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A Darszon

Publications and source records attributed to A Darszon.

At least 37 records · Page 2Linked to original sources
PubMed1998

Site directed mutants of Noxiustoxin reveal specific interactions with potassium channels.

Several site directed mutations were introduced into a synthetic Noxiustoxin (NTX) gene. Alanine scanning of the nonapeptide at the N-terminal segment of NTX (threonine 1 (T1) to serine 9 (S9)) was constructed and the recombinant products were obtained in pure form. Additionally, lysine 28 (K28) was changed to arginine (R) or glutamic acid (E), cysteine 29 was changed to alanine, and residues 37-39 (Tyr-Asn-Asn) of the carboxyl end were deleted. The recombinant mutants were tested for their ability to displace 125I-NTX from rat brain synaptosome membranes, as well as for their efficiency in blocking the activity of Kv1.1 K+ channels expressed in Xenopus laevis oocytes. The main results indicate that residues K6, T8 at the amino end, and K28 and the tripeptide YNN at the carboxyl end are involved in specific interactions of NTX with rat brain and/or Kv1.1 K+ channels.

Animals↗
PubMed1998

Mouse sperm patch-clamp recordings reveal single Cl- channels sensitive to niflumic acid, a blocker of the sperm acrosome reaction.

Ion channels lie at the heart of gamete signaling. Understanding their regulation will improve our knowledge of sperm physiology, and may lead to novel contraceptive strategies. Sperm are tiny (approximately 3 microm diameter) and, until now, direct evidence of ion channel activity in these cells was lacking. Using patch-clamp recording we document here, for the first time, the presence of cationic and anionic channels in mouse sperm. Anion selective channels were blocked by niflumic acid (NA) (IC50 = 11 microM). The blocker was effective also in inhibiting the acrosome reaction induced by the zona pellucida, GABA or progesterone. These observations suggest that Cl- channels participate in the sperm acrosome reaction in mammals.

Acrosome↗
PubMed1998

Acrosome reaction inactivation in sea urchin sperm.

Acrosome reaction inactivation (ARI) is a process that renders sperm irreversibly refractory to the egg jelly (the natural inducer of the acrosome reaction, AR). This process triggered by the egg jelly, is associated with an increase in [Ca2+]i. However, we show here that a rise in [Ca2+]i alone is not sufficient to induce ARI, since artificially increasing [Ca2+]i with either an ionophore or rising external pH, does not trigger ARI. Contrary to the AR which strictly requires Ca2+, ARI can be triggered almost equally well by Sr2+. On the other hand, Mn2+ inhibits ARI and, as we showed earlier, does not affect AR. These observations indicate that the mechanisms involved in ARI differ from those leading to AR. In addition, we report here that high external pH (a non-physiological inducer of AR) triggers the AR in previously inactivated sperm by opening the same Ca2+ channels activated by the egg jelly. Considering that the opening of Ca2+ channels is one of the earliest responses triggered by the egg jelly and that ARI requires the egg jelly receptor to be activated, we have concluded that ARI involves the uncoupling between the egg jelly receptor and Ca2+ channels. Furthermore, intracellular pH (pHi) determinations, in the presence or absence of ionomycin to substitute for the uncoupled Ca2+ channels, indicate that pHi regulation is also impaired in inactivated sperm. In conclusion, ARI is a manifestation of the uncoupling of the egg jelly receptor from the different ion transport systems required for the acrosome reaction.

Acrosome↗
PubMed1998

Localisation of inositol trisphosphate and ryanodine receptors during mouse spermatogenesis: possible functional implications.

During spermatogenesis the activity of intracellular Ca(2+)-release channels is likely to play an important role in different specific cellular functions. Accordingly, messenger RNAs for the three inositol 1,4,5-trisphosphate receptor (IP3R) subtypes were found to be present throughout spermatogenesis. Immunocytochemical analysis revealed distinct distribution patterns of the mature IP3Rs during sperm differentiation. At early stages, IP3Rs are distributed throughout the cytoplasm, and as differentiation proceeds they become selectively localised to the Golgi complex. Consistently, spermatogonia underwent large intracellular Ca2+ release in response to thapsigargin (TG), while smaller responses were detected in late spermatocytes and spermatids. The distribution of IP3Rs and the larger Ca(2+)-release responses found in spermatogonia, suggest that IP3Rs may be involved in cell proliferation at this stage. This notion is supported by our observations in a spermatogenic cell line that depletion of intracellular Ca2+ pools using TG inhibits cell division, and that incubation with an IP3R-I antisense oligonucleotide completely inhibited proliferation. Furthermore, the three genes encoding ryanodine receptor proteins (RyRs) are expressed at all stages of spermatogenesis. However, immunocytochemical studies with specific antibodies against each of the RyR subtypes detected types 1 and 3 in spermatogenic cells and only type 3 in mature sperm. In contrast to IP3Rs, RyRs remain scattered in the cytoplasm throughout differentiation. Functional responses to caffeine and ryanodine were absent in spermatogenic cells and in mature sperm. These findings suggest that IP3Rs have significantly more important roles in spermatogenesis than RyRs, and that one of these roles is crucial for cell proliferation.

Animals↗
PubMed1998

Properties of a novel pH-dependent Ca2+ permeation pathway present in male germ cells with possible roles in spermatogenesis and mature sperm function.

Rises of intracellular Ca2+ ([Ca2+]i) are key signals for cell division, differentiation, and maturation. Similarly, they are likely to be important for the unique processes of meiosis and spermatogenesis, carried out exclusively by male germ cells. In addition, elevations of [Ca2+]i and intracellular pH (pHi) in mature sperm trigger at least two events obligatory for fertilization: capacitation and acrosome reaction. Evidence implicates the activity of Ca2+ channels modulated by pHi in the origin of these Ca2+ elevations, but their nature remains unexplored, in part because work in individual spermatozoa are hampered by formidable experimental difficulties. Recently, late spermatogenic cells have emerged as a model system for studying aspects relevant for sperm physiology, such as plasmalemmal ion fluxes. Here we describe the first study on the influence of controlled intracellular alkalinization on [Ca2+]i on identified spermatogenic cells from mouse adult testes. In BCECF [(2',7')-bis(carboxymethyl)- (5, 6)-carboxyfluorescein]-AM-loaded spermatogenic cells, a brief (30-60 s) application of 25 mM NH4Cl increased pHi by approximately 1.3 U from a resting pHi approximately 6.65. A steady pHi plateau was maintained during NH4Cl application, with little or no rebound acidification. In fura-2-AM-loaded cells, alkalinization induced a biphasic response composed of an initial [Ca2+]i drop followed by a two- to threefold rise. Maneuvers that inhibit either Ca2+ influx or intracellular Ca2+ release demonstrated that the majority of the Ca2+ rise results from plasma membrane Ca2+ influx, although a small component likely to result from intracellular Ca2+ release was occasionally observed. Ca2+ transients potentiated with repeated NH4Cl applications, gradually obliterating the initial [Ca2+]i drop. The pH-sensitive Ca2+ permeation pathway allows the passage of other divalents (Sr2+, Ba2+, and Mn2+) and is blocked by inorganic Ca2+ channel blockers (Ni2+ and Cd2+), but not by the organic blocker nifedipine. The magnitude of these Ca2+ transients increased as maturation advanced, with the largest responses being recorded in testicular sperm. By extrapolation, these findings suggest that the pH-dependent Ca2+ influx pathway could play significant roles in mature sperm physiology. Its pharmacology and ion selectivity suggests that it corresponds to an ion channel different from the voltage-gated T-type Ca2+ channel also present in spermatogenic cells. We postulate that the Ca2+ permeation pathway regulated by pHi, if present in mature sperm, may be responsible for the dihydropyridine-insensitive Ca2+ influx required for initiating the acrosome reaction and perhaps other important sperm functions.

Acrosome↗
PubMed1997

Aminopeptidase dependent pore formation of Bacillus thuringiensis Cry1Ac toxin on Trichoplusia ni membranes.

The insecticidal Bacillus thuringiensis Cry1Ac delta-endotoxin specifically binds to a 120 kDa aminopeptidase N (APN) in the midgut of susceptible insects such as Manduca sexta, Heliothis virescens, Lymantria dispar and Plutella xylostella. The 120 kDa APN has a glycosylphosphatidylinositol (GPI) anchor susceptible to the action of GPI-specific phospholipase C (PIPLC). Here we show that Cry1Ac pore-forming activity depends on the amount of APN present on brush border membrane vesicles (BBMV) from Trichoplusia ni larvae. Inhibition of APN activity with bestatin did not affect Cry1Ac pore formation, suggesting that Cry1Ac action depends on the presence of APN, but not on its enzymatic activity. N-Acetyl-D-galactosamine blocks the action of the toxin, indicating that this sugar is also directly involved in the Cry1Ac toxin-receptor interaction. Membrane potential measurements using PIPLC treated and non-treated BBMV suggest that both APN could participate as Cry1Ac receptor. The kinetic characterization of PIPLC sensitive and resistant APN indicates that they could be different isoforms. Finally, we show that in the presence of 200 mM Cs+ intrinsic BBMV T. ni channel permeability is not observed, while the toxin induced permeability is not affected, allowing an accurate analysis of the effect of the Cry1Ac toxin on T. ni midgut membranes.

Acetylgalactosamine↗
PubMed1997

Inositol triphosphate receptors in sea urchin sperm.

Inositol 1,4,5-triphosphate (Ins(1,4,5)P3) is a second messenger that regulates Ca2+ channels in many important cell signalling pathways. In sea urchin sperm the outer investment of the egg triggers the acrosome reaction (AR) that involves Ins(1,4,5)P3 production and the opening of two Ca2+ channels. Here we have sought to identify a high-affinity Ins(1,4,5)P3 receptor in Strongylocentrotus purpuratus sperm. An Ins(1,4,5)P3 binding component was affinity-purified 12-fold from sperm extracts. It displayed similar characteristics to the Ins(1,4,5)P3 receptor from other sources: pH-dependent high affinity for Ins(1,4,5)P3 (KD = 261 nM), a tau1/2 of association and dissociation of 50 and 40 s, respectively, specificity (IC50 > 5 microM for Ins(1)P1, Ins(1,4)P2 and Ins(1,3,4,5)P4), and pharmacological sensitivity (10 and 100 microg heparin/ml inhibited 75% and 100% binding respectively). An antibody against the carboxy-terminal of the type I Ins(1,4,5)P3 receptor of somatic cells recognised a plasma membrane component in the sperm head and less intensely in the flagella. This antibody also recognised a 240 kDa band from isolated head plasma membranes, and weakly in flagellar membrane. This IP3 receptor-like protein may mediate the sustained uptake of Ca2+ through the second Ca2+ channel opened during the AR.

Animals↗
PubMed1996

Refolding of triosephosphate isomerase in low-water media investigated by fluorescence resonance energy transfer.

The refolding and reassociation of rabbit muscle triosephosphate isomerase (TPI) monomers unfolded with guanidine hydrochloride (GdnHCl) was studied in aqueous media and in reverse micelles (RM) formed with hexadecyltrimethylammonium bromide and n-octane/hexanol. Fluorescence resonance energy transfer (FRET) studies were carried out using TPI labeled at Cys-217 with 5-((2-((iodoacetyl)-amino)ethyl)amino)naphthalene-1-sulfonic acid (1,5-IAEDANS) and TPI labeled at one of the free amino groups with fluorescein 5'-isothiocyanate (FITC). Efficient FRET between monomers of AEDANS-TPI and FITC-TPI in aqueous media was detected 2-3 min after denaturant dilution and remained constant for hours. The distance between AEDANS and FITC in a labeled, renatured hetero-TPI dimer calculated from FRET results was 48 A, in reasonable agreement with estimates based on the crystal structure of TPI. In RM, recovery of enzyme activity during renaturation correlates with the appearance of a high-intrinsic fluorescence intermediate believed to be a partially folded monomer (Fernández-Velasco et al., 1995). Nevertheless, when AEDANS- and FITC-labeled monomers were mixed in RM, FRET occurred as soon as GdnHCl was diluted (FRET efficiency = 0.36), preceding the changes in TPI intrinsic fluorescence and reactivation. Thereafter, the efficiency of FRET increased during the next hour up to approximately 0.50, where it remained after 24 h, when 80% of the enzyme activity was recovered. The high initial FRET seen in RM could indicate the formation of an inactive dimer within the first minutes after denaturant dilution. The further increase in FRET observed over the next hour could reflect conformational rearrangements of the protein and transition from the inactive to the active dimer.

Animals↗
PubMed1996

T-type Ca2+ channels and alpha1E expression in spermatogenic cells, and their possible relevance to the sperm acrosome reaction.

There is pharmacological evidence that Ca2+ channels play an essential role in triggering the mammalian sperm acrosome reaction, an exocytotic process required for sperm to fertilize the egg. Spermatozoa are small terminally differentiated cells that are difficult to study by conventional electrophysiological techniques. To identify the members of the voltage-dependent Ca2+ channel family possibly present in sperm, we have looked for the expression of the alpha 1A, alpha 1B, alpha 1C, alpha 1D and alpha 1E genes in mouse testis and in purified spermatogenic cell populations with RT-PCR. Our results indicate that all 5 genes are expressed in mouse testis, and in contrast only alpha 1E, and to a minor extent alpha 1A, are expressed in spermatogenic cells. In agreement with these findings, only T-type Ca2+ channels sensitive to the dihydropyridine nifedipine were observed in patch-clamp recordings of pachytene spermatocytes. These results suggest that low-threshold Ca2+ channels are the dihydropyridine-sensitive channels involved in the sperm acrosome reaction.

Acrosome↗
PubMed1996

Membrane potential regulates sea urchin sperm adenylylcyclase.

Adenylylcyclase (AC) from sea urchin sperm does not appear to be regulated by G proteins [Hildebrandt, J. D., Tash, J. S., Kirchick, H. J., Lipschunits, L., Secra, R. D., & Birmbaumer, L. (1985) Endocrinology 116, 1357-1366]. During sperm activation and the acrosome reaction, membrane potential changes and cAMP increases. Here we explore if membrane potential can modulate the sperm AC. Hyperpolarization of Lytechinus pictus sea urchin sperm either with valinomycin in artificial sea water (ASW) without K+ or with dilution in ASW without Na+ increased the [c-AMP] (2.2- and 5.8-fold, respectively). This increase also occurred in the absence of extracellular Ca2+ (1.9- and 3.1-fold, respectively) and was enhanced by 100 microM IBMX, a phosphodiesterase inhibitor. It has been suggested that sea urchin sperm AC is stimulated by increases in intracellular Ca2+ and intracellular pH. In ASW without Na+ and Ca2+ (0Na0CaASW), sea urchin sperm intracellular pH decreases, and intracellular Ca2+ cannot increase. Therefore, these observations taken together indicate that AC in these cells in modulated by membrane potential. Dilution of Strogylocentrotus purpuratus sperm in 0Na0CaASW hyperpolarized them and increased their cAMP levels (1.3-fold). This stimulation was enhanced by IBMX (1.6-fold). Addition of the egg peptide speract under this condition further hyperpolarized S. purpuratus sperm and synergistically increased [cAMP] above 0Na0CaASW. This stimulation became larger in the presence of IBMX (from 1.6- to 5.2-fold). Since speract cannot elevate intracellular pH or [Ca2+] in 0Na0CaASW, the increase in [cAMP] it causes must be due to sperm hyperpolarization.

1-Methyl-3-isobutylxanthine↗
PubMed1996

A cAMP regulated K+-selective channel from the sea urchin sperm plasma membrane.

Ion channels are deeply involved in sperm physiology. In sea urchin sperm cyclic nucleotide levels increase during quimotaxis and in the acrosome reaction (AR). Although cyclic nucleotides are second messengers known to directly or indirectly modulate ion channels, it is not clear how they modulate sperm responses to the egg outer layer. Here, we describe a cAMP regulated K+-selective channel from sea urchin sperm plasma membranes fused into planar bilayers that may have a role during sea urchin sperm quimotaxis and/or the AR. Its single channel conductance in 100 mM KCl is 103 pS. In bi-ionic experiments, the channel displayed a K+/Na+ permeability ratio (PK+/PNa+) of approximately 5. Thus, in sea water its reversal potential would be approximately -13 mV and channel opening would depolarize spermatozoa. The channel has low open probability (Po = 0.8 +/- 0.2% at 0 mV applied voltage) and weak voltage dependence. Channel activity is reversibly up-regulated by cAMP in the cis bilayer side, but not by cGMP. This modulation followed a single Langmuir isotherm with an apparent kd of 200 microM. At this concentration the channel open probability at 0 mV increased up to 11- fold. TEA+ blocked the channel only from the trans side. Also Ba2+ in trans blocked the channel in a voltage-dependent manner.

Animals↗
PubMed1996

Structural and functional comparison of toxins from the venom of the scorpions Centruroides infamatus infamatus, Centruroides limpidus limpidus and Centruroides noxius.

Two novel toxins containing 66 amino acid residues each were isolated from the venom of the scorpions Centruroides infamatus infamatus and Centruroides limpidus limpidus, respectively. Their full amino acid sequences were determined. Comparison of primary structures showed that they share 97% similarity among themselves and 83% to that of toxin 2 from Centruroides noxius. The three toxins studied compete with each other for the same binding sites on membranes prepared from rat brain synaptosomes, suggesting that they are all beta-scorpion toxins. Toxin action was assayed into the microI-2 rat skeletal muscle Na+ channel heterologously expressed into Xenopus oocytes. All three toxins block this Na+ channel in a similar fashion, without affecting inactivation, and showed IC50 values in the micromolar concentration range.

Amino Acid Sequence↗
PubMed1996

A dihydropyridine-sensitive T-type Ca2+ current is the main Ca2+ current carrier in mouse primary spermatocytes.

Ca2+ entry through Ca2+ channels is likely to play an important role in the differentiation of male germ cells as well as in fertilization by mature sperm. Here we present a detailed analysis of Ca2+ currents expressed in acutely dissociated mouse primary spermatocytes. Patch-clamp recordings demonstrated that the only voltage-gated Ca2+ channels present belong to the family of T-type Ca2+ currents. Accordingly, Ni2+ (200 microM) and amiloride (500 microM) reduced current amplitude by 75 and 62%, respectively. To our knowledge, this is the first report of a system where T-type Ca2+ channels are expressed in isolation. Unexpectedly, 5 and 10 microM nifedipine also reduced peak currents by 38 and 53%, respectively significant inhibition of the Ca2+ current occurred at concentrations as low as 2 microM. Because mature sperm cells are unable to synthesize new proteins, these Ca2+ channels are also likely to be present in these cells, where they may contribute to the Ca2+ influx required to trigger the acrosome reaction. This notion is supported by the fact that concentrations of Ni2+ and nifedipine, which block these Ca2+ currents, also inhibit the acrosome reaction. Because these channels represent the primary pathway for voltage-gated Ca2+ entry in mouse spermatocytes, they may also participate in regulating meiotic cell division and sperm differentiation.

Acrosome↗
PubMed1995

Mouse sperm membrane potential: changes induced by Ca2+.

Mouse sperm resting membrane potential (Er) (-42 +/- 8.8 mV), determined with a potential sensitive dye, depended on extracellular K+ and, in the absence of extracellular Ca2+ ([Ca2+]e), on external Na+ ([Na+]e). Ca2+ addition (> 5 microM) to sperm in Ca-free media induced a transient hyperpolarization (Ca-ith) which strongly depended on [Na+]e and less on external Cl- ([Cl-]e). Cd2+ and Mn2+ (microM) mimicked the Ca2+ effect, but not Ba2+. The Ca-ith was partially inhibited by ouabain (74%, IC50 = 5.8 microM) and niflumic acid (38%, IC50 = 240 microM), indicating the participation of the Na-K ATPase and Cl- channels. In Ca-free low-Na+ media, Ca2+ addition caused a depolarization sensitive to: nimodipine (25 microM), trifluoperazine (12.5 microM) and Mg2+ (1.2 mM), suggesting the participation of Ca2+ channels. Since some inhibitors of the sperm Ca-ith block the acrosome reaction (AR), both processes may share transport systems.

Animals↗
PubMed1995

Delta-endotoxins induce cation channels in Spodoptera frugiperda brush border membranes in suspension and in planar lipid bilayers.

Membrane potential measurements using a fluorescent dye indicated that two specific toxins active against Spodoptera frugiperda larvae (CryIC and CryID) cause immediate permeability changes in midgut epithelial brush border membrane vesicles (BBMV). The initial response and the sustained permeability change are cationic, not very K+ selective, and occur at in vivo lethal doses (nM). The toxin response has a different ion selectivity and is more sensitive to Ba2+ than the intrinsic cation permeability of BBMV. Experiments incorporating BBMV into planar lipid bilayers (PLB) demonstrated that these vesicles contain cation channels (31, 47 and 76 pS). A 2-40 fold conductance increase was induced by nM concentrations of toxin in PLB containing BBMV. Cationic single channel transitions of 50, 106, 360 and 752 pS were resolved. Thus, Bacillus thuringiensis delta-endotoxins induce an increase in cation membrane permeability involving ion channels in BBMV-containing functional receptors.

Animals↗
PubMed1995

Water requirements in monomer folding and dimerization of triosephosphate isomerase in reverse micelles. Intrinsic fluorescence of conformers related to reactivation.

The possibility of using reverse micelles to stabilize monomers prior to formation of dimeric triosephosphate isomerase (TPI) from rabbit muscle was studied. TPI denatured with guanidine hydrochloride undergoes reactivation in reverse micelles formed with n-octane, hexanol, cetyltrimethylammonium bromide, and water. Reactivation of around 80% is observed at TPI concentrations of about 2 micrograms/mL of reverse micelles and water concentrations above 4.0%. With 3.0% water, reactivation is about 10%. If denatured TPI is incubated for a few seconds in reverse micelles with 5.0% water (or higher) followed by incubation in 3.0% water, reactivation is between 35% and 50%. That is, a brief exposure of denatured TPI to reverse micelles with a relatively high water concentration yielded a significant amount of structures competent for formation of catalytically active dimers. As evidenced by kinetic data, these structures correspond to monomers of TPI [Garza-Ramos, G. Tuena de Gómez-Puyou, M., Gómez-Puyou, A., & Gracy R. W. (1992) Eur. J. Biochem. 208, 389-395]. After a 5-2.0% water transition, competent monomers were stabilized for at least 30 min; a subsequent rise in water concentration led to dimerization and appearance of activity. By changes in the amount of water, it was possible to determine in reverse micelles the amount of water required for monomer folding and dimerization; i.e., less water was required in the dimerization step. Experiments with a model system, trypsin and the soybean inhibitor, showed that, in reverse micelles with 2.0% water, protein-protein interactions readily take place.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Ion channels from the mouse sperm plasma membrane in planar lipid bilayers.

Fusion of purified mouse sperm plasma membranes to planar lipid bilayers resulted in the insertion of three ion channel types. They could be discerned on the basis of their selectivity, conductance, gating and voltage-dependent properties. The presence of a previously reported large, Ca2+ -selective channel was confirmed. Here, it is reported that the Ca2+ -selective channel from mouse sperm plasma membrane displayed a PNa+/PK+ = 1.6 +/- 0.2 (n = 4) and was blocked by micromolar concentrations of ruthenium red. Fusion yielded also a cation-selective channel (PNa+/PK+ = 2.5 +/- 0.3, n = 3) with a main open conductance substate of 103 pS and a smaller open substate of 51 pS (600 mM K+ cis/100 mM Na+ trans). The channel inserted into bilayers in two stable fashions: a high-activity mode (open probability = 0.57 +/- 0.02, n = 3), and a low activity mode (open probability < 1%, n = 4). In high mode, the channel displayed bursting kinetics and burst length was voltage independent. In addition, a perfectly anion-selective channel, with a slope conductance of 83 pS (600 KCl cis/100 KCl trans), was identified. It displayed a high, nearly constant open probability (approximately 0.90) in the 0 to -80 mV range.

Animals↗