No abstract

Fertilization is a matter of life or death. In animals of sexual reproduction, the appropriate communication between mature and competent male and female gametes determines the generation of a new individual. Ion channels are key elements in the dialogue between sperm, its environment, and the egg. Components from the outer layer of the egg induce ion permeability changes in sperm that regulate sperm motility, chemotaxis, and the acrosome reaction. Sperm are tiny differentiated terminal cells unable to synthesize protein and difficult to study electrophysiologically. Thus understanding how sperm ion channels participate in fertilization requires combining planar bilayer techniques, in vivo measurements of membrane potential, intracellular Ca2+ and intracellular pH using fluorescent probes, patch-clamp recordings, and molecular cloning and heterologous expression. Spermatogenic cells are larger than sperm and synthesize the ion channels that will end up in mature sperm. Correlating the presence and cellular distribution of various ion channels with their functional status at different stages of spermatogenesis is contributing to understand their participation in differentiation and in sperm physiology. The multi-faceted approach being used to unravel sperm ion channel function and regulation is yielding valuable information about the finely orchestrated events that lead to sperm activation, induction of the acrosome reaction, and in the end to the miracle of life.

Two vesicle pools, readily releasable (RRP) and reserve (RP) pools, are present at Drosophila neuromuscular junctions. Using a temperature-sensitive mutant, shibire(ts), we studied pool sizes and vesicle mobilization rates. In shibire(ts), due to lack of endocytosis at nonpermissive temperatures, synaptic currents continuously declined during tetanic stimulation until they ceased as the result of vesicle depletion. By then, approximately 84,000 quanta were released. Vesicles were mobilized from RP at a rate 1/7-1/10 of RRP. Cytochalasin D inhibited mobilization of vesicles from RP, allowing us to estimate the size of RRP as 14%-19% of all vesicles. Vesicle recycling supports synaptic transmission during prolonged tetanic stimulation and the maximum recycling rate was 1000 vesicles/s.

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.

To fertilize, mammalian sperm must complete a maturational process called capacitation. It is thought that the membrane potential of sperm hyperpolarizes during capacitation, possibly due to the opening of K(+) channels, but electrophysiological evidence is lacking. In this report, using patch-clamp recordings obtained from isolated mouse spermatogenic cells we document the presence of a novel K(+)-selective inwardly rectifying current. Macroscopic current activated at membrane potentials below the equilibrium potential for K(+) and its magnitude was dependent on the external K(+) concentration. The channels selected K(+) over other monovalent cations. Current was virtually absent when external K(+) was replaced with Na(+) or N-methyl-D-glucamine. Addition of Cs(+) or Ba(2+) (IC(50) of approximately 15 microM) to the external solution effectively blocked K(+) current. Dialyzing the cells with a Mg(2+)-free solution did not affect channel activity. Cytosolic acidification reversibly inhibited the current. We verified that the resting membrane potential of mouse sperm changed from -52 +/- 6 to -66 +/- 9 mV during capacitation in vitro. Notably, application of 0.3-1 mM Ba(2+) during capacitation prevented this hyperpolarization and decreased the subsequent exocytotic response to zona pellucida. A mechanism is proposed whereby opening of inwardly rectifying K(+) channels may produce hyperpolarization under physiological conditions and contribute to the cellular changes that give rise to the capacitated state in mature sperm.

Identification of the minimum number of ways in which open and closed states communicate is a crucial step in defining the gating kinetics of multistate channels. We used certain correlation functions to extract information about the pathways connecting the open and closed states of the cation channel of the purified nicotinic acetylcholine receptor and of the chloride channel of Torpedo californica electroplax membranes. Single channel currents were recorded from planar lipid bilayers containing the membrane channel proteins under investigation. The correlation functions are conveniently computed from single channel current records and yield information on E, the minimum number of entry/exit states into the open or closed aggregates. E gives a lower limit on the numbers of transition pathways between open and closed states. For the acetylcholine receptor, the autocorrelation analysis shows that there are at least two entry/exit states through which the open and closed aggregates communicate. The chloride channel fluctuates between three conductance substates, here indentified as C, M, and H for closed, intermediate, and high conductance, respectively. Correlation analysis shows that E is greater than or equal to 2 for the M aggregate, indicating that there are at least two distinct entry/exit states in the M aggregate. In contrast, there is no evidence for the existence of more than one entry/exit state in the C or H aggregates. Thus, these correlation functions provide a simple and general strategy to extract information on channel gating kinetics.

A voltage-dependent, K+-selective ionic channel from sarcoplasmic reticulum of rabbit skeletal muscle has been studied in a planar phospholipid bilayer membrane. The purpose of this work is to study the mechanism by which the channel undergoes transitions between its conducting and nonconducting states. Thermodynamic studies show that the "open" and "closed" states of the channel exist in a voltage-dependent equilibrium, and that the channel displays only a single open state; the channel conductance is 120 pmho in 0.1 M K +. The channel's gating process follows single exponential kinetics at all voltages tested, and the individual opening and closing rate constants are exponentially dependent on voltage. The individual rate constants may also be determined from a stochastic analysis of channel fluctuations among multiple conductance levels. Neither the thermodynamic nor the kinetic parameters of gating depend on the absolute concentration of channels in the bilayer. The results are taken as evidence that the channel gates by an unusually simple twostate conformational mechanism in which the equivalent of 1.1 net charges are moved across the membrane during the formation of the open channel.Of the events leading to the contraction of vertebrate skeletal muscle, those involved in the release of Ca ++ from the sarcoplasmic reticulum (SR) membrane are the least understood. It is clear that the permeability of the SR membrane to Ca ++ is greatly increased during the release process (Endo, 1977), but it is not known to what extent other ions participate in any voltage and conductance changes that may occur during Ca ++ release. One obvious question to arise from any consideration of Ca ++ movements is: what other ions move across the SR membrane to maintain electroneutrality and, hence, to permit the rapid, massive fluxes of Ca ++ into and out of the SR throughout the contraction-relaxation cycle? Indirect approaches have yielded estimates of the overall SR conductance (Vergara et al., 1978), but the ionic basis of this is entirely unknown. In particular, the role of K +, the overwhelmingly abundant ion on both sides of the SR membrane in vivo (Somlyo et al., 1977)) is obscure.

Sarcoplasmic reticulum (SR) vesicles from frog leg muscle were fused with a planar phospholipid bilayer by a method described previously for rabbit SR. As a result of the fusion, K+-selective conduction channels are inserted into the bilayer. Unlike the two-state rabbit channel, the frog channel displays three states: a nonconducting ("closed") state and two conducting states "alpha" and "beta". In 0.1 M K+ the single-channel conductances are 50 and 150 pS for alpha and beta, respectively. The probabilities of appearance of the three states are voltage-dependent, and transitions between the closed and beta states proceed through the alpha state. Both open states follow a quantitatively identical selectivity sequence in channel conductance: K+ greater than NH4+ greater than Rb+ greater than Na+ greater than Li+ greater than Cs+. Both open states are blocked by Cs+ asymmetrically in a voltage-dependent manner. The zero-voltage dissociation constant for blocking is the same for both open states, but the voltage-dependences of the Cs+ block for the two states differ in a way suggesting that the Cs+ blocking site is located more deeply inside the membrane in the beta than in the alpha state.