Zn induces hyperpolarization by activation of a K channel and increases intracellular Ca and pH in sea urchin spermatozoa

Zinc (Zn²⁺) has been recently recognized as a crucial element for male gamete function in many species although its detailed mechanism of action is poorly understood. In sea urchin spermatozoa, Zn²⁺ was reported as an essential trace ion for efficient sperm motility initiation and the acrosome reaction by modulating intracellular pH (pH_i). In this study we found that submicromolar concentrations of free Zn²⁺ change membrane potential (Em) and increase the concentration of intracellular Ca²⁺ ([Ca²⁺]_i) and cAMP in Lytechinus pictus sperm. Our results indicate that the Zn²⁺ response in sperm of this species mainly involves an Em hyperpolarization caused by K⁺ channel activation. The pharmacological profile of the Zn²⁺-induced hyperpolarization indicates that the cGMP-gated K⁺ selective channel (tetraKCNG/CNGK), which is crucial for speract signaling, is likely a main target for Zn²⁺. Considering that Zn²⁺ also induces [Ca²⁺]_i fluctuations, our observations suggest that Zn²⁺ activates the signaling cascade of speract, except for an increase in cGMP, and facilitates sperm motility initiation upon spawning. These findings provide new insights about the role of Zn²⁺ in male gamete function.     

Iberiotoxin and clofilium regulate hyperactivation,acrosome reaction,and ion homeostasis synergistically during human sperm capacitation

Potassium channels play essential roles in the regulation of male fertility. However, potassium channels mediating K⁺ currents in human sperm (I_KSper) remain controversial. Besides SLO3, the SLO1 potassium channel is a potential candidate for human sperm KSper. This study intends to elucidate the function of SLO1 potassium channel during human sperm capacitation. Human sperm were treated with iberiotoxin (IbTX, a SLO1 specific inhibitor) and clofilium (SLO3 inhibitor) separately or simultaneously during in vitro capacitation. A computer-assisted sperm analyzer was used to assess sperm motility. The sperm acrosome reaction (AR) was analyzed using fluorescein isothiocyanate-conjugated Pisum sativum agglutinin staining. Sperm protein tyrosine phosphorylation was studied using western blotting. Intracellular Ca²⁺, K⁺, Cl⁻, and pH were analyzed using ion fluorescence probes. Independent inhibition with IbTX or clofilium decreased the sperm hyperactivation, AR, and protein tyrosine phosphorylation, and was accompanied by an increase in [K⁺]_i, [Cl⁻]_i, and pH_i, but a decrease in [Ca²⁺]_i. Simultaneously inhibition with IbTX and clofilium lower sperm hyperactivation and AR more than independent inhibition. The increase in [K⁺]_i, [Cl⁻]_i, and pH_i, and the decrease in [Ca²⁺]_i were more pronounced. This study suggested that the SLO1 potassium channel may have synergic roles with SLO3 during human sperm capacitation.     

Characterization of the tissue‐level Ca2+ signals in spontaneously contracting human myometrium

In the labouring uterus, millions of myocytes forming the complex geometrical structure of myometrium contract in synchrony to increase intrauterine pressure, dilate the cervix and eventually expel the foetus through the birth canal. The mechanisms underlying the precise coordination of contractions in human myometrium are not completely understood. In the present study, we have characterized the spatio‐temporal properties of tissue‐level [Ca²⁺]_i transients in thin slices of intact human myometrium. We found that the waveform of [Ca²⁺]_i transients and isotonic contractions recorded from thin slices was similar to the waveform of isometric contractions recorded from the larger strips in traditional organ bath experiments, suggesting that the spatio‐temporal information obtained from thin slices is representative of the whole tissue. By comparing the time course of [Ca²⁺]_i transients in individual cells to that recorded from the bundles of myocytes we found that the majority of myocytes produce rapidly propagating long‐lasting [Ca²⁺]_i transients accompanied by contractions. We also found a small number of cells showing desynchronized [Ca²⁺]_i oscillations that did not trigger contractions. The [Ca²⁺]_i oscillations in these cells were insensitive to nifedipine, but readily inhibited by the T‐type Ca²⁺ channel inhibitor NNC55‐0396. In conclusion, our data suggest that the spread of [Ca²⁺]_i signals in human myometrium is achieved via propagation of long‐lasting action potentials. The propagation was fast when action potentials propagated along bundles of myocytes and slower when propagating between the bundles of uterine myocytes.     

Pharmacological Targeting of Native CatSper Channels Reveals a Required Role in Maintenance of Sperm Hyperactivation

The four sperm-specific CatSper ion channel proteins are required for hyperactivated motility and male fertility, and for Ca²⁺ entry evoked by alkaline depolarization. In the absence of external Ca²⁺, Na⁺ carries current through CatSper channels in voltage-clamped sperm. Here we show that CatSper channel activity can be monitored optically with the [Na⁺]_i-reporting probe SBFI in populations of intact sperm. Removal of external Ca²⁺ increases SBFI signals in wild-type but not CatSper2-null sperm. The rate of the indicated rise of [Na⁺]_i is greater for sperm alkalinized with NH₄Cl than for sperm acidified with propionic acid, reflecting the alkaline-promoted signature property of CatSper currents. In contrast, the [Na⁺]_i rise is slowed by candidate CatSper blocker HC-056456 (IC₅₀ ∼3 µM). HC-056456 similarly slows the rise of [Ca²⁺]_i that is evoked by alkaline depolarization and reported by fura-2. HC-056456 also selectively and reversibly decreased CatSper currents recorded from patch-clamped sperm. HC-056456 does not prevent activation of motility by HCO₃ ⁻ but does prevent the development of hyperactivated motility by capacitating incubations, thus producing a phenocopy of the CatSper-null sperm. When applied to hyperactivated sperm, HC-056456 causes a rapid, reversible loss of flagellar waveform asymmetry, similar to the loss that occurs when Ca²⁺ entry through the CatSper channel is terminated by removal of external Ca²⁺. Thus, open CatSper channels and entry of external Ca²⁺ through them sustains hyperactivated motility. These results indicate that pharmacological targeting of the CatSper channel may impose a selective late-stage block to fertility, and that high-throughput screening with an optical reporter of CatSper channel activity may identify additional selective blockers with potential for male-directed contraception.     

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 (pH_i) in mature sperm trigger at least two events obligatory for fertilization: capacitation and acrosome reaction. Evidence implicates the activity of Ca²⁺ channels modulated by pH_i 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 pH_i by ∼1.3 U from a resting pH_i ∼6.65. A steady pH_i 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 pH_i, 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.     

CRISP1 as a novel CatSper regulator that modulates sperm motility and orientation during fertilization

Ca²⁺-dependent mechanisms are critical for successful completion of fertilization. Here, we demonstrate that CRISP1, a sperm protein involved in mammalian fertilization, is also present in the female gamete and capable of modulating key sperm Ca²⁺ channels. Specifically, we show that CRISP1 is expressed by the cumulus cells that surround the egg and that fertilization of cumulus–oocyte complexes from CRISP1 knockout females is impaired because of a failure of sperm to penetrate the cumulus. We provide evidence that CRISP1 stimulates sperm orientation by modulating sperm hyperactivation, a vigorous motility required for penetration of the egg vestments. Moreover, patch clamping of sperm revealed that CRISP1 has the ability to regulate CatSper, the principal sperm Ca²⁺ channel involved in hyperactivation and essential for fertility. Given the critical role of Ca²⁺ for sperm motility, we propose a novel CRISP1-mediated fine-tuning mechanism to regulate sperm hyperactivation and orientation for successful penetration of the cumulus during fertilization.     

Mouse Sperm Membrane Potential Hyperpolarization Is Necessary and Sufficient to Prepare Sperm for the Acrosome Reaction

Mammalian sperm are unable to fertilize the egg immediately after ejaculation; they acquire this capacity during migration in the female reproductive tract. This maturational process is called capacitation and in mouse sperm it involves a plasma membrane reorganization, extensive changes in the state of protein phosphorylation, increases in intracellular pH (pH_i) and Ca²⁺ ([Ca²⁺]_i), and the appearance of hyperactivated motility. In addition, mouse sperm capacitation is associated with the hyperpolarization of the cell membrane potential. However, the functional role of this process is not known. In this work, to dissect the role of this membrane potential change, hyperpolarization was induced in noncapacitated sperm using either the ENaC inhibitor amiloride, the CFTR agonist genistein or the K⁺ ionophore valinomycin. In this experimental setting, other capacitation-associated processes such as activation of a cAMP-dependent pathway and the consequent increase in protein tyrosine phosphorylation were not observed. However, hyperpolarization was sufficient to prepare sperm for the acrosome reaction induced either by depolarization with high K⁺ or by addition of solubilized zona pellucida (sZP). Moreover, K⁺ and sZP were also able to increase [Ca²⁺]_i in non-capacitated sperm treated with these hyperpolarizing agents but not in untreated cells. On the other hand, in conditions that support capacitation-associated processes blocking hyperpolarization by adding valinomycin and increasing K⁺ concentrations inhibited the agonist-induced acrosome reaction as well as the increase in [Ca²⁺]_i. Altogether, these results suggest that sperm hyperpolarization by itself is key to enabling mice sperm to undergo the acrosome reaction.     

Effect of low extracellular Ca2+ on growth spreading area,cytoplasmic Ca2+ concentration,and intracellular pH in normal and transformed human fibroblasts

The transformation of certain cells reduces the requirement of extracellular Ca²⁺ for growth. The SV-40 transformed human lung fibroblasts, WI-38 VA13, require less Ca²⁺ than normal WI-38 cells. Spreading area of normal cells decreases when cultured in 10 μM Ca²⁺ medium. Intracellular calcium concentration ([Ca²⁺]_i), of the normal and transformed cells cultured in 10μM and 2 mM Ca²⁺ media was measured by the fluorescence microscope technique using fura-2 as a probe. The [Ca²⁺], is measured in the resting state and during mobilization by serum or bradykinin stimulation. The lowering of extracellular calcium concentration results in a decrease in the resting state [Ca²⁺],_i of both normal and transformed cells. Although the total decrease in [Ca²⁺]_i is the same for both cell, the rate of decrease is much faster in normal cells than in transformed cells. Low extracellular Ca²⁺ reduces the number of cells responsive to the serum or bradykinin stimulation and decreases the peak [Ca²⁺]_i value in both cells. In addition, we investigated, using BCECF as a fluorecent probe, the intracellular pH (pH_i) of normal and transformed cells maintained at low and normal Ca²⁺. The low Ca²⁺ condition makes pH_i acidic in normal cells but not in transformed cells. The acidification of the normal cell is accompanied by a decrease in the spreading area of the cells. The decrease of the cell attacment, followed by the reduced spreading area, induced the acidic pH_i. These results suggest that the reduced Ca²⁺ requirement of transformed cells for growth is related to the mechanism of pH_i regulation rather than Ca²⁺ homeostasis and, possibly, to the anchorage-independent growth, which is a unique feature of transformed cells. © 1993 Wiley-Liss, Inc.     

Proteolytic processing of phospholipase Czeta and [Ca2+]i oscillations during mammalian fertilization

Phospholipase Cζ (PLCζ) is a sperm-specific PLC capable of causing repetitive intracellular Ca²⁺ ([Ca²⁺]_i) release ([Ca²⁺]_i oscillations) in mammalian eggs. Accumulating evidence suggests that PLCζ is the sperm factor responsible for inducing egg activation. Nevertheless, some sperm fractions devoid of 72-kDa PLCζ showed [Ca²⁺]_i oscillation-inducing and PLCζ-like PLC activity (Kurokawa et al., (2005) Dev. Biol. 285, 376-392). Here, we report that PLCζ remains functional after proteolytic cleavage at the X-Y linker region. We found that N-terminal (33 and 37 kDa) and C-terminal fragments (27 kDa), presumably the result of PLCζ cleavage at the X-Y linker region, were present in fresh sperm as well as in sperm extracts and remained associated as functional complexes. Protease V8 cleaved 72-kDa PLCζ into 33/37 and 27 kDa fragments, while PLC activity and [Ca²⁺]_i oscillation-inducing activity persisted until degradation of the fragments. Immunodepletion or affinity depletion of these fragments abolished PLC activity and [Ca²⁺]_i oscillation-inducing activity from sperm extracts. Lastly, co-expression of cRNAs encoding residues 1-361 and 362-647 of mouse PLCζ, mimicking cleavage at the X-Y linker region, induced [Ca²⁺]_i oscillations and embryo development in mouse eggs. Our results support the hypothesis that PLCζ is the sole mammalian sperm factor and that its linker region may have important regulatory functions during mammalian fertilization.     

Activation of Ca2+channels during the acrosome reaction of sea urchin sperm is inhibited by inhibitors of chymotrypsin-like proteases

Probable participation of sperm protease in the acrosome reaction was investigated using several inhibitors and substrates. Among those examined, L-l-tosylamide-2-phenylethyl chloromethyl ketone (TPCK) and chymostatin, chymotrypsin inhibitors, p-nitrophenyl-p′-guanidinobenzoate (NPGB), a serine protease inhibitor, and N-benzoyl-L-tyrosine ethyl ester (BTEE), a chymotrypsin substrate, inhibited the egg jelly-induced acrosome reaction of Strongylocentrotus intermedius. TPCK and BTEE, however, did not inhibit the reaction caused by ionophores, A23187, or nigericin. To know the mechanism of inhibition by chymotrypsin inhibitors and substrates of the egg jelly-induced acrosome reaction, intraccllular Ca²⁺ concentration ([Ca²⁺]_i) and pH (pH_i) were measured with fura-2 and 2′,7′-bis (carboxy-ethyl)carboxyfluorescein (BCECF), respectively. Egg jelly caused increase of [Ca²⁺]_i which was depressed by BTEE. Egg jelly also caused a transient rise of pH_i, which was not depressed by BTEE. In the presence of verapamil, the acrosome reaction by egg jelly was significantly inhibited concomitant with depressed increase of [Ca²⁺]_i. The rise of pHj was not depressed by verapamil. Thus, modes of action of BTEE and of verapamil are similar to each other. Bringing these findings together, the authors present a view that a chymotrypsin-like protease of sea urchin sperm activates verapamil-sensitive Ca²⁺ channels, which take part in the acrosome reaction.     

Increases in Intracellular pH and Ca2+ are Essential for K+ Channel Activation After Modest `Physiological' Swelling in Villus Epithelial Cells

We studied the relationship between changes in intracellular pH (pH _i ), intracellular Ca²⁺([Ca²⁺] _i ) and charybdotoxin sensitive (CTX) maxi-K⁺ channels occurring after modest `physiological' swelling in guinea pig jejunal villus enterocytes. Villus cell volume was assessed by electronic cell sizing, and pH <sub>i</sub> and [Ca<sup>2+</sup>] <sub>i</sub> by fluorescence spectroscopy with 2,7, biscarboxyethyl-5-6-carboxyfluorescein and Indo-1, respectively. In a slightly (0.93 × isotonic) hypotonic medium, villus cells swelled to the same size they would reach during d-glucose or l-alanine absorption; the subsequent Regulatory Volume Decrease (RVD) was prevented by CTX. After the large volume increase in a more hypotonic (0.80 × isotonic) medium, RVD was unaffected by CTX. After modest swelling associated with 0.93 × isotonic dilution, the pH <sub>i</sub> alkalinized but N-5-methyl-isobutyl amiloride (MIA) prevented this ΔpH <sub>i</sub> and the subsequent RVD. Even in the presence of MIA, alkalinization with added NH<sub>4</sub>Cl permitted complete RVD which could be inhibited by CTX. The rate of <sup>86</sup>Rb efflux which also increased after this 0.93 × isotonic dilution was inhibited an equivalent amount by CTX, MIA or Na<sup>+</sup>-free medium. Modest swelling transiently increased [Ca<sup>2+</sup>] <sub>i</sub> and Ca<sup>2+</sup>-free medium or blocking alkalinization by MIA or Na<sup>+</sup>-free medium diminished this transient increase an equivalent amount. RVD after modest swelling was prevented in Ca<sup>2+</sup>-free medium but alkalinization still occurred. After large volume increases, alkalinization of cells increased [Ca<sup>2+</sup>] <sub>i</sub> and volume changes became sensitive to CTX. We conclude that both alkalinization of pH <sub>i</sub> and increased [Ca<sup>2+</sup>] <sub>i</sub> observed with `physiological' volume increase are essential for the activation of CTX-sensitive maxi-K⁺ channels required for RVD. Received: 30 March 1999/Revised: 6 July 1999     

Contributions of extracellular and intracellular Ca2+ to regulation of sperm motility: Release of intracellular stores can hyperactivate CatSper1 and CatSper2 null sperm

In order to fertilize, mammalian sperm must hyperactivate. Hyperactivation is triggered by increased flagellar Ca(2+), which switches flagellar beating from a symmetrical to an asymmetrical pattern by increasing bending to one side. Thimerosal, which releases Ca(2+) from internal stores, induced hyperactivation in mouse sperm within seconds, even when extracellular Ca(2+) was buffered with BAPTA to approximately 30 nM. In sperm from CatSper1 or CatSper2 null mice, which lack functional flagellar alkaline-activated calcium currents, 50 microM thimerosal raised the flagellar bend amplitudes from abnormally low levels to normal pre-hyperactivated levels and, in 20-40% of sperm, induced hyperactivation. Addition of 1 mM Ni(2+) diminished the response. This suggests that intracellular Ca(2+) is abnormally low in the null sperm flagella. When intracellular Ca(2+) was reduced by BAPTA-AM in wild-type sperm, they exhibited flagellar beat patterns more closely resembling those of null sperm. Altogether, these results indicate that extracellular Ca(2+) is required to supplement store-released Ca(2+) to produce maximal and sustained hyperactivation and that CatSper1 and CatSper2 are key elements of the major Ca(2+) entry pathways that support not only hyperactivated motility but possibly also normal pre-hyperactivated motility.     

Age-associated potency decline in bovine oocytes is delayed by blocking extracellular Ca2+ influx

Oocyte aging due to delayed fertilization is associated with declining quality and developmental potential. Intracellular calcium (Ca²⁺) concentration ([Ca²⁺]_i) regulates oocyte growth, maturation, and fertilization and has also been implicated in aging. Using bovine oocytes, we tested the hypothesis that oocyte aging could be delayed by reducing [Ca²⁺]_i via blocking the influx of extracellular Ca²⁺ or chelating ooplasmic free Ca²⁺. After IVM, cumulus–oocyte complexes or denuded oocytes were cultured in medium supplemented with 1-octanol, phorbol 12-myristate 13-acetate, or 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis-acetoxymethyl ester (BAPTA-AM) to manipulate [Ca²⁺]_i. Addition of 1-mM 1-octanol increased blastocyst development rates in the cumulus–oocyte complexes aged for 6 hours by IVF and for 6, 12, and 24 hours by parthenoactivation, and this effect was independent of the presence of cumulus cells. The intracellular levels of ATP, Glutathione, and Glutathione disulfide were not affected by 1-octanol, but [Ca²⁺]_i was significantly decreased. When oocytes were cultured in Ca²⁺-free medium for 12 hours, the blastocyst development rate was greater and the beneficial effects of 1-octanol on oocyte aging were abolished. However, when the medium was supplemented with phorbol 12-myristate 13-acetate, [Ca²⁺]_i increased and the blastocyst development rate decreased. Moreover, BAPTA-AM reduced [Ca²⁺]_i and increased blastocyst development rates after IVF or parthenoactivation. We conclude that the age-associated developmental potency decline was delayed by blocking the influx of extracellular Ca²⁺ or reducing ooplasmic free Ca²⁺. 1-Octanol, BAPTA-AM, or Ca²⁺-free medium could be used to lengthen the fertilization windows of aged bovine oocytes.     

Suppression of programmed neuronal death by a thapsigargin-induced Ca2+ influx

Rat sympathetic neurons undergo programmed cell death (PCD) in vitro and in vivo when they are deprived of nerve growth factor (NGF). Chronic depolarization of these neurons in cell culture with elevated concentrations of extracellular potassium ([K⁺]_o) prevents this death. The effect of prolonged depolarization on neuronal survival is thought to be mediated by a rise of intracellular calcium concentration ([Ca²⁺]_i) caused by Ca²⁺ influx through voltage-gated channels. In this report we investigate the effects of chronic treatment of rat sympathetic neurons with thapsigargin, an inhibitor of intracellular Ca²⁺ sequestration. In medium containing a normal concentration of extracellular Ca²⁺ ([Ca²⁺]_o), thapsigargin caused a sustained rise of intracellular Ca²⁺ concentration and partially blocked death of NGF-deprived cells. Elevating [Ca²⁺]_o in the presence of thapsigargin further increased [Ca²⁺]_i, suggesting that the sustained rise of [Ca²⁺]_i was caused by a thapsigargin-induced Ca²⁺ influx. This treatment potentiated the effect of thapsigargin on survival. The dihydropyridine Ca²⁺ channel antagonist, nifedipine, blocked both a sustained elevation of [Ca²⁺]_i and enhanced survival caused by depolarization with elevated [K⁺]_o, suggesting that these effects are mediated by Ca²⁺ influx through L-type channels. Nifedipine did not block the sustained rise of [Ca²⁺]_i or enhanced survival caused by thapsigargin treatment, indicating that these effects were not mediated by influx of Ca²⁺ through L-type channels. These results provide additional evidence that increased [Ca²⁺]_i can suppress neuronal PCD and identify a novel method for chronically raising neuronal [Ca²⁺]_i for investigation of this and other Ca²⁺-dependent phenomena. © 1995 John Wiley & Sons, Inc.     

Ca2+ Influx via the Na+/Ca2+ Exchanger Is Enhanced in Malignant Hyperthermia Skeletal Muscle

Malignant hyperthermia (MH) is potentially fatal pharmacogenetic disorder of skeletal muscle caused by intracellular Ca²⁺ dysregulation. NCX is a bidirectional transporter that effluxes (forward mode) or influxes (reverse mode) Ca²⁺ depending on cellular activity. Resting intracellular calcium ([Ca²⁺]_r) and sodium ([Na⁺]_r) concentrations are elevated in MH susceptible (MHS) swine and murine muscles compared with their normal (MHN) counterparts, although the contribution of NCX is unclear. Lowering [Na⁺]_e elevates [Ca²⁺]_r in both MHN and MHS swine muscle fibers and it is prevented by removal of extracellular Ca²⁺ or reduced by t-tubule disruption, in both genotypes. KB-R7943, a nonselective NCX3 blocker, reduced [Ca²⁺]_r in both swine and murine MHN and MHS muscle fibers at rest and decreased the magnitude of the elevation of [Ca²⁺]_r observed in MHS fibers after exposure to halothane. YM-244769, a high affinity reverse mode NCX3 blocker, reduces [Ca²⁺]_r in MHS muscle fibers and decreases the amplitude of [Ca²⁺]_r rise triggered by halothane, but had no effect on [Ca²⁺]_r in MHN muscle. In addition, YM-244769 reduced the peak and area under the curve of the Ca²⁺ transient elicited by high [K⁺]_e and increased its rate of decay in MHS muscle fibers. siRNA knockdown of NCX3 in MHS myotubes reduced [Ca²⁺]_r and the Ca²⁺ transient area induced by high [K⁺]_e. These results demonstrate a functional NCX3 in skeletal muscle whose activity is enhanced in MHS. Moreover reverse mode NCX3 contributes to the Ca²⁺ transients associated with K⁺-induced depolarization and the halothane-triggered MH episode in MHS muscle fibers.     

A Model of CatSper Channel Mediated Calcium Dynamics in Mammalian Spermatozoa

CatSpers are calcium (Ca²⁺) channels that are located along the principal piece of mammalian sperm flagella and are directly linked to sperm motility and hyperactivation. It has been observed that Ca²⁺ entry through CatSper channels triggers a tail to head Ca²⁺ propagation in mouse sperm, as well as a sustained increase of Ca²⁺ in the head. Here, we develop a mathematical model to investigate this propagation and sustained increase in the head. A 1-d reaction-diffusion model tracking intracellular Ca²⁺ with flux terms for the CatSper channels, a leak flux, and plasma membrane Ca²⁺ clearance mechanism is studied. Results of this simple model exhibit tail to head Ca²⁺ propagation, but no sustained increase in the head. Therefore, in this model, a simple plasma membrane pump-leak system with diffusion in the cytosol cannot account for these experimentally observed results. It has been proposed that Ca²⁺ influx from the CatSper channels induce additional Ca²⁺ release from an internal store. We test this hypothesis by examining the possible role of Ca²⁺ release from the redundant nuclear envelope (RNE), an inositol 1,4,5-trisphosphate (IP₃) gated Ca²⁺ store in the neck. The simple model is extended to include an equation for IP₃ synthesis, degradation, and diffusion, as well as flux terms for Ca²⁺ in the RNE. When IP₃ and the RNE are accounted for, the results of the model exhibit a tail to head Ca²⁺ propagation as well as a sustained increase of Ca²⁺ in the head.     

Trifluoperazine enhancement of Ca2+-dependent inactivation of L-type Ca2+ currents inHelix aspersa neurons

The effects of trifluoperazine hydrochloride (TFP), a calmodulin antagonist, on L-type Ca²⁺ currents (L-type ICa²⁺) and their Ca²⁺-dependent inactivation, were studied in identifiedHelix aspersa neurons, using two microelectrode voltage clamp. Changes in [Ca²⁺]_i were measured in unclamped fura-2 loaded neurons. Bath applied TFP produced a reversible and dose-dependent reduction in amplitude of L-type ICa²⁺ (IC₅₀=28 μM). Using a double-pulse protocol, we found that TFP enhances the efficacy of Ca²⁺-dependent inactivation of L-type ICa²⁺. Trifluoperazine sulfoxide (50 μM), a TFP derivative with low calmodulin-antagonist activity, did not have any effects on either amplitude or inactivation of L-type ICa²⁺. TFP (20 μM) increased basal [Ca²⁺]_i from 147±37 nM to 650±40nM (N=7). The increase in [Ca²⁺]_i was prevented by removal of external Ca²⁺ and curtailed by depletion of caffeine-sensitive intracellular Ca²⁺ stores. Since TFP may also block protein kinase C (PKC), we tested the effect of a PKC activator (12-O-tetradecanoyl-phorbol-13-acetate) on L-type Ca²⁺ currents. This compound produced an increase in L-type ICa²⁺ without enhancing Ca²⁺-dependent inactivation. The results show that 1) TFP reduces L-type ICa²⁺ while enhancing the efficacy of Ca²⁺-dependent inactivation. 2) TFP produces an increase in basal [Ca²⁺]_i which may contribute to the enhancement of Ca²⁺-dependent inactivation. 3) PKC up-regulates L-type ICa²⁺ without altering the efficacy of Ca²⁺ dependent inactivation. 4) The TFP effects cannot be attributed to its action as PKC blocker.