Donner F. Babcock

Dominant role of mitochondria in clearance of large Ca2+ loads from rat adrenal chromaffin cells.

Cytosolic Ca2+ (Ca2+c) clearance from adrenal chromaffin cells was studied by whole-cell patch clamp and indo-1 Ca2+ photometry after influx of Ca2+ through voltage-dependent Ca2+ channels. We isolated the rates of Ca2+c clearance by several mechanisms using combinations of the following agents (with their expected targets): Li+ or TEA substituted for Na+ (Na(+)-Ca2+ exchange), 1 mM La3+ applied after the depolarization (Na(+)-Ca2+ exchange and plasma membrane Ca(2+)-ATPase), 1 microM thapsigargin (pumping into reticular stores), and 2 microM carbonyl cyanide m-chlorophenylhydrazone (uptake into mitochondria). Remarkably, whenever [Ca2+]c rose above approximately 500 nM, Ca2+c clearance by mitochondria exceeded clearance by either Na(+)-Ca2+ exchange or the Ca2+ pumps of the plasma and reticular membranes. As [Ca2+]c fell again, Ca2+ reemerged from mitochondria, prolonging the final return to basal levels.

CatSper1 required for evoked Ca2+ entry and control of flagellar function in sperm

CatSper family proteins are putative ion channels expressed exclusively in membranes of the sperm flagellum and required for male fertility. Here, we show that mouse CatSper1 is essential for depolarization-evoked Ca2+ entry and for hyperactivated movement, a key flagellar function. CatSper1 is not needed for other developmental landmarks, including regional distributions of CaV1.2, CaV2.2, and CaV2.3 ion channel proteins, the cAMP-mediated activation of motility by \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{HCO}}_{3}^{-}\end{equation*}\end{document}, and the protein phosphorylation cascade of sperm capacitation. We propose that CatSper1 functions as a voltage-gated Ca2+ channel that controls Ca2+ entry to mediate the hyperactivated motility needed late in the preparation of sperm for fertilization.

Activation of voltage-dependent calcium channels of mammalian sperm is required for zona pellucida-induced acrosomal exocytosis

Previous work indicates that antagonists of the L-type voltage-dependent Ca2+ channel (VDCC) prevent the Ca(i) increase in mammalian sperm that is promoted by incubation in alkaline, K(+)-based media. Here, were provide additional evidence that sperm possess VDCC and show that their activation is required for the Ca2+ entry that mediates acrosomal exocytosis in both the presence and the absence of egg agonists. Specifically, we report that: (1) Sperm membrane potential changes, Ca(i) elevation, and acrosomal exocytosis have similar K+ dose dependencies, consistent with a characteristic requirement of a large depolarization for activation of the sperm VDCC; (2) High affinity binding sites (Kd approximately 0.35 +/- 0.03 and 0.45 +/- 0.06 nM; Bmax = 16.0 +/- 1.4 and 5.8 +/- 0.8 fmole/mg protein) for the VDCC antagonist, PN200-110, respectively, are present in membrane preparations from sperm of the ram and bull; (3) PN200-110 and the other VDCC antagonists nitrendipine, nisoldipine, verapamil, diltiazem, Ni2+, or Co2+ inhibit (IC50 = 0.1, 0.4, 0.6, 0.8, 1.0, 60, and 110 microM, respectively) the acrosomal exocytosis produced by combined elevation of pH0 and membrane depolarization; (4) Exocytosis induced by the ZP3 agonist of the mammalian egg also is inhibited by VDCC antagonists with similar dose dependencies; (5) Depolarizing treatments that presumably activate the sperm VDCC bypass the blockade of ZP3-induced exocytosis imposed by pertussis toxin. These results indicate that activation of the sperm VDCC is sufficient to induce sperm acrosomal exocytosis and that VDCC activation is necessary in the ZP3 signal transduction pathway. They also indicate that the presumed G-protein targets of pertussis toxin probably produce a required but indirect activation of the putative sperm VDCC. Possible intervening events include alteration of the voltage sensitivity of the VDCC, membrane depolarization, or both. We suggest that the depolarization-induced acrosome reaction may provide a useful system to investigate subsequent events in the exocytotic process.

An adhesion-associated agonist from the zona pellucida activates G protein-promoted elevations of internal Ca2+ and pH that mediate mammalian sperm acrosomal exocytosis☆

Solubilized oocyte zonae pellucidae promoted acrosomal exocytosis in fura-2- or carboxyfluorescein-loaded, mature bovine sperm. Associated elevations of internal [Ca2+] and pH in sperm suspensions were first detectable at 2-5 min, without apparent temporal resolution, and increased monotonically thereafter. Video imaging of fura-2-loaded, single cells identified a responsive subpopulation, destined to undergo exocytosis, that displayed no early transient but manifested lags of 1-7 min then sustained elevations of internal [Ca2+]. Both the zona-induced exocytosis and dye responses were diminished for functionally immature sperm and for mature sperm treated preliminarily with pertussis toxin. Together, these results indicate that a developmentally regulated mechanism of signal transduction employs G protein(s) to couple the physiological (zona) agonist to alterations of the internal ionic mediators of acrosomal exocytosis.

Mitochondrial oversight of cellular Ca2+ signaling

Mitochondria, the metabolic powerhouses of the cell, can sequester and release large amounts of Ca2+. This import and export of Ca2+ helps to adjust energy production to cellular needs. Recent advances show that mitochondrial Ca2+ fluxes play a major role in normal Ca2+ signaling.

Bicarbonate actions on flagellar and Ca2+ -channel responses: initial events in sperm activation.

At mating, mammalian sperm are diluted in the male and female reproductive fluids, which brings contact with HCO3- and initiates several cellular responses. We have identified and studied two of the most rapid of these responses. Stop-motion imaging and flagellar waveform analysis show that for mouse epididymal sperm in vitro, the resting flagellar beat frequency is 2-3 Hz at 22-25°C. Local perfusion with HCO3- produces a robust, reversible acceleration to 7 Hz or more. At 15 mM the action of HCO3- begins within 5 seconds and is near-maximal by 30 seconds. The half-times of response are 8.8±0.2 seconds at 15 mM HCO3- and 17.5±0.4 seconds at 1 mM HCO3-. Removal of external HCO3- allows a slow return to basal beat frequency over ∼10 minutes. Increases in beat symmetry accompany the accelerating action of HCO3-. As in our past work, HCO3- also facilitates opening of voltagegated Ca2+ channels, increasing the depolarization-evoked rate of rise of intracellular Ca2+ concentration by more than fivefold. This action also is detectable at 1 mM HCO3- and occurs with an apparent halftime of ∼60 seconds at 15 mM HCO3-. The dual actions of HCO3- respond similarly to pharmacological intervention. Thus, the phosphodiesterase inhibitor IBMX promotes the actions of HCO3- on flagellar and channel function, and the protein kinase A inhibitor H89 blocks these actions. In addition, a 30 minute incubation with 60 μM cAMP acetoxylmethyl ester increases flagellar beat frequency to nearly 7 Hz and increases the evoked rates of rise of intracellular Ca2+ concentration from 17±4 to 41±6 nM second-1. However, treatment with several other analogs of cAMP produces only scant evidence of the expected mimicry or blockade of the actions of HCO3-, perhaps as a consequence of limited permeation. Our findings indicate a requirement for cAMP-mediated protein phosphorylation in the enhancement of flagellar and channel functions that HCO3- produces during sperm activation.

Calcium Clearance Mechanisms of Mouse Sperm

The spermatozoon is specialized for a single vital role in fertilization. Past studies show that Ca2+ signals produced by the opening of plasma membrane entry channels initiate several events required for the sperm to reach and enter the egg but reveal little about how resting [Ca2+]i is maintained or restored after elevation. We examined these homeostatic mechanisms by monitoring the kinetics of recovery from depolarizing stimuli under conditions intended to inhibit candidate mechanisms for sequestration or extrusion of Ca2+ from the cytosol. We found that the Ca2+-ATPase pump of the plasma membrane performs the major task of Ca2+ clearance. It is essential in the final stages of recovery to achieve a low resting [Ca2+]i. With immunomethods we found a ∼130-kD plasma membrane Ca2+-ATPase protein on Western blots of whole sperm extracts and showed immunolocalization to the proximal principal piece of the flagellum. The plasma membrane Na+-Ca2+ exchanger also exports Ca2+ when [Ca2+]i is elevated. Simultaneous inhibition of both mechanisms of extrusion revealed an additional contribution to clearance from a CCCP-sensitive component, presumably sequestration by the mitochondria. Involvement of SERCA pumps was not clearly detected. Many aspects of the kinetics of Ca2+ clearance observed in the presence and absence of inhibitors were reproduced in a mathematical model based on known and assumed kinetic parameters. The model predicts that when cytosolic [Ca2+] is at 1 μM, the rates of removal by the Ca2+-ATPase, Na+-Ca2+-exchanger, mitochondrial uniporter, and SERCA pump are ∼1.0, 0.35, 0.33, and 0 μmole l−1 s−1, rates substantially slower than those reported for other cells studied by similar methods. According to the model, the Na+-Ca2+ exchanger is poised so that it may run in reverse at resting [Ca2+]i levels. We conclude that the essential functions of sperm do not require the ability to recover rapidly from globally elevated cytosolic [Ca2+].

Ca2+ clearance mechanisms in isolated rat adrenal chromaffin cells.

1. Intracellular Ca2+ clearance mechanisms were studied in rat adrenal chromaffin cells, by measuring slow tail currents through small‐conductance Ca(2+)‐activated K+ channels and using indo‐1 photometry following depolarization‐induced Ca2+ loading. 2. Following several‐hundred millisecond depolarizations, [Ca2+]i decayed in three phases. An initial fast decay was followed by a long‐lasting, low plateau, then [Ca2+]i returned to the resting level slowly. 3. Replacement of external Na+ moderately slowed [Ca2+]i decay, indicating a contribution of plasma membrane Na(+)‐Ca2+ exchange. 4. Raising external pH or application of extracellular Eosin of La3+ prolonged slow tail currents, indicating a contribution of plasma membrane Ca(2+)‐ATPase to Ca2+ clearance. 5. Ca(2+)‐induced Ca2+ release from caffeine‐sensitive stores occurred during depolarization. 6. Inhibition of endoplasmic reticulum Ca(2+)‐ATPase had little effect on Ca2+ clearance. 7. Slow tail currents and [Ca2+]i decay following 0.2 ‐ 2 s depolarizations were much prolonged by mitochondrial inhibition with carbonyl cyanide m‐chlorophenylhydrazone (CCCP) or Ruthenium Red, which abolished the initial rapid decay and plateau of [Ca2+]i. 8. In conclusion, mitochondrial Ca2+ uptake plays a major role in Ca2+ clearance by rapidly and reversibly sequestering Ca2+ during depolarization‐evoked Ca2+ loads.

Stimulation of exocytosis without a calcium signal

More than 30 years ago, Douglas ( Douglas & Rubin, 1961 ; Douglas, 1968 ) proposed that intracellular Ca2+ controls stimulus‐secretion coupling in endocrine cells, and Katz & Miledi ( 1967 ; Katz, 1969 ) proposed that intracellular Ca2+ ions control the rapid release of neurotransmitters from synapses. These related hypotheses have been amply confirmed in subsequent years and for students of excitable cells, they dominate our teaching and research. Calcium controls regulated exocytosis. On the other hand, many studies of epithelial and blood cell biology emphasize Ca2+‐independent regulation of secretion of mucin, exocytotic delivery of transporters and degranulation. The evidence seems good. Are these contrasting conclusions somehow mistaken, or are the dominant factors controlling exocytosis actually different in different cell types? In this essay, we try to reconcile these ideas and consider classes of questions to ask and hypotheses to test in seeking a more integrated understanding of excitation‐secretion coupling. Our review is conceptual and narrowly selective of a few examples rather than referring to a broader range of useful studies in the extensive literature. The examples are taken from mammals and are documented principally by citing other reviews and two of our own studies. The evidence shows that protein phosphorylation by kinases potentiates Ca2+‐dependent exocytosis and often suffices to induce exocytosis by itself. Apparently, protein phosphorylation is the physiological trigger in a significant number of examples of regulated exocytosis. We conclude that although sharing many common properties, secretory processes in different cells are specialized and distinct from each other.

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.