Arturo Liévano

T-type Ca2+ channels and α1E 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 α 1A, α 1B, α 1C, α 1D and α 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 α 1E, and to a minor extent α 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. The results suggest that low‐threshold Ca2+ channels are the dihydropyridine‐sensitive channels involved in the sperm acrosome reaction.

δ-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, notvery 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 δ‐endotoxins induce an increase in cation membrane permeability involving ion channels in BBMV‐containing functional receptors.

A high-conductance voltage-dependent multistate Ca2' channel found in sea urchin and mouse spermatozoa

Ion fluxes through poorly understood channel‐mediated mechanisms participate in the interaction between spermatozoa and egg. Previously, we reported the characterization in planar bilayers of a high conductance Ca2+‐selective, voltage‐dependent multistate channel from S. purpuratus sea urchin sperm plasma membranes [14]. Here we show that this ion channel can be directly transferred to planar lipid bilayers upon sperm addition, from sea urchin [S. purpuratus and L. pictus) and from mouse. We found that spermatozoa from these species posses a conspicuous Ca2+‐selective, high conductance, multi‐state, voltage‐dependent channel, which displays similar voltage dependence and equal PBa2+/PK + ~4 in the three species. The presence of this Ca2+ channel in such diverse species suggests it plays a relevant role in sperm physiology. The high sensitivity of planar bilayers to detect single ion channels can now be used to study ion channel regulation and gamete interaction.

4 Ion Channels: Key Elements in Gamete Signaling

Publisher Summary This chapter discusses the participation of ion channels in the information exchange between gametes themselves and with the environment. Since the best known sperm ion transport systems have been described in the sea urchin, a marine invertebrate, and in mouse, bull, and pig in mammals, these species will be referred to more extensively. The success of fertilization depends on gamete information processing from the environment. There are long- and short-range signals, emitted by the egg, that influence sperm function and lead to proper gamete interaction, and finally to fertilization. Although the factors that mediate the sperm-egg dialogue have been studied for close to a century, the detailed molecular mechanisms involved in these events remain elusive. However, there is growing evidence that ion channels are deeply involved in gamete signaling. For instance, in echinoderm, fish, and mammalian spermatozoa, the acrosome reaction (AR), a necessary process for fertilization in many species, is inhibited by ion channel blockers.

Single-channel activity of bilayers derived from sea urchin sperm plasma membranes at the tip of a patch-clamp electrode☆

Changes in the plasma membrane permeability of echinoderm sperm play a fundamental role in the acrosome reaction. During the reaction there is an increase in intracellular Ca2+ and Na+ and an efflux of H+ and K+. We have formed bilayers at the tip of patch pipets from a mixture of lipid vesicles and sea urchin sperm plasma membranes (12-50 microgram protein/ml). We observed three types of K+ channels (conductances: 22, 46, and 82 pS), two of which are partially blocked by TEA, and one Cl- channel (148 pS). The presence of K+ channels in sperm plasma membranes is consistent with the inhibition by TEA of the acrosome reaction in whole sperm and the membrane potential change that occurs during the reaction.

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.

Ionic bases of the membrane potential and intracellular pH changes induced by speract in swollen sea urchin sperm.

Signal transduction initiated by the egg peptide, speract, in sea urchin sperm is not fully understood. Hypotonically swollen sperm are a suitable model to study peptide signal transduction. Ion substitution experiments now indicate (i) that the permeability to Na+, Ca2+, and Mg2+ contributes to the sperm resting membrane potential; (ii) the repolarization induced by nM concentrations of speract is Na+ dependent and mediated by an as yet unidentified channel; (iii) the depolarization triggered by nM concentrations of speract involves Ca2+ channels since it is Ca2+‐dependent and blocked by Co2+ and Ni2+, two Ca2+ channel blockers; (iv) hyperpolarizing swollen sperm with valinomycin increases intracellular pH (pHi) in the same way as speract, thus the speract‐induced hyperpolarization may be responsible for the pHi increase.

Purification and reconstitution of potassium channel proteins from squid axon membranes

Voltage‐dependent K+ channels are responsible for repolarization of the cell membrane during the late phase of the action potential. Here we report the purification of proteins from squid axon membranes which bind the K+‐channel blocker noxiustoxin (NTX), and their subsequent functional reconstitution in planar bilayers. The NXT‐affinity purified proteins had Mr values of 60000 ± 6000, 160000 ± 15000 and 220000 ± 20000. Their incorporation into bilayers resulted in single‐channel currents with three conductances, the most frequent one of 11 pS in 300/100 mM KCl (cis/trans). The voltage dependence, reversal potential and bursting behavior suggest that these are the K+ channels involved in the squid axon action potential.

Incorporation of ionic channels from yeast plasma membranes into black lipid membranes

Recently, patch-clamping of yeast protoplasts has revealed the presence of plasma membrane K+ channels (Gustin, M. C., B. Martinac, Y. Saimi, M. R. Culberston, and C. Kung. 1986. Science (Wash. DC). 233:1195-1197). In this work we show that fusion of purified plasma membranes into planar bilayers allows the study of the yeast channels. The main cationic conductances detected were of 64 and 116 pS, however, larger and smaller conductances have been observed. The two main conductances were sensitive to the K+ channels blockers tetraethylammonium (TEA+) and Ba2+. Bionic experiments indicated that both conductances were K+ selective.

Ion Channels in Sperm

Publisher Summary This chapter discusses the ion channels in sperm. The chapter mentions that the flow of ions through the plasma membrane of sperm, particularly Ca2+, participates crucially in the events leading to fertilization. Cell communication involves molecular mechanisms as they play a key role in determining the behavior of organisms. The chapter discusses sperm responses to egg components and sperm ion channels. Sperm responses to egg components discuss sea urchin sperm, and mammalian sperm. Sperm ion channels discuss sea urchin sperm ion channels, and ion channels of mammalian sperm. Sea urchin sperm further discusses responses to egg peptides and acrosome reaction. Capacitation and acrosome reaction are also discussed under mammalian sperm. It is mentioned that mammalian sperm capacitation can be accomplished in vitro by incubating ejaculated sperm in defined medium. The three key components required for mammalian sperm capacitation in mouse sperm are Ca2+, NaHCO3, and serum albumin. Successful gamete interactions that require cell signaling and the crucial participation of ion channels determine the propagation of life. Ionic fluxes play a fundamental role in activation of respiration and motility, chemotaxis, the sperm acrosome reaction (AR), and therefore in fertilization. Sperm are excitable cells that quickly respond to components from the outer layer of the egg, the jelly, with fast changes in their plasma membrane permeability. Model membranes formed from sperm components and patch-clamp techniques in whole cells have been used to detect, for the first time, the activity of single channels in the plasma membrane of sea urchin and mouse sperm. Swollen sperm can be much more easily patch-clamped than normal sperm, thus, providing new avenues to study ionic channels and their regulation by egg factors and second messengers. An alternative to identifying and functionally studying ion channels in sperm is to look for their expression in spermatogenic cells, combining molecular biological strategies and electrophysiology. This will allow a deeper understanding of the finely arranged events that lead to sperm activation, induction of the acrosome reaction, and in the end to the generation of a new individual.