How to study a highly toxic protein to bacteria: A case of voltage sensor domain of mouse sperm-specific sodium/proton exchanger.

Heterologous expression systems have been used as a powerful experimental strategy to study the function of many proteins, particularly ion transporters. For this experiment, it is fundamental to prepare an expression vector encoding a protein of interest. However, we encountered problems in vector preparation of the voltage sensor domain (VSD) of murine sperm-specific Na+/H+ exchanger (sNHE) due to its severe toxicity to bacteria. We overcame the problems by insertion of an amber stop codon or a synthetic intron into the coding sequence of the VSD in the expression vectors. Both methods allowed us to express the protein of interest in HEK293 cells (combined with a stop codon suppression system for amber codon). The VSD of mouse sNHE generates voltage-dependent outward ionic currents, which is a probable cause of toxicity to bacteria. We propose these two strategies as practical solutions to study the function of any protein toxic to bacteria.

CaV3.1 channel pore pseudo-symmetry revealed by selectivity filter mutations in its domains I/II.

There is growing evidence indicating that the pore structure of voltage-gated ion channels (VGICs) influences gating besides their conductance. Regarding low voltage-activated (LVA) Ca2+ channels, it has been demonstrated that substitutions of the pore aspartate (D) by a glutamate (D-to-E substitution) in domains III and IV alter channel gating properties such as a positive shift in the channel activation voltage dependence. In the present report, we evaluated the effects of E-to-D substitution in domains I and II on the CaV3.1 channel gating properties. Our results indicate that substitutions in these two domains differentially modify the gating properties of CaV3.1 channels. The channel with a single mutation in domain I (DEDD) presented slower activation and faster inactivation kinetics and a slower recovery from inactivation, as compared with the WT channel. In contrast, the single mutant in domain II (EDDD) presented a small but significant negative shift of activation voltage dependence with faster activation and slower inactivation kinetics. Finally, the double mutant channel (DDDD) presented somehow intermediate properties with respect to the two single mutants but with fastest deactivation kinetics. Overall, our results indicate that single amino acid modification of the selectivity filter of LVA Ca2+ channels in distinct domains differentially influence their gating properties, supporting a pore pseudo-symmetry.

Differences and Similarities: The Richness of Comparative Sperm Physiology.

Species preservation depends on the success of fertilization. Sperm are uniquely equipped to fulfill this task, and, although several mechanisms are conserved among species, striking functional differences have evolved to contend with particular sperm-egg environmental characteristics. This review highlights similarities and differences in sperm strategies, with examples within internal and external fertilizers, pointing out unresolved issues.

Nonenzymatically oxidized arachidonic acid regulates T-type Ca2+ currents in mouse spermatogenic cells.

During spermatogenesis, fatty acids play an important role both as structural components and messengers that trigger male germ cell line differentiation. The spontaneous oxidation of fatty acids causes a decrease in mammalian fertility. Here, we examine the effects of nonenzymatically oxidized arachidonic acid (AAox ) on mouse spermatogenic T-type Ca2+ currents (ICaT ) due to their physiological relevance during spermatogenesis. AAox is 25-fold more potent than AA at inhibiting ICaT and it left shifts the I-V curve peak and both activation and steady-state inactivation curves. In addition, ICaT deactivation kinetics and their recovery from inactivation are slower in the presence of AAox . Therefore, the fraction of inactivated Ca2+ channels is increased. AAox -induced ICaT inhibition could contribute to male infertility affecting Ca2+ regulation in spermatogenic cells.

Robust evaluation of intermolecular FRET using a large Stokes shift fluorophore as a donor.

Fluorescence (or Förster) resonance energy transfer (FRET) is a straightforward and sensitive technique to evaluate molecular interactions. However, most of the popular FRET pairs suffer cross-excitation of the acceptor, which could lead to false positives. To overcome this problem, we selected a large Stokes shift (LSS) fluorophore as a FRET donor. As a successful example, we employed a new FRET pair mAmetrine (an LSS yellow fluorescence protein)/DY-547 (a cyanine derivative) to substitute CFP/fluorescein that we previously employed to study molecular interactions between cyclic nucleotide-binding domains and cyclic nucleotides. The new FRET pair is practically free of cross-excitation of the acceptor. Namely, a change in the fluorescence spectral shape implies evidence of FRET without other control experiments.

Cd(2+) sensitivity and permeability of a low voltage-activated Ca(2+) channel with CatSper-like selectivity filter.

CatSper is a sperm-specific Ca(2+) channel that plays an essential role in the male fertility. However, its biophysical properties have been poorly characterized mainly due to its deficient heterologous expression. As other voltage-gated Ca(2+) channels (CaVs), CatSper possesses a conserved Ca(2+)-selective filter motif ([T/S]x[D/E]xW) in the pore region. Interestingly, CatSper conserves four aspartic acids (DDDD) as the negatively charged residues in this motif while high voltage-activated CaVs have four glutamic acids (EEEE) and low voltage-activated CaVs possess two glutamic acids and two aspartic acids (EEDD). Previous studies based on site-directed mutagenesis of L- and T-type channels showed that the number of D seems to have a negative correlation with their cadmium (Cd(2+)) sensitivity. These results suggest that CatSper (DDDD) would have low sensitivity to Cd(2+). To explore Cd(2+)-sensitivity and -permeability of CatSper, we performed two types of experiments: 1) Electrophysiological analysis of heterologously expressed human CaV3.1 channel and three pore mutants (DEDD, EDDD and DDDD), 2) Cd(2+) imaging of human spermatozoa with FluoZin-1. Electrophysiological studies showed a significant increase in Cd(2+) and manganese (Mn(2+)) currents through the CaV3.1 mutants as well as a reduction in the inhibitory effect of Cd(2+) on the Ca(2+) current. In fluorescence imaging with human sperm, we observed an increase in Cd(2+) influx potentiated by progesterone, a potent activator of CatSper. These results support our hypothesis, namely that Cd(2+)-sensitivity and -permeability are related to the absolute number of D in the Ca(2+)-selective filter independently to the type of the Cav channels.

Regulation of Spermatogenic Cell T-Type Ca(2+) Currents by Zn(2+): Implications in Male Reproductive Physiology.

Zn(2+) is a trace metal which is important for spermatogenesis progression; its deficiency causes atrophy or malignant growth of the testis. Although testis, epididymis, and prostate contain high Zn(2+) concentrations, the molecular entities which are modulated by this metal are still under study. Interestingly, spermatogenic cells mainly express CaV 3.2-encoded T-type Ca(2+) currents (ICaT) which are positively or negatively modulated by Zn(2+) in other tissues. To explore whether ICaT could be regulated by Zn(2+) and albumin, its main physiological carrier, we performed whole cell electrophysiological recordings of spermatogenic cell ICaT in the absence or presence of different Zn(2+) concentrations. Zn(2+) decreased ICaT in a concentration-dependent manner (IC50 = 2 µM) and this inhibition could only be completely removed in presence of albumin. Differently to previous reports, ICaT did not show a tonic inhibition by Zn(2+) . Further analysis showed that Zn(2+) did not affect the voltage dependency or the kinetics of current activation, but right shifted the steady-state inactivation curve and slowed inactivation and deactivation kinetics. Recovery from inactivation was also altered. However, these apparent alterations in gating properties are not enough to explain the strong ICaT reduction. Using non-stationary fluctuation analysis, we found that Zn(2+) mainly reduced the number of available Ca(2+) channels without changing the single channel current amplitude. ICaT modulation by Zn(2+) could be relevant for spontaneous Ca(2+) oscillations during spermatogenesis and in pathophysiological conditions such as diabetes.

Pharmacology of hSlo3 channels and their contribution in the capacitation-associated hyperpolarization of human sperm.

Slo3 channels (mSlo3) primarily mediate mouse sperm K(+) currents and are essential for the capacitation-associated hyperpolarization (CAH). Whether Slo3 and/or Slo1, two Slo family K(+) channels are functionally expressed in human sperm is controversial. Our recent pharmacological studies of the human sperm CAH suggested the participation of both. Lack of a detailed pharmacology of heterologously expressed human Slo3 (hSlo3) prevented precisely identifying the K(+) channel(s) involved. In the present report, we compare the pharmacological profile of expressed hSlo3 in CHO cells with that of the CAH to advance this matter. Whole-cell patch-clamp recordings showed that hSlo3 currents are inhibited: significantly by progesterone, Ba(2+) and quinidine; partially by Penitrem A and Charybdotoxin; and poorly by Iberiotoxin and Slotoxin. Surprisingly, hSlo3 currents were resistant to Clofilium and 60 mM TEA(+) which inhibit mSlo3. Pharmacological comparison of the CAH and hSlo3 profiles indicates in addition to hSlo3, other K(+) channels, possibly Slo1, may participate in CAH. The pharmacological profile of heterologously expressed hSlo3 channels differs from that of mSlo3 K(+) channels, consistent with species-specific differences observed among other sperm ion channels. While the pharmacological correlation analysis of the hSlo3 currents and the CAH confirmed the participation of hSlo3 channels, it suggests that additional K(+) channels may be involved, in particular Slo1 channels.

Are TRP channels involved in sperm development and function?

Spermatozoa must translate information from their environment and the egg to achieve fertilization in sexually reproducing animals. These tasks require decoding a variety of signals in the form of intracellular Ca(2+) changes. As TRP channels constitute a large family of versatile multi-signal transducers, they are interesting subjects in which to explore their possible participation in sperm function. Here, we review the evidence for their presence and involvement in sperm motility, maturation, and the acrosome reaction, an exocytotic process required for sperm-egg fusion. Since store-operated Ca(2+) entry (SOCE) has been proposed to play an important role in these three functions, the main proteins responsible for this transport (STIM and ORAI) and their interaction with TRPs are also discussed. Improving our tools to solve infertility, improve animal breeding, and preserve biodiversity requires a better understanding of how Ca(2+) is regulated in spermatozoa.

Synphilin suppresses α-synuclein neurotoxicity in a Parkinson's disease Drosophila model.

Parkinson's disease (PD) is the second most common neurodegenerative disorder in humans. It affects 1% of the population over 65-years old. Its causes are environmental and genetic. As the world population ages, there is an urgent need for better and more detailed animal models for this kind of disease. In this work we show that the use of transgenic Drosophila is comparable to more complicated and costly animal models such as mice. The Drosophila model behaves very similar to the equivalent transgenic mice model. We show that both Synphilin-1 and α-synuclein are toxic by themselves, but when co-expressed, they suppress their toxicity reciprocally. Importantly, the symptoms induced in the fly can be treated and partially reverted using standard PD pharmacological treatments. This work showcases Drosophila as a detailed and multifaceted model for Parkinson's disease, providing a convenient platform in which to study and find new genetic modifiers of PD. genesis 49:392-402, 2011.

Enzymatic synthesis of capsaicin analogs and their effect on the T-type Ca2+ channels.

Capsaicin (Cap) and its analogs (CAPanalogs) have diverse effects in sensory neurons including analgesia, implying they modulate other cellular targets besides the TRPV1 Cap receptor. Since Cap and CAPanalogs are not largely available and their chemical synthesis is cumbersome, they have been obtained through a direct lipase-catalyzed reaction. Capsiate, the ester CAPanalog, was synthesized using a novel enzymatic transacylation one-pot strategy. Five different CAPanalogs were synthesized by amidation in 2-methyl-2-butanol with higher yields than previously reported. Voltage-dependent Ca(2+) channels (Ca(v)s) are among the main Ca(2+) entry paths into cells. They are classified as high-voltage-activated Ca(2+) channels (HVA) and low-voltage-activated Ca(2+) channels (LVA) constituted only by T-type channels. Though HVA Ca(v)s are Cap sensitive, it is not known if capsaicinoids inhibit LVA Ca(v)s which participate in the primary sensory neuron pain pathway. Here we first report that Cap, dihydrocapsaicin, N-VAMC(8), N-VAMC(9), and N-VAMC(10) can directly and partially reversibly inhibit T-type Ca(v)s, whereas olvanil, capsiate, and vanillylamine cannot. The Cap inhibition of T-type Ca(v)s was independent of TRPV1 activation.

Oviduct contraction in Drosophila is modulated by a neural network that is both, octopaminergic and glutamatergic.

Fertility is a highly complex and regulated phenomenon essential for the survival of any species. To identify Drosophila fertility-specific neural networks, we used a GAL4/UAS enhancer trap genetic screen that selectively inactivates groups of neurons. We identified a GAL4 line (bwktqs) that has a female sterile phenotype only when it expresses the tetanus toxin light chain (TeTxLC). These flies lack oviduct contraction, lay almost no eggs, sperm accumulate in the oviducts, and fewer than normal are seen in the storage organs. In insects, two neuroactive substances are important for oviduct contraction: octopamine (OA), a monoamine that inhibits oviduct contraction, and glutamate (Glu), a neurotransmitter that induces contraction. It is known that octopaminergic neurons of the thoracic abdominal ganglion (TAG) modulate oviduct contraction, however, the glutamatergic neurons that innervate the oviduct have not been identified yet and the interaction between these two neuroactive substances is not well understood. Immunostaining experiments revealed that the bwktqs line trapped an octopaminergic neural network that innervates the genital tract. We show that wt like oviduct contraction in TeTxLC-inactivated flies can only be rescued by simultaneous application of Glu and OA suggesting that the abdominal bwktqs neurons are both octopaminergic and glutamatergic, the use of an agonist and an antagonist for Glu receptors as well as their direct visualization confirmed its participation in this phenomenon. Our work provides the first evidence that adult abdominal type II visceral innervations co-express Glu and OA and allows us to re-evaluate the previous model of neuronal network controlling insect oviduct contraction.

Sodium and epithelial sodium channels participate in the regulation of the capacitation-associated hyperpolarization in mouse sperm.

In a process called capacitation, mammalian sperm gain the ability to fertilize after residing in the female tract. During capacitation the mouse sperm plasma membrane potential (E(m)) hyperpolarizes. However, the mechanisms that regulate sperm E(m) are not well understood. Here we show that sperm hyperpolarize when external Na(+) is replaced by N-methyl-glucamine. Readdition of external Na(+) restores a more depolarized E(m) by a process that is inhibited by amiloride or by its more potent derivative 5-(N-ethyl-N-isopropyl)-amiloride hydrochloride. These findings indicate that under resting conditions an electrogenic Na(+) transporter, possibly involving an amiloride sensitive Na(+) channel, may contribute to the sperm resting E(m). Consistent with this proposal, patch clamp recordings from spermatogenic cells reveal an amiloride-sensitive inward Na(+) current whose characteristics match those of the epithelial Na(+) channel (ENaC) family of epithelial Na(+) channels. Indeed, ENaC-alpha and -delta mRNAs were detected by reverse transcription-PCR in extracts of isolated elongated spermatids, and ENaC-alpha and -delta proteins were found on immunoblots of sperm membrane preparations. Immunostaining indicated localization of ENaC-alpha to the flagellar midpiece and of ENaC-delta to the acrosome. Incubations known to produce capacitation in vitro or induction of capacitation by cell-permeant cAMP analogs decreased the depolarizing response to the addition of external Na(+). These results suggest that increases in cAMP content occurring during capacitation may inhibit ENaCs to produce a required hyperpolarization of the sperm membrane.

Cavbeta-subunit displacement is a key step to induce the reluctant state of P/Q calcium channels by direct G protein regulation.

P/Q Ca(2+) channel activity is inhibited by G protein-coupled receptor activation. Channel inhibition requires a direct Gbetagamma binding onto the pore-forming subunit, Ca(v)2.1. It is characterized by biophysical changes, including current amplitude reduction, activation kinetic slowing, and an I-V curve shift, which leads to a reluctant mode. Here, we have characterized the contribution of the auxiliary beta(3)-subunit to channel regulation by G proteins. The shift in I-V to a P/Q reluctant mode is exclusively observed in the presence of beta(3). Along with the observation that Gbetagamma has no effect on the I-V curve of Ca(v)2.1 alone, we propose that the reluctant mode promoted by Gbetagamma corresponds to a state in which the beta(3)-subunit has been displaced from its channel-binding site. We validate this hypothesis with a beta(3)-I-II(2.1) loop chimera construct. Gbetagamma binding onto the I-II(2.1) loop portion of the chimera releases the beta(3)-binding domain and makes it available for binding onto the I-II loop of Ca(v)1.2, a G protein-insensitive channel. This finding is extended to the full-length Ca(v)2.1 channel by using fluorescence resonance energy transfer. Gbetagamma injection into Xenopus oocytes displaces a Cy3-labeled beta(3)-subunit from a GFP-tagged Ca(v)2.1 channel. We conclude that beta-subunit dissociation from the channel complex constitutes a key step in P/Q calcium channel regulation by G proteins that underlies the reluctant state and is an important process for modulating neurotransmission through G protein-coupled receptors.

Repositioning of charged I-II loop amino acid residues within the electric field by beta subunit as a novel working hypothesis for the control of fast P/Q calcium channel inactivation.

We have investigated the contribution of the Ca(v)beta subunits to the process of inactivation dependent of the I-II loop of Ca(v)alpha(2.1). Two amino acid residues located in the alpha1 interaction domain (AID) of the I-II loop of Ca(v)alpha(2.1) (Arg(387) and Glu(388)) have been directly implicated in voltage-dependent inactivation of this channel. Various point mutations of these residues disrupt the interaction between the I-II loop and the III-IV loop, and thereby modify the inactivation properties of the channel by accelerating its kinetics and shifting the steady-state inactivation curve towards hyperpolarized potentials. A similar disruption is produced by Ca(v)beta(4) subunit association with the I-II loop. Moreover, in the presence of Ca(v)beta(4) subunit, introducing negatively charged residues at positions 387 or 388 slows inactivation kinetics down, whereas introducing positive charges has the opposite effect. The shift of the steady-state inactivation curve is also amino acid charge-dependent. In contrast, mutation of Arg(387) or Glu(388) does not alter the differential regulation of the different Ca(v)beta isoforms on inactivation. These results suggest that the expression of Ca(v)beta(4) alters the contribution of charged residues at positions 387 and 388 to inactivation. We discuss these results with regard to the actual hypotheses on the mechanisms of calcium channel inactivation. We introduce the working concept that Ca(v)beta-subunits produce a conformational repositioning of charged AID residues within the electric field.

Expression and differential cell distribution of low-threshold Ca(2+) channels in mammalian male germ cells and sperm.

Numerous sperm functions including the acrosome reaction (AR) are associated with Ca(2+) influx through voltage-gated Ca(2+) (Ca(V)) channels. Although the electrophysiological characterization of Ca(2+) currents in mature sperm has proven difficult, functional studies have revealed the presence of low-threshold (Ca(V)3) channels in spermatogenic cells. However, the molecular identity of these proteins remains undefined. Here, we identified by reverse transcription polymerase chain reaction the expression of Ca(V)3.3 mRNA in mouse male germ cells, an isoform not previously described in these cells. Immunoconfocal microscopy revealed the presence of the three Ca(V)3 channel isoforms in mouse spermatogenic cells. In mature mouse sperm only Ca(V)3.1 and Ca(V)3.2 were detected in the head, suggesting its participation in the AR. Ca(V)3.1 and Ca(V)3.3 were found in the principal and the midpiece of the flagella. All Ca(V)3 channels are also present in human sperm, but only to a minor extent in the head. These findings were corroborated by immunogold transmission electron microscopy. Tail localization of Ca(V)3 channels suggested they may participate in motility, however, mibefradil and gossypol concentrations that inhibit Ca(V)3 channels did not significantly affect human sperm motility. Only higher mibefradil doses that can block high-threshold (HVA) Ca(V) channels caused small but significant motility alterations. Antibodies to HVA channels detected Ca(V)1.3 and Ca(V)2.3 in human sperm flagella.

A maurotoxin with constrained standard disulfide bridging: innovative strategy of chemical synthesis, pharmacology, and docking on K+ channels.

Maurotoxin (MTX) is a 34-residue toxin that has been isolated initially from the venom of the scorpion Scorpio maurus palmatus. It presents a large number of pharmacological targets, including small conductance Ca2+-activated and voltage-gated K+ channels. Contrary to other toxins of the alpha-KTx6 family (Pi1, Pi4, Pi7, and HsTx1), MTX exhibits a unique disulfide bridge organization of the type C1-C5, C2-C6, C3-C4, and C7-C8 (instead of the conventional C1-C5, C2-C6, C3-C7, and C4-C8, herein referred to as Pi1-like) that does not prevent its folding along the classic alpha/beta scaffold of scorpion toxins. Here, we developed an innovative strategy of chemical peptide synthesis to produce an MTX variant (MTXPi1) with a conventional pattern of disulfide bridging without any alteration of the toxin chemical structure. This strategy was used solely to address the impact of half-cystine pairings on MTX structural properties and pharmacology. The data indicate that MTXPi1 displays some marked changes in affinities toward the target K+ channels. Computed docking analyses using molecular models of both MTXPi1 and the various voltage-gated K+ channel subtypes (Shaker B, Kv1.2, and Kv1.3) were found to correlate with MTXPi1 pharmacology. A functional map detailing the interaction between MTXPi1 and Shaker B channel was generated in line with docking experiments.

Scorpion toxins that block T-type Ca2+ channels in spermatogenic cells inhibit the sperm acrosome reaction.

The acrosome reaction (AR) is a Ca(2+)-dependent event required for sperm to fertilize the egg. The activation of T-type voltage-gated Ca(2+) channels plays a key role in the induction of this process. This report describes the actions of two toxins from the scorpion Parabuthus granulatus named kurtoxin-like I and II (KLI and KLII, respectively) on sperm Ca(2+) channels. Both toxins decrease T-type Ca(2+) channel activity in mouse spermatogenic cells and inhibit the AR in mature sperm. Saturating concentrations of the toxins inhibited at most approximately 70% of the whole-cell Ca(2+) current, suggesting the presence of a toxin-resistant component. In addition, both toxins inhibited approximately 60% of the AR, which is consistent with the participation of T-type Ca(2+) channels in the sperm AR.

Two new scorpion toxins that target voltage-gated Ca2+ and Na+ channels.

This report describes the isolation, primary structure determination, and functional characterization of two similar toxins from the scorpion Parabuthus granulatus named kurtoxin-like I and II (KLI and KLII, respectively). KLII from P. granulatus is identical to kurtoxin from Parabuthus transvaalicus (a 63 amino-acid long toxin) whereas KLI is a new peptide containing 62 amino acid residues closely packed by four disulfide bridges with a molecular mass of 7244. Functional assays showed that both toxins, KLI and kurtoxin from P. granulatus, potently inhibit native voltage-gated T-type Ca(2+) channel activity in mouse male germ cells. In addition, KLI was shown to significantly affect the gating mechanisms of recombinant Na(+) channels and weakly block alpha(1)3.3Ca(V) channels expressed in Xenopus oocytes. KLI and kurtoxin from P. granulatus represent new probes to study the role of ion channels in germ cells, as well as in cardiac and neural tissue.