Internalization of plasma membrane Ca2+-ATPase during Xenopus oocyte maturation

A transient increase in intracellular Ca²⁺ is the universal signal for egg activation at fertilization. Eggs acquire the ability to mount the specialized fertilization-specific Ca²⁺ signal during oocyte maturation. The first Ca²⁺ transient following sperm entry in vertebrate eggs has a slow rising phase followed by a sustained plateau. The molecular determinants of the sustained plateau are poorly understood. We have recently shown that a critical determinant of Ca²⁺ signaling differentiation during oocyte maturation is internalization of the plasma membrane calcium ATPase (PMCA). PMCA internalization is representative of endocytosis of several integral membrane proteins during oocyte maturation, a requisite process for early embryogenesis. Here we investigate the mechanisms regulating PMCA internalization. To track PMCA trafficking in live cells we cloned a full-length cDNA of Xenopus PMCA1, and show that GFP-tagged PMCA traffics in a similar fashion to endogenous PMCA. Functional data show that MPF activation during oocyte maturation is required for full PMCA internalization. Pharmacological and co-localization studies argue that PMCA is internalized through a lipid raft endocytic pathway. Deletion analysis reveal a requirement for the N-terminal cytoplasmic domain for efficient internalization. Together these studies define the mechanistic requirements for PMCA internalization during oocyte maturation.     

Ca(2+)- and volume-sensitive chloride currents are differentially regulated by agonists and store-operated Ca2+ entry

Using patch-clamp and calcium imaging techniques, we characterized the effects of ATP and histamine on human keratinocytes. In the HaCaT cell line, both receptor agonists induced a transient elevation of [Ca2+]i in a Ca(2+)-free medium followed by a secondary [Ca2+]i rise upon Ca2+ readmission due to store-operated calcium entry (SOCE). In voltage-clamped cells, agonists activated two kinetically distinct currents, which showed differing voltage dependences and were identified as Ca(2+)-activated (I(Cl(Ca))) and volume-regulated (I(Cl, swell)) chloride currents. NPPB and DIDS more efficiently inhibited I(Cl(Ca)) and I(Cl, swell), respectively. Cell swelling caused by hypotonic solution invariably activated I(Cl, swell) while regulatory volume decrease occurred in intact cells, as was found in flow cytometry experiments. The PLC inhibitor U-73122 blocked both agonist- and cell swelling-induced I(Cl, swell), while its inactive analogue U-73343 had no effect. I(Cl(Ca)) could be activated by cytoplasmic calcium increase due to thapsigargin (TG)-induced SOCE as well as by buffering [Ca2+]i in the pipette solution at 500 nM. In contrast, I(Cl, swell) could be directly activated by 1-oleoyl-2-acetyl-sn-glycerol (OAG), a cell-permeable DAG analogue, but neither by InsP3 infusion nor by the cytoplasmic calcium increase. PKC also had no role in its regulation. Agonists, OAG, and cell swelling induced I(Cl, swell) in a nonadditive manner, suggesting their convergence on a common pathway. I(Cl, swell) and I(Cl(Ca)) showed only a limited overlap (i.e., simultaneous activation), although various maneuvers were able to induce these currents sequentially in the same cell. TG-induced SOCE strongly potentiated I(Cl(Ca)), but abolished I(Cl, swell), thereby providing a clue for this paradox. Thus, we have established for the first time using a keratinocyte model that I(Cl, swell) can be physiologically activated under isotonic conditions by receptors coupled to the phosphoinositide pathway. These results also suggest a novel function for SOCE, which can operate as a "selection" switch between closely localized channels.     

Essential role of the N-terminus of murine Orai1 in store-operated Ca2+ entry

Store-operated Ca(2+) entry (SOCE) is a physiologically important process that is triggered by intracellular Ca(2+) depletion. Recently, human Orai1 (the channel-forming subunit) and STIM1 (the calcium sensor) were identified as essential molecules for SOCE. Here, we report the cloning and functional analysis of three murine orthologs of Orai1, termed Orai1, 2, and 3. Among the genes identified, Orai1 contains a distinctive proline- and arginine-rich N-terminal cytoplasmic sequence. Co-expression of STIM1 with Orai1 produced a marked effect on SOCE, while co-expression with Orai2 or Orai3 had little effect. Expression of Orai1 without its N-terminal tail had a marginal effect on SOCE, while chimeric Orai2 containing the Orai1 N-terminus produced a marked increase in SOCE. In addition, a truncated version of Orai1 containing the N-terminus without the pore-forming transmembrane domain had a dominant negative effect on SOCE. These results reveal the essential role of Orai1 and its N-terminal sequence in SOCE.     

Increased sensitivity and clustering of elementary Ca2+ release events during oocyte maturation

The universal signal for egg activation at fertilization is a rise in cytoplasmic Ca(2+) with defined spatial and temporal kinetics. Mammalian and amphibian eggs acquire the ability to produce such Ca(2+) signals during a maturation period that precedes fertilization and encompasses resumption of meiosis and progression to metaphase II. In Xenopus, immature oocytes produce fast, saltatory Ca(2+) waves that can be oscillatory in nature in response to IP(3). In contrast, mature eggs produce a single continuous, sweeping Ca(2+) wave in response to IP(3) or sperm fusion. The mechanisms mediating the differentiation of Ca(2+) signaling during oocyte maturation are not well understood. Here, I characterized elementary Ca(2+) release events (Ca(2+) puffs) in oocytes and eggs and show that the sensitivity of IP(3)-dependent Ca(2+) release is greatly enhanced during oocyte maturation. Furthermore, Ca(2+) puffs in eggs have a larger spatial fingerprint, yet are short lived compared to oocyte puffs. Most interestingly, Ca(2+) puffs cluster during oocyte maturation resulting in a continuum of Ca(2+) release sites over space in eggs. These changes in the spatial distribution of elementary Ca(2+) release events during oocyte maturation explain the continuous nature and slower speed of the fertilization Ca(2+) wave.     

STIM1 regulates store-operated Ca entry in oocytes

The single transmembrane-spanning Ca²⁺-binding protein, STIM1, has been proposed to function as a Ca²⁺ sensor that links the endoplasmic reticulum to the activation of store-operated Ca²⁺ channels. In this study, the presence, subcellular localization and function of STIM1 in store-operated Ca²⁺ entry in oocytes was investigated using the pig as a model. Cloning and sequence analysis revealed the presence of porcine STIM1 with a coding sequence of 2058 bp. In oocytes with full cytoplasmic Ca²⁺ stores, STIM1 was localized predominantly in the inner cytoplasm as indicated by immunocytochemistry or overexpression of human STIM1 conjugated to the yellow fluorescent protein. Depletion of the Ca²⁺ stores was associated with redistribution of STIM1 along the plasma membrane. Increasing STIM1 expression resulted in enhanced Ca²⁺ influx after store depletion and subsequent Ca²⁺ add-back; the influx was inhibited when the oocytes were pretreated with lanthanum, a specific inhibitor of store-operated Ca²⁺ channels. When STIM1 expression was suppressed using siRNAs, there was no change in cytosolic free Ca²⁺ levels in the store-depleted oocytes after Ca²⁺ add-back. The findings suggest that in oocytes, STIM1 serves as a sensor of Ca²⁺ store content that after store depletion moves to the plasma membrane to stimulate store-operated Ca²⁺ entry.     

Ca(2+) response to cADPr during maturation and fertilization of starfish oocytes.

During the reinitiation of the meiotic cycle (maturation) induced by the hormone 1-methyladenine (1-MA), starfish oocytes undergo structural and biochemical changes in preparation for successful fertilization. Previous work has shown that the sensitivity of internal Ca(2+) stores to InsP(3) increases during maturation of the oocytes. Since Astropecten auranciacus oocytes also respond to cADPr, we have studied whether the response to cADPr also changes during maturation. We have found that the photoactivation of injected cADPr in immature oocytes immediately induces multiple patches of Ca(2+) release in the cortical region. The Ca(2+) signal then spreads from these initial points of increase to the entire cell. In mature oocytes, the uncaging of cADPr induces instead a single (or at most a dual) initial point of Ca(2+) release, which is immediately followed by the formation of a cortical Ca(2+) flash and then by the globalization of the wave and by the elevation of the fertilization envelope. External Ca(2+) plays a role in the Ca(2+) responses. Inhibition of L-type Ca(2+) channels does not affect the initial Ca(2+) release, but abolishes the cortical flash and impairs the elevation of the fertilization envelope. External Ca(2+) has other effects, as shown by the irregular appearance of the surface of oocytes incubated in Ca(2+)-free sea water. The sequence of Ca(2+) responses induced by cADPr in mature oocytes mimics those seen at fertilization, i.e., a first localized Ca(2+) increase followed by a cortical flash and by the globalization of the Ca(2+) signal. As in the case of maturation, L-type Ca(2+) channel blockers abolish the sperm induced cortical flash.     

Calcium signaling differentiation during Xenopus oocyte maturation

Ca(2+) is the universal signal for egg activation at fertilization in all sexually reproducing species. The Ca(2+) signal at fertilization is necessary for egg activation and exhibits specialized spatial and temporal dynamics. Eggs acquire the ability to produce the fertilization-specific Ca(2+) signal during oocyte maturation. However, the mechanisms regulating Ca(2+) signaling differentiation during oocyte maturation remain largely unknown. At fertilization, Xenopus eggs produce a cytoplasmic Ca(2+) (Ca(2+)(cyt)) rise that lasts for several minutes, and is required for egg activation. Here, we show that during oocyte maturation Ca(2+) transport effectors are tightly modulated. The plasma membrane Ca(2+) ATPase (PMCA) is completely internalized during maturation, and is therefore unable to extrude Ca(2+) out of the cell. Furthermore, IP(3)-dependent Ca(2+) release is required for the sustained Ca(2+)(cyt) rise in eggs, showing that Ca(2+) that is pumped into the ER leaks back out through IP(3) receptors. This apparent futile cycle allows eggs to maintain elevated cytoplasmic Ca(2+) despite the limited available Ca(2+) in intracellular stores. Therefore, Ca(2+) signaling differentiates in a highly orchestrated fashion during Xenopus oocyte maturation endowing the egg with the capacity to produce a sustained Ca(2+)(cyt) transient at fertilization, which defines the egg's competence to activate and initiate embryonic development.     

Heteromeric TRPV4-C1 channels contribute to store-operated Ca(2+) entry in vascular endothelial cells

There is controversy as to whether TRP channels participate in mediating store-operated current (I_SOC) and store-operated Ca²⁺ entry (SOCE). Our recent study has demonstrated that TRPC1 forms heteromeric channels with TRPV4 in vascular endothelial cells and that Ca²⁺ store depletion enhances the vesicle trafficking of heteromeric TRPV4-C1 channels, causing insertion of more channels into the plasma membrane in vascular endothelial cells. In the present study, we determined whether the enhanced TRPV4-C1 insertion to the plasma membrane could contribute to SOCE and I_SOC. We found that thapsigargin-induced SOCE was much lower in aortic endothelial cells derived from trpv4^−/− or trpc1^−/− knockout mice when compared to that of wild-type mice. In human umbilical vein endothelial cells (HUVECs), thapsigargin-induced SOCE was markedly reduced by knocking down the expression of TRPC1 and/or TRPV4 with respective siRNAs. Brefeldin A, a blocker of vesicular translocation, inhibited the SOCE. These results suggest that an enhanced vesicular trafficking of heteromeric TRPV4-C1 channels contributes to SOCE in vascular endothelial cells. Vascular tension studies suggest that such an enhanced trafficking of TRPV4-C1 channels may play a role in thapsigargin-induced vascular relaxation in rat small mesenteric arteries.     

2-Aminoethyl diphenylborinate analogues: selective inhibition for store-operated Ca2+ entry

Capacitative calcium entry (CCE), the mechanism that replenishes the internal Ca2+ stores with Ca2+ from the extracellular milieu in response to depletion of the store, is mediated by Ca2+ channels in the plasma membrane generally referred to as store-operated channels (SOCs). However, the roles of SOCs in the more physiological context have been fully elucidated. 2-Aminoethyl diphenylborinate (2-APB) strongly inhibits SOCs, as well as inositol-1,4,5 trisphosphate (IP3) receptors. In the present study, we screened a library of 166 2-APB analogues for effects on CCE and IP3-induced Ca2+ release in order to discover specific SOC inhibitors, and found that some blocked both store-operated and receptor-operated Ca2+ influx more strongly and selectively than 2-APB. Indeed, these new compounds ceased the prolonged intracellular Ca2+ oscillations induced by a low concentration of ATP in CHO-K1 cells. These novel SOC inhibitors will be valuable pharmacological and biochemical tools for elucidating the physiological roles.     

Molecular evolution and structural analysis of the Ca(2+) release-activated Ca(2+) channel subunit, Orai

Depletion of intracellular Ca(2+) stores evokes Ca(2+) entry across the plasma membrane by inducing Ca(2+) release-activated Ca(2+) (CRAC) currents in many cell types. Recently, Orai and STIM proteins were identified as the molecular identities of the CRAC channel subunit and the endoplasmic reticulum Ca(2+) sensor, respectively. Here, extensive database searching and phylogenetic analysis revealed several lineage-specific duplication events in the Orai protein family, which may account for the evolutionary origins of distinct functional properties among mammalian Orai proteins. Based on similarity to key structural domains and essential residues for channel functions in Orai proteins, database searching also identifies a putative primordial Orai sequence in hyperthermophilic archaeons. Furthermore, modern Orai appears to acquire new structural domains as early as Urochodata, before divergence into vertebrates. The evolutionary patterns of structural domains might be related to distinct functional properties of Drosophila and mammalian CRAC currents. Interestingly, Orai proteins display two conserved internal repeats located at transmembrane segments 1 and 3, both of which contain key amino acids essential for channel function. These findings demonstrate biochemical and physiological relevance of Orai proteins in light of different evolutionary origins and will provide novel insights into future structural and functional studies of Orai proteins.     

The anti-anginal drug fendiline elevates cytosolic Ca(2+) in rabbit corneal epithelial cells

The effect of the anti-anginal drug fendiline on intracellular free Ca(2+) levels ([Ca(2+)](i)) in a rabbit corneal epithelial cell line (SIRC) was explored using fura-2 as a fluorescent Ca(2+) indicator. At a concentration above 1 microM, fendiline increased [Ca(2+)](i) in a concentration-dependent manner with an EC(50) value of 7 microM. The [Ca(2+)](i) response consisted of an immediate rise and an elevated phase. Extracellular Ca(2+) removal decreased half of the [Ca(2+)](i )signal. Fendiline induced quench of fura-2 fluorescence by Mn(2+) (50 microM), suggesting the presence of Ca(2+) influx across the plasma membrane. This Ca(2+) influx was abolished by La(3+) (50 microM), but was insensitive to dihydropyridines, verapamil and diltiazem. Fendiline (10 microM)-induced store Ca(2+) release was largely reduced by pretreatment with thapsigargin (1 microM) (an endoplasmic reticulum Ca(2+) pump inhibitor) to deplete the endoplasmic reticulum Ca(2+). Conversely, pretreatment with 10 microM fendiline abolished thapsigargin-induced Ca(2+) release. Fendiline (10 microM)-induced Ca(2+) release was not altered by inhibiting phospholipase C with 2 microM 1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122). Cumulatively, this study shows that fendiline induced concentration-dependent [Ca(2+)](i )increases in corneal epithelial cells by releasing the endoplasmic reticulum Ca(2+) in a phospholipase C-independent manner, and by causing Ca(2+) influx.     

Regulation of store-operated Ca entry in pulmonary artery smooth muscle cells

Store-operated Ca²⁺ entry (SOCE) is an important mechanism for Ca²⁺ influx in smooth muscle cells; however the activation and regulation of this influx pathway are incompletely understood. In the present study we have examined the effect of several protein kinases in regulating SOCE in pulmonary artery smooth muscle cells (PASMCs) of the rat. Inhibition of protein kinase C with chelerythrine (3 μM) potentiated SOCE by 47 ± 2%, while the tyrosine kinase inhibitors genistein (100 μM) and tyrphostin 23 (100 μM) caused a significant reduction in SOCE of 55 ± 9% and 43 ± 7%, respectively. It has been proposed that Ca²⁺-insensitive phospholipase A₂ (iPLA₂) is involved in the activation of SOCE in many different cell types. The iPLA₂ inhibitor, bromoenol lactone had no effect on SOCE, suggesting that this mechanism was not involved in the activation of the pathway. The calmodulin antagonists, calmidazolium (CMZ) (10 μM) and W-7 (10 μM) appeared to potentiate SOCE in PASMCs. Further investigation established that CMZ was actually activating a Ca²⁺ influx pathway that was independent of the filling state of the sarcoplasmic reticulum. The CMZ-activated Ca²⁺ influx was blocked by Gd³⁺ (10 μM), but unaffected by 2-APB (75 μM), indicating a pharmacological profile distinct from the classical SOCE pathway.     

'Quantal' Ca(2+) release reassessed--a clue to oscillation and synchronization

Ca(2+) release from intracellular Ca(2+) stores, a pivotal event in Ca(2+) signaling, is a 'quantal' process; it terminates after a rapid release of a fraction of stored Ca(2+). To explain the 'quantal' nature, 'all-or-none' model and 'steady-state' model were proposed. This article shortly reviews these hypotheses and considers a recently proposed mechanism, 'luminal potential' model, in which the membrane potential of Ca(2+) store regulates Ca(2+) efflux. By reassessing the 'quantal' nature, other important features of Ca(2+) signaling, oscillation and synchronization, are highlighted. The mechanism for 'quantal' Ca(2+) release may underlie the temporal and spatial control of Ca(2+) signaling.     

A store-operated calcium channel in Drosophila S2 cells

Using whole-cell recording in Drosophila S2 cells, we characterized a Ca(2+)-selective current that is activated by depletion of intracellular Ca2+ stores. Passive store depletion with a Ca(2+)-free pipette solution containing 12 mM BAPTA activated an inwardly rectifying Ca2+ current with a reversal potential >60 mV. Inward currents developed with a delay and reached a maximum of 20-50 pA at -110 mV. This current doubled in amplitude upon increasing external Ca2+ from 2 to 20 mM and was not affected by substitution of choline for Na+. A pipette solution containing approximately 300 nM free Ca2+ and 10 mM EGTA prevented spontaneous activation, but Ca2+ current activated promptly upon application of ionomycin or thapsigargin, or during dialysis with IP3. Isotonic substitution of 20 mM Ca2+ by test divalent cations revealed a selectivity sequence of Ba2+ > Sr2+ > Ca2+ > Mg2+. Ba2+ and Sr2+ currents inactivated within seconds of exposure to zero-Ca2+ solution at a holding potential of 10 mV. Inactivation of Ba2+ and Sr2+ currents showed recovery during strong hyperpolarizing pulses. Noise analysis provided an estimate of unitary conductance values in 20 mM Ca2+ and Ba2+ of 36 and 420 fS, respectively. Upon removal of all external divalent ions, a transient monovalent current exhibited strong selectivity for Na+ over Cs+. The Ca2+ current was completely and reversibly blocked by Gd3+, with an IC50 value of approximately 50 nM, and was also blocked by 20 microM SKF 96365 and by 20 microM 2-APB. At concentrations between 5 and 14 microM, application of 2-APB increased the magnitude of Ca2+ currents. We conclude that S2 cells express store-operated Ca2+ channels with many of the same biophysical characteristics as CRAC channels in mammalian cells.     

Store-operated Ca2+ entry in astrocytes: different spatial arrangement of endoplasmic reticulum explains functional diversity in vitro and in situ

Ca(2+) signaling is the astrocyte form of excitability and the endoplasmic reticulum (ER) plays an important role as an intracellular Ca(2+) store. Since the subcellular distribution of the ER influences Ca(2+) signaling, we compared the arrangement of ER in astrocytes of hippocampus tissue and astrocytes in cell culture by electron microscopy. While the ER was usually located in close apposition to the plasma membrane in astrocytes in situ, the ER in cultured astrocytes was close to the nuclear membrane. Activation of metabotropic receptors linked to release of Ca(2+) from ER stores triggered distinct responses in cultured and in situ astrocytes. In culture, Ca(2+) signals were commonly first recorded close to the nucleus and with a delay at peripheral regions of the cells. Store-operated Ca(2+) entry (SOC) as a route to refill the Ca(2+) stores could be easily identified in cultured astrocytes as the Zn(2+)-sensitive component of the Ca(2+) signal. In contrast, such a Zn(2+)-sensitive component was not recorded in astrocytes from hippocampal slices despite of evidence for SOC. Our data indicate that both, astrocytes in situ and in vitro express SOC necessary to refill stores, but that a SOC-related signal is not recorded in the cytoplasm of astrocytes in situ since the stores are close to the plasma membrane and the refill does not affect cytoplasmic Ca(2+) levels.     

Dependence of the resting [Ca2+]cyt of RBL-1 cells on Ca2+ entry

The cytoplasmic Ca²⁺ concentration ([Ca²⁺]_cyt) in resting cells in an equilibrium between several influx and efflux mechanisms. Here we address the question of whether capacitative Ca²⁺ entry to some extent is active at resting conditions and therefore is part of processes that guarantee a constant [Ca²⁺]_cyt. We measured changes of [Ca²⁺]_cyt in RBL-1 cells with fluorometric techniques. An increase of the extracellular [Ca²⁺] from 1.3 mM to 5 mM induced an incrase in [Ca²⁺]_cyt from 105±10 nM to 145±8.5 nM. This increase could be inhibited by 10 μM Gd³⁺, 10 μM La³⁺ or 50 μM 2-aminoethoxydiphenyl borate, blockers of capacitative Ca²⁺ entry. Application of those blockers to a resting cell in a standard extracellular solution (1.3 mM Ca²⁺) resulted in a decrease of [Ca²⁺]_cyt from 105±10 nM to 88.5±10 nM with La³⁺, from 103±12 to 89±12 nM with Gd³⁺ and from 102±12 nM to 89.5±5 nM with 2-aminoethoxydiphenyl borate. From these data, we conclude that capacitative Ca²⁺ entry beside its function in Ca²⁺ signaling contributes to the regulation of resting [Ca²⁺]_cyt.     

Arachidonic acid inhibits capacitative Ca2+ entry and activates non-capacitative Ca2+ entry in cultured astrocytes

Arachidonic acid (AA) plays important physiological or pathophysiological roles. Here, we show in cultured rat astrocytes that: (i) endothelin-1 or thapsigargin (Tg) induces store-depleted activated Ca²⁺ entry (CCE), which is inhibited by 2-aminoethoxydiphenyl borane (2-APB) or La³⁺; (ii) AA (10 μM) and other unsaturated fatty acids (8,11,14-eicosatrienoic acid and γ-linoleic acid) have an initial inhibitory effect on the CCE, due to AA- or fatty acid-induced internal acid load; (iii) after full activation of CCE, AA induces a further Ca²⁺ influx, which is not inhibited by 2-APB or La³⁺, indicating that AA activates a second Ca²⁺ entry pathway, which coexists with CCE; and (iv) Tg or AA activates two independent and co-existing non-selective cation channels and the Tg-induced currents are initially inhibited by addition of AA or weak acids. A possible pathophysiological effect of the AA-induced [Ca]_i overload is to cause delayed cell death in astrocytes.     

Purification and identification of a Ca(2+)-pectate binding peroxidase from Arabidopsis leaves

A protein fraction was obtained from Arabidopsis (Arabidopsis thaliana, L.) leaf extract by affinity chromatography through a Ca(2+)-pectate/polyacrylamide gel. Further purification by preparative isoelectric focusing and SDS PAGE allowed the separation of a peroxidase that was identified as being peroxidase AtPrx34 (AtprxCb, accession number X71794) by N-terminal amino acid microsequencing. AtPrx34 belongs to a group of five Arabidopsis sequences encoding putative pectin-binding peroxidases. An expression study showed that it is expressed in root, stem, flower and leaf. It was produced by Escherichia coli and tested for its ability to bind to Ca(2+)-pectate. The identity of the amino acids involved in the interaction between the peroxidase and the Ca(2+)-pectate structure is discussed.     

Release of the Ca(2+) oscillation-inducing sperm factor during mouse fertilization

A cytosolic sperm protein(s), referred to as the sperm factor (SF), is thought to induce intracellular calcium ([Ca(2+)](i)) oscillations during fertilization in mammalian eggs. These oscillations, which are responsible for inducing complete egg activation, persist for several hours. Nevertheless, whether a protracted release of SF is responsible for the duration of the oscillations is unknown. Using a combination of intracytoplasmic sperm injection (ICSI), in vitro fertilization (IVF), sperm removal, reinjection of the withdrawn sperm, and [Ca(2+)](i) monitoring, we determined that 30 min was necessary for establishing oscillations. Importantly, a significant portion of the Ca(2+) activity became dissociated from the sperm within 15-60 min after entry, and by 120 min post-ICSI or IVF, sperm were unable to induce oscillations. The initiation of oscillations coincided with exposure and solubilization of the perinuclear theca (PT), as evidenced by transmission electron microscopy, although disassembly of the PT was not required for commencement of the [Ca(2+)](i) responses. Remarkably, despite its complete release into the ooplasm, SF associated with nuclear structures at the time of pronuclear formation. Lastly, release of SF was not affected by the cell cycle. We conclude that mouse sperm serves as a carrier for SF, which is rapidly and completely solubilized to establish [Ca(2+)](i) oscillations.     

Elimination of the BK(Ca) channel's high-affinity Ca(2+) sensitivity

We report here a combination of site-directed mutations that eliminate the high-affinity Ca(2+) response of the large-conductance Ca(2+)-activated K(+) channel (BK(Ca)), leaving only a low-affinity response blocked by high concentrations of Mg(2+). Mutations at two sites are required, the "Ca(2+) bowl," which has been implicated previously in Ca(2+) binding, and M513, at the end of the channel's seventh hydrophobic segment. Energetic analyses of mutations at these positions, alone and in combination, argue that the BK(Ca) channel contains three types of Ca(2+) binding sites, one of low affinity that is Mg(2+) sensitive (as has been suggested previously) and two of higher affinity that have similar binding characteristics and contribute approximately equally to the power of Ca(2+) to influence channel opening. Estimates of the binding characteristics of the BK(Ca) channel's high-affinity Ca(2+)-binding sites are provided.