Dynamics of intracellular calcium induced by lactate and glucose in rat pachytene spermatocytes and round spermatids.

Glycolytic metabolism in meiotic and post-meiotic spermatogenic cells shows differentiation-related changes. The developmental and physiological significance of these metabolic changes is not known. The aim of the present study was to test the hypothesis that glucose and lactate metabolism can modulate intracellular calcium [Ca2+](i) in spermatogenic cells in an opposing and dynamic manner. Fluorescent probes were used to measure [Ca2+](i) and pH(i), and HPLC was used to measure intracellular adenine nucleotides and mitochondrial sensing of ATP turnover. [Ca2+](i) in pachytene spermatocytes and round spermatids was modulated by changes in lactate and glucose concentrations in the media. The kinetics and magnitude of the [Ca2+](i) changes induced by lactate and glucose were different in meiotic and post-meiotic spermatogenic cells. The presence of glucose in the medium induced a decrease in pH(i) in spermatogenic cells. This glucose-induced pH(i) decrease occurred later than the changes in [Ca2+](i), which were also observed when the pH(i) decrease was inhibited, indicating that the glucose-induced [Ca2+](i) increase was not a consequence of pH(i) changes. Hexose phosphorylation in glycolysis was part of the mechanism by which glucose metabolism induced a [Ca2+](i) increase in spermatogenic cells. The sensitivity of [Ca2+](i) to carbohydrate metabolism was higher in round spermatids than in pachytene spermatocytes. Thus, differentiation-related changes in carbohydrate metabolism in spermatogenic cells determine a dynamic and differential modulation of their [Ca2+](i) by glucose and lactate, two substrates secreted by the Sertoli cells.

Sea urchin sperm cation-selective channels directly modulated by cAMP.

Components of the sea urchin outer egg jelly layer such as speract drastically change second messenger levels and membrane permeability in sperm. Ion channels are deeply involved in the sperm-egg dialogue in sea urchin and other species. Yet, due to the small size of sperm, studies of ion channels and their modulation by second messengers in sperm are scarce. In this report we offer the first direct evidence that cation-selective channels upwardly regulated by cAMP operate in sea urchin sperm. Due to their poor selectivity among monovalent cations, channel activation in seawater could contribute to sperm membrane repolarization during the speract response.

Inwardly rectifying K(+) channels in spermatogenic cells: functional expression and implication in sperm capacitation.

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.

Size of vesicle pools, rates of mobilization, and recycling at neuromuscular synapses of a Drosophila mutant, shibire.

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.

Ion channels in sperm physiology.

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.

Pore accessibility during C-type inactivation in Shaker K+ channels.

Shaker K+ channels inactivate through two distinct molecular mechanisms: N-type, which involves the N-terminal domain and C-type that appears to involve structural modifications at the external mouth of the channel. We have tested pore accessibility of the Shaker K+ channel during C-type inactivation using Ba2+ as a probe. We determined that external Ba2+ binds to C-type inactivated channels forming an extremely stable complex; i.e. there is Ba2+ trapping by C-type inactivated channels. The structural changes Shaker channels undergo during C-type inactivation create high energy barriers that hinder Ba2+ exit to either the extracellular solution or to the intracellular solution.

Synaptic defects and compensatory regulation of inositol metabolism in inositol polyphosphate 1-phosphatase mutants.

Phosphoinositides function as important second messengers in a wide range of cellular processes. Inositol polyphosphate 1-phosphatase (IPP) is an enzyme essential for the hydrolysis of the 1-phosphate from either Ins(1,4)P2 or Ins(1,3,4)P3. This enzyme is Li+ sensitive, and is one of the proposed targets of Li+ therapy in manic-depressive illness. Drosophila ipp mutants accumulate IP2 in their system and are incapable of metabolizing exogenous Ins(1,4)P2. Notably, ipp mutants demonstrate compensatory upregulation of an alternative branch in the inositol-phosphate metabolism tree, thus providing a means of ensuring continued availability of inositol. We demonstrate that ipp mutants have a defect in synaptic transmission resulting from a dramatic increase in the probability of vesicle release at larval neuromuscular junctions. We also show that Li+ phenocopies this effect in wild-type synapses. Together, these results support a role for phosphoinositides in synaptic vesicle function in vivo and mechanistically question the "lithium hypothesis."

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.

Outward currents in Drosophila larval neurons: dunce lacks a maintained outward current component downregulated by cAMP.

Outward current modulation by cAMP was investigated in wild type (wt) and dunce (dnc) Drosophila larval neurons. dnc is deficient in a cAMP phosphodiesterase and has altered memory. Outward current modulation by cAMP was investigated by acute or chronic exposure to cAMP analogs. The analysis included a scrutiny of outward current modulation by cAMP in neurons from the mushroom bodies (mrb). In Drosophila, the mrb are the centers of olfactory acquisition and retention. Based on outward current patterns, neurons were classified into four types. Downmodulation of outward currents induced by acute application of cAMP analogs was reversible and found only in type I and type IV neurons. In the general wt neuron population, approximately half of neurons exhibited cAMP-modulated, 4-aminopyridine (4-AP)-sensitive currents. On the other hand, a significantly larger fraction of mrb neurons in wt (70%) was endowed with cAMP-modulated, 4-AP-sensitive currents. Only 30% of the dnc neurons displayed outward currents modulated by cAMP. The deficit of cAMP-modulated outward currents was most severe in neurons derived from the mrb of dnc individuals. Only 4% of the mrb neurons of dnc were cAMP-modulated. The dnc defect can be induced by chronic exposure of wt neurons to cAMP analogs. These results document for the first time a well defined electrophysiological neuron phenotype in correlation with the dnc defect. Moreover, this study demonstrates that in dnc mutants such a deficiency affects most severely neurons in brain centers of acquisition and retention.

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.

Ion channel classes in purified olfactory cilia membranes: planar lipid bilayer studies.

Ciliary membrane fragment fusion to planar lipid bilayers resulted in the insertion of four ion channel types. cAMP-activated, cation-selective channels could be detected only in the absence of Ca2+ and had a conductance of 23 pS. They exhibited an apparent dissociation constant (Kd) for the cyclic nucleotide of approximately 30 microM and an estimated permeability ratio (PNa/PK) of 2.4. The cAMP cation-selective channel coinserted with a K(+)-selective channel refractory to cAMP, Ca2+, and D-myo-inositol 1,4,5-trisphosphate. This K+ channel was voltage independent and exhibited open-conductance substates of 60 and 112 pS. cAMP was also found to modulate a novel K+ channel with a Kd = 140 microM. It displayed three nearly equally spaced open substates with conductances of 34, 80, and 130 pS. In the absence and in the presence of cAMP the probability of occurrence of the open substates was binomially distributed. A fourth channel type was a Ca(2+)-activated K+ channel with a conductance of 240 pS. It was blocked by charybdotoxin at nanomolar concentrations (Kd = 3 nM). These results add support to the idea that, besides cAMP-activated cation-selective channels, vertebrate chemosensory olfactory membranes possess an arrangement of ion channels.

Selectivity and gating properties of a cAMP-modulated, K(+)-selective channel from Drosophila larval muscle.

The selectivity and gating properties of cAMP-modulated, voltage-independent, K(+)-selective channel from Drosophila larval muscle were investigated using the patch-clamp technique. In symmetrical 115 mM K+ the channel displayed a linear current-voltage relation with slope conductance of 43 pS. Under biionic conditions (115 mM K+ pipette/115 mM X+ cytoplasmic) the permeability sequence was K+ > Rb+ > NH4+ >> Cs+,Na+. The channel was impermeable to Ca2+ (PCa/PK < 0.02). Under steady-state conditions and regardless [cAMP], open dwell times showed a double exponential distribution. [cAMP] did not affect the time constants of the two components of open times, or their relative amplitudes. Moreover, successive openings were correlated in open time. Closed dwell times were made of at least three exponential components. Fast application of cAMP to the cytoplasmic side of the channel induced a transient increase in open probability that relaxed to a lower value within seconds. This last result suggests that cAMP can activate and desensitize this cAMP-modulated, K(+)-selective channel.

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.

Molecular determinants of ion conduction and inactivation in K+ channels.

K+ channel-forming proteins can be grouped into three families that differ by the number of potential membrane-spanning segments. The largest of these families is composed of tetrameric channels with subunits containing six putative membrane-spanning segments (S1-S6). Inward rectifiers comprise a second family of K+ channels with subunits having two transmembrane domains (M1, M2). Monomers in the third family are proteins containing only one membrane-spanning segment, and they give origin to minK+ channels. Joining together segments S5 and S6 in the case of voltage-gated K+ channels and M1 and M2 in inward rectifiers, there is a highly conserved region with a hairpin shape called the H5 or P region. The P region, the loop connecting the S4 and S5 domains and the S6 transmembrane segment in Shaker-type K+ channels and the COOH-terminal in inward rectifiers, appears to play crucial roles in ion conduction. In Shaker K+ channels the NH2-terminal has been identified as responsible for fast inactivation (N-type inactivation). If the fast-inactivation gate is removed, a slower inactivation process persists, and its rate can be altered by mutations of amino acid residues forming part of the region in the neighborhood of the COOH-terminal (C-type inactivation). In this review we discuss the strategies followed to identify the different structures of K+ channels involved in ion conduction and inactivation processes and how they interplay.

A ciliary K+ conductance sensitive to charibdotoxin underlies inhibitory responses in toad olfactory receptor neurons.

In olfactory neurons from Caudiverbera caudiverbera, a mixture of putrid odorants trigger an inhibitory, K(+)-selective current and a hyperpolarizing receptor potential. The current-voltage relation resembles that of a Ca(2+)-activated K+ conductance; their amplitude depends on extracellular Ca2+. 10 nM charibdotoxin, a blocker of K(+)-selective channels, including Ca(2+)-activated ones, reversibly abolished inhibitory currents and receptor potentials. Focal stimulation demonstrates that the underlying transduction mechanism is confined to the cilia. This represents the first evidence for inhibitory responses in vertebrate olfactory cells mediated by a ciliary CTX-sensitive K+ conductance, most likely a Ca(2+)-activated one.

Inhibitory K+ current activated by odorants in toad olfactory neurons.

Odorant responses of isolated olfactory neurons from the toad Caudiverbera caudiverbera were monitored by using patch-clamp techniques. Depending on the stimulus, the same neuron responded with an increase or a decrease in action potential firing. Odorants that activate the cAMP cascade in olfactory cilia increased electrical activity, caused membrane depolarization, and triggered inward currents. In contrast, odorants that do not activate the cAMP cascade inhibited electrical activity, produced membrane hyperpolarization, and activated outward currents in a dose-dependent fashion. Such currents were carried by K+ and blocked by tetraethylammonium. Similar currents were recorded from Xenopus laevis. Our results suggest that this K+ current is responsible for odorant-induced inhibition of action potential firing in olfactory neurons.

Shaker mutants lack post-tetanic potentiation at motor end-plates.

The two-electrode voltage clamp technique was employed to measure end-plate currents in larval neuromuscular junctions of wild-type (Canton-S) and of three different Drosophila Shaker mutants: ShakerKS133, Shaker102 and f5Shaker5. In the Shaker mutants, nerve-evoked end-plate currents (neepc) were 4-5-fold larger than those measured in Canton-S. Shaker motor end-plates were found to lack post-tetanic potentiation (PTP), but could undergo facilitation. Moreover, PTP but not facilitation was lost in wild-type larvae if the neuromuscular junction was exposed to 4-aminopyridine (4-AP), a blocker of Shaker A-type K+ currents. End-plate currents were depressed by Ca2+ channel blockers like Mg2+, at millimolar concentrations, and Co2+ and Cd2+, at micromolar concentrations, but not by nifedipine (100 nM) and verapamil (100 nM). After exposure to Ca2+ channel blockers, Shaker end-plates exhibited PTP. In particular, Cd2+ was most effective in depressing neepcs and in restoring PTP in all Shaker mutants. The results obtained indicate the abnormal function of Shaker K+ channels at motor nerves specifically abolishes PTP in Drosophila larval neuromuscular junctions.

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 Ca(2+)-selective, voltage-dependent multistate channel from S. purpuratus sea urchin sperm plasma membranes. 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 possess a conspicuous Ca(2+)-selective, high conductance, multi-state, voltage-dependent channel, which displays similar voltage dependence and equal PBa2+/PK+ approximately 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.

Properties of whole cell currents in isolated olfactory neurons from the chilean toad Caudiverbera caudiverbera.

Isolated olfactory neurons from the chilean toad Caudiverbera caudiverbera were found to possess a same set of currents. Outward currents, made of a delayed rectifier and a Ca(2+)-dependent component, were blocked by replacing K+ by Cs+ in the patch pipette, in the presence of millimolar concentrations of tetraethylammonium and 4-aminopyridine in the external solution. Inward currents were made of a transient and a maintained component. The transient was abolished in the absence of external Na+ and was blocked by tetrodotoxin, with an apparent dissociation constant (KDapp) of 25.4 +/- 0.3 nM. The maintained inward currents were suppressed on removing external Ca2+, could be carried also by Ba2+, and were selectively blocked by Cd2+ (KDapp = 3.2 +/- 1.3 microM). A variety of agents found to block the maintained Ca2+ inward currents, including Co2+ and Ni2+, at millimolar concentrations, and nifedipine, verapamil, amiloride, and the amiloride analogue benzamil, at micromolar concentrations, were also effective in either modifying the gating of, or in blocking, the transient inward currents.

K(+)-channel blockers restore synaptic plasticity in the neuromuscular junction of dunce, a Drosophila learning and memory mutant.

The effects of K(+)-channel blockers on synaptic transmission in dunce (dnc), a Drosophila learning and memory mutant, were investigated. Larvae dnc mutants lack facilitation and post-tetanic potentiation (PTP) at their motor end-plates; dnc mutants are also deficient in a form of phosphodiesterase, and exhibit abnormally high levels of cyclic adenosine 3',5'-monophosphate (cAMP). A two-microelectrode voltage-clamp was used to record end-plate currents and spontaneous end-plate currents from longitudinal ventrolateral third-instar larval muscle. The K(+)-channel blockers 3,4-diaminopyridine (3,4-DAP) and tetraethylammonium (TEA), at micromolar concentrations, caused a reversible decrease in end-plate current amplitudes both in wild-type and mutant end-plates. In the presence of blockers, a period of high-frequency stimulation (tetanus) of the nerve gave way to a transient increase in the end-plate currents of dnc mutants resembling facilitation and PTP in normal end-plates; 3,4-DAP and TEA also restored facilitation and PTP in normal end-plates after incubation with a non-hydrolysable analogue of cAMP (8Br-cAMP). It is suggested that a specific K+ conductance might be relevant to the lack of synaptic plasticity at the dnc neuromuscular synapses.