose 4 μg/100 g, subsequent doses 0.05 μg/100 g, im, 7 days) or vehicle. Lead treatment reduced the contractile response of aortic rings to phenylephrine (PHE) without changing the vasodilator response to acetylcholine (ACh) or sodium nitroprusside (SNP). Furthermore, this treatment increased basal O₂⁻ production, and apocynin (0.3 μM), superoxide dismutase (150 U/mL) and catalase (1000 U/mL) reduced the response to PHE only in the treated group. Lead also increased aortic functional NKA activity evaluated by K⁺-induced relaxation curves. Ouabain (100 μM) plus L-NAME (100 μM), aminoguanidine (50 μM) or tetraethylammonium (TEA, 2 mM) reduced the K⁺-induced relaxation only in lead-treated rats. When aortic rings were precontracted with KCl (60 mM/L) or preincubated with TEA (2 mM), 4-aminopyridine (4-AP, 5 mM), iberiotoxin (IbTX, 30 nM), apamin (0.5 μM) or charybdotoxin (0.1 μM), the ACh-induced relaxation was more reduced in the lead-treated rats. Additionally, 4-AP and IbTX reduced the relaxation elicited by SNP more in the lead-treated rats. Results suggest that lead treatment promoted NKA and K⁺ channels activation and these effects might contribute to the preservation of aortic endothelial function against oxidative stress.     

Functional expression of a recombinant toxin - rPnTx2-6 - active in erectile function in rat

In the current study, the putative cDNA for PnTx2-6 toxin of the Phoneutria nigriventer spider venom was cloned and expressed as tioredoxin fusion protein in the cytoplasm of Escherichia coli. The fusion protein was purified from the bacterial extracts by combination of immobilized Ni-ion affinity and gel filtration chromatographies. Then, it was cleaved by enterokinase and the generated recombinant PnTx2-6 (rPnTx2-6) was further purified by reverse-phase HPLC. Likewise the native toxin purified from the spider venom, rPnTx2-6 potentiates the erectile function when injected in rats. This result indicates that the production of functional recombinant PnTx2-6 might be an alternative to provide this basic and valuable tool for study, as well as for further understanding such complex physiological system, including its correlation with the central nervous system and local tissue factors.     

Dual action of a dinoflagellate-derived precursor of Pacific ciguatoxins (P-CTX-4B) on voltage-dependent K and Na channels of single myelinated axons

The effects of Pacific ciguatoxin-4B (P-CTX-4B, also named gambiertoxin), extracted from toxic Gambierdiscus dinoflagellates, were assessed on nodal K⁺ and Na⁺ currents of frog myelinated axons, using a conventional voltage-clamp technique. P-CTX-4B decreased, within a few minutes, both K⁺ and Na⁺ currents in a dose-dependent manner, without inducing any marked change in current kinetics. The toxin was more effective in blocking K⁺ than Na⁺ channels. P-CTX-4B shifted the voltage-dependence of Na⁺ conductance by about 14 mV towards more negative membrane potentials. This effect was reversed by increasing Ca²⁺ in the external solution. A negative shift of about 16 mV in the steady-state Na⁺ inactivation-voltage curve was also observed in the presence of the toxin. Unmodified and P-CTX-4B-modified Na⁺ currents were similarly affected by the local anaesthetic lidocaine. The decrease of the two currents by lidocaine was dependent on both the concentration and the membrane potential during pre-pulses. In conclusion, P-CTX-4B appears about four times more effective than P-CTX-1B to affect K⁺ channels, whereas it is about 50 times less efficient to affect Na⁺ channels of axonal membranes. These actions may be related to subtle differences between the two chemical structures of molecules.     

Structural analysis of the venom glands of the armed spider Phoneutria nigriventer (Keyserling, 1891): microanatomy, fine structure and confocal observations

Spiders belonging to the genus Phoneutria (Perty, 1833), most commonly known as 'armed' spiders, are among the most dangerous species in Brazil due to high toxicity of their venom, associated with their habit of invading domestic or specific areas such as banana plantations. The venom of Phoneutria spiders is secreted by a pair of venom glands located inside their cephalothoraxes and connected to the chelicerae by two independent ducts. In the present study, the microanatomy and histological structure of the venom glands of Phoneutria nigriventer (Keyserling, 1891) were examined in detail by histochemical and conventional stains with laser confocal, scanning and transmission electron microscopies. The analysis confirmed the bulbous-shaped organ previously observed by others. The venom glands of P. nigriventer are covered externally by a double layer of striated muscles, which are arranged in a spiral fashion. This disposition of the external muscle fibers might provide the contraction movement of the venom gland to release their contents during a sting aggression. The presence of pore-like openings between the muscle fibers that cover the venom glands of P. nigriventer was considered quite remarkable. The presence of axon-like structures between the muscle fibers seen in the gland surface was also quite remarkable. The secretory epithelium of P. nigriventer invaginates into the gland lumen, contributing to the increase of the secretory surface area and also accommodating a higher number of secretory cells. Our observation of histological sections and SEM showed that the secretory cells in the venom gland of P. nigriventer form complex structures, secretory units, which originate at the base near the muscular layer and that extend into the central area until the gland lumen. Our study also identified a possible holocrine secretory mechanism of P. nigriventer venom gland, at least in the first venom milking, since we were able to see nuclei stained on confocal laser microscopy. However, our observation cannot disregard other possible types of secretory pathways in subsequent milkings, since we found no nuclei in the second and in the third venom secretions.     

ATP-sensitive K+ channels in the kidney

ATP-sensitive K⁺ channels (K_ATP channels) form a link between the metabolic state of the cell and the permeability of the cell membrane for K⁺ which, in turn, is a major determinant of cell membrane potential. K_ATP channels are found in many different cell types. Their regulation by ATP and other nucleotides and their modulation by other cellular factors such as pH and kinase activity varies widely and is fine-tuned for the function that these channels have to fulfill. In most excitable tissues they are closed and open when cell metabolism is impaired; thereby the cell is clamped in the resting state which saves ATP and helps to preserve the structural integrity of the cell. There are, however, notable exceptions from this rule; in pancreatic -cells, certain neurons and some vascular beds, these channels are open during the normal functioning of the cell.In the renal tubular system, K_ATP channels are found in the proximal tubule, the thick ascending limb of Henle's loop and the cortical collecting duct. Under physiological conditions, these channels have a high open probability and play an important role in the reabsorption of electrolytes and solutes as well as in K⁺ homeostasis. The physiological role of their nucleotide sensitivity is not entirely clear; one consequence is the coupling of channel activity to the activity of the Na-K-ATPase (pump-leak coupling), resulting in coordinated vectorial transport. In ischemia, however, the reduced ATP/ADP ratio would increase the open probability of the K_ATP channels independently from pump activity; this is particularly dangerous in the proximal tubule, where 60 to 70% of the glomerular ultrafiltrate is reabsorbed.The pharmacology of K_ATP channels is well developed including the sulphonylureas as standard blockers and the structurally heterogeneous family of channel openers. Blockers and openers, exemplified by glibenclamide and levcromakalim, show a wide spectrum of affinities towards the different types of K_ATP channels. Recent cloning efforts have solved the mystery about the structure of the channel: the K_ATP channels in the pancreatic -cell and in the principal cell of the renal cortical collecting duct are heteromultimers, composed of an inwardly rectifying K⁺ channel and sulphonylurea binding subunit(s) with unknown stoichiometry. The proteins making up the K_ATP channel in these two cell types are different (though homologous), explaining the physiological and pharmacological differences between these channel subtypes.     

In vitro effect of the Phoneutria nigriventer spider venom on cell viability, paracellular barrier function and transcellular transport in cultured cell lines.

Phoneutria nigriventer spider venom (PNV) induces, in rats, local edema as result of an increased vascular permeability, as well as causes blood-brain barrier (BBB) breakdown by altering transendothelial transport routes in hippocampal microvessels. In this work we investigated the in vitro effects of PNV on cell viability and cellular transport routes using three cell lines, the ECV304 endothelial-, the C6 glioma- and the MDCK epithelial cells. We showed that PNV (14.6 and 292 microg crude venom/ml culture medium) had no direct cytotoxic effect on both the ECV304 and the MDCK cell lines but slightly reduced the viability of C6 glioma cells (P<0.05) at the highest concentration, as revealed by the cellular neutral red uptake assay. The PNV effects on cell transport were evaluated in MDCK cell line. PNV seems do not cause any disturbance in the paracellular barrier function of the cultured MDCK cells, as shown by the lack of a significant change in the distribution and expression of the junctional proteins, ZO-1, occludin, E-cadherin and the cytoskeletal F-actin. In contrast, PNV-treated MDCK monolayers showed an enhancement in the transepithelial electrical resistance and a tendency towards an increased occludin expression. In addition, the PNV significantly increased the apical endocytosis of HRP, which was not followed by an equivalent exocytosis at the basal side, as revealed by biochemical and ultrastructural methods. We conclude that the venom of P. nigriventer displays a relatively low cytotoxicity in vitro as well as activates directly the endocytic transport pathway in MDCK cells without disrupting the paracellular route.     

Toxin modulators and blockers of hERG K channels

The K⁺ channel encoded by the Ether-á-go-go-Related Gene (ERG) is expressed in different tissues of different animal species. There are at least three subtypes of this channel, being the sub-type 1 (ERG1) crucial in the repolarization phase of the cardiac action potential. Mutations in this gene can affect the properties of the channel producing the type II long QT syndrome (LQTS2) and many drugs are also known to affect this channel with a similar side effect. Various scorpion, spider and sea anemone toxins affect the ERG currents by blocking the ion-conducting pore from the external side or by modulating channel gating through binding to the voltage-sensor domain. By doing so, these toxins become very useful tools for better understanding the structural and functional characteristics of these ion channels. This review discusses the interaction between the ERG channels and the peptides isolated from venoms of these animals. Special emphasis is placed on scorpion toxins, although the effects of several spider venom toxins and anemone toxins will be also revised.     

Effects of U-37883A on intracellular Ca2+ -activated large-conductance K+ channels in pig proximal urethral myocytes

Kinetic studies of U-37883A (4-morpholinecarboximidine-N-1-adamantyl-N'-cyclohexyl-hydrochloride), a vascular ATP-sensitive K+ channel (KATP channel) blocker, were performed on pig urethral myocytes to investigate inhibitory effects on large-conductance intracellular Ca2+ -sensitive K+ channels (i.e., BKCa channels; 225 pS K+ channels) by use of single-channel recordings (outside-out and inside-out configuration). BKCa channels in pig urethral smooth muscles showed extracellular iberiotoxin (300 nM) sensitivity and voltage dependency. The alpha subunit of BKCa channel proteins was detected in the membrane fraction by use of Western blot technique. Application of U-37883A (> or =10 microM) reduced the activity of BKCa channels in a concentration-dependent manner, not only by decreasing mean openlife time but also by prolonging the mean closed time. These results shows that U-37883A affects channels other than the vascular KATP channel, and demonstrates how it inhibits the activities of BKCa channels in urethral smooth muscles.     

Gambierol Blocks a K+ Current Fraction without Affecting Catecholamine Release in Rat Fetal Adrenomedullary Cultured Chromaffin Cells

Gambierol inhibits voltage-gated K⁺ (K_V) channels in various excitable and non-excitable cells. The purpose of this work was to study the effects of gambierol on single rat fetal (F19–F20) adrenomedullary cultured chromaffin cells. These excitable cells have different types of K_V channels and release catecholamines. Perforated whole-cell voltage-clamp recordings revealed that gambierol (100 nM) blocked only a fraction of the total outward K⁺ current and slowed the kinetics of K⁺ current activation. The use of selective channel blockers disclosed that gambierol did not affect calcium-activated K⁺ (K_Ca) and ATP-sensitive K⁺ (K_ATP) channels. The gambierol concentration necessary to inhibit 50% of the K⁺ current-component sensitive to the polyether (IC₅₀) was 5.8 nM. Simultaneous whole-cell current-clamp and single-cell amperometry recordings revealed that gambierol did not modify the membrane potential following 11s depolarizing current-steps, in both quiescent and active cells displaying repetitive firing of action potentials, and it did not increase the number of exocytotic catecholamine release events, with respect to controls. The subsequent addition of apamin and iberiotoxin, which selectively block the K_Ca channels, both depolarized the membrane and enhanced by 2.7 and 3.5-fold the exocytotic event frequency in quiescent and active cells, respectively. These results highlight the important modulatory role played by K_Ca channels in the control of exocytosis from fetal (F19–F20) adrenomedullary chromaffin cells.     

Inhibition of minK protein induced K+ channels in Xenopus oocytes by estrogens

Previously it was shown that minK protein expression in uterus is regulated by estrogen. In the present study, we were interested in putative direct effects of estrogen on minK protein induced K⁺ currents (I_minK) in Xenopus oocytes. Superfusion with 17--estradiol (1 M) resulted in an inhibition of minK-induced currents, but had no appreciable effects on the delayed rectifier and inward rectifier K⁺ channels Kv1.1 and Kir2.1, respectively. The inhibition of I_minK by 17--estradiol was concentration-dependent, with an IC₅₀ of approximately 0.5 M. In the presence of 17--estradiol, the conductance-voltage relationship was shifted to more depolarized potentials. I_minK inhibition occurred also in the presence of the estrogen-receptor antagonist tamoxifen, suggesting that a mechanism independent of estrogen receptors is involved. The synthetic estrogen diethylstilbestrol (DES) also inhibited I_minK but with a lower affinity (IC₅₀ of 4.5 M), while cortisol and progesterone had only weak effects on I_minK. In summary, the results indicate that estrogens directly inhibit I_minK.