articlesThe building block of synaptic transmission is the quantum, the minimal increment of postsynaptic signals 1 . At vertebrate neuromuscular junctions, the quantum may be equated with spontaneous signals obtained in the absence of presynaptic action potentials, called miniature currents (or potentials) and believed to be due to release of one neurotransmitter vesicle. For central synapses, this issue remains an open question, as large miniature currents are suggested to arise from the concerted release of several presynaptic vesicles and to be the sum of several quanta 2-5 . Such multivesicular miniature events could reflect tetrodotoxinresistant action potentials in presynaptic terminals 6 . Another explanation comes from evidence for functional intracellular Ca 2+ stores in presynaptic terminals. First, inositoltrisphosphate (InsP 3 ) receptors are immunolocalized in presynaptic terminals of the deep cerebellar nuclei and retina 7,8 . Second, at the frog neuromuscular junction, agents that affect ryanodine-sensitive Ca 2+ stores also regulate presynaptic intracellular Ca 2+ (Ca 2+ i ) rises and acetylcholine release during high-frequency stimulation 9,10 . Third, action-potential-evoked release of acetylcholine at synapses in Aplysia buccal ganglia is inhibited by ryanodine and augmented by presynaptic injection of cyclic ADP ribose 11 . Fourth, caffeine and/or ryanodine modify presynaptic Ca 2+ i signals in autonomic ganglia 12,13 and in photoreceptors 14 . Finally, in hippocampal pyramidal cells, caffeine or thapsigargin can increase the frequency of miniature IPSCs 15 . Hence, spontaneous Ca 2+ release from presynaptic Ca 2+ stores may provide the synchronization mechanism that leads to multivesicular miniatures. However, except for one study that gave negative results in cultured retinal amacrine cells 16 , this possibility has not been tested systematically.To assess the contribution of intracellular Ca 2+ stores to neurotransmitter release, we monitored the amplitude distribution of miniature synaptic currents while manipulating potential presynaptic Ca 2+ stores. Using cerebellar interneuron-Purkinje cell synapses, in which large miniature synaptic currents are prominent, we found that the largest mIPSCs result from multivesicular release and depend on Ca 2+ mobilization from ryanodine-sensitive presynaptic stores. Further, two-photon confocal microscopy showed ryanodine-sensitive intracellular Ca 2+ i transients highly localized to presumed release sites, which may underlie large miniature currents. RESULTSMiniature IPSCs recorded in cerebellar Purkinje cells, at -60 mV under symmetrical Cl -concentrations and in the presence of tetrodotoxin (TTX) and ionotropic glutamate receptor blockers, showed mean amplitudes of 125 ± 9 pA (n = 28 cells), larger than for most neurons (Fig. 1a). Amplitude histograms had a distinct peak for values less than 200 pA, followed by a long tail with amplitudes up to 1500 pA (Fig. 1b). Spurious summation of independent events did not contribute to the generation of lar...

Abstract-Hyperphosphorylation of the cardiac Ca 2ϩ release channel (ryanodine receptor, RyR2) by protein kinase A (PKA) at serine-2808 has been proposed to be a key mechanism responsible for cardiac dysfunction in heart failure (HF). However, the sites of PKA phosphorylation in RyR2 and their phosphorylation status in HF are not well defined. Here we used various approaches to investigate the phosphorylation of RyR2 by PKA. Mutating serine-2808, which was thought to be the only PKA phosphorylation site in RyR2, did not abolish the phosphorylation of RyR2 by PKA. Two-dimensional phosphopeptide mapping revealed two major PKA phosphopeptides, one of which corresponded to the known serine-2808 site. Another, novel, PKA phosphorylation site, serine 2030, was identified by Edman sequencing. Using phospho-specific antibodies, we showed that the novel serine-2030 site was phosphorylated in rat cardiac myocytes stimulated with isoproterenol, but not in unstimulated cells, whereas serine-2808 was considerably phosphorylated before and after isoproterenol treatment. We further showed that serine-2030 was stoichiometrically phosphorylated by PKA, but not by CaMKII, and that mutations of serine-2030 altered neither the FKBP12.6-RyR2 interaction nor the Ca 2ϩ dependence of [ 3 H]ryanodine binding. Moreover, the levels of phosphorylation of RyR2 at serine-2030 and serine-2808 in both failing and non-failing canine hearts were similar. Together, our data indicate that serine-2030 is a major PKA phosphorylation site in RyR2 responding to acute ␤-adrenergic stimulation, and that

Abstract-Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited disease characterized by life threatening arrhythmias and mutations in the gene encoding the ryanodine receptor (RyR2). Disagreement exists on whether (1) RyR2 mutations induce abnormal calcium transients in the absence of adrenergic stimulation; (2) decreased affinity of mutant RyR2 for FKBP12.6 causes CPVT; (3) K201 prevent arrhythmias by normalizing the FKBP12.6-RyR2 binding. We studied ventricular myocytes isolated from wild-type (WT) and knock-in mice harboring the R4496C mutation (RyR2 R4496Cϩ/Ϫ ). Pacing protocols did not elicit delayed afterdepolarizations (DADs) (nϭ20) in WT but induced DADs in 21 of 33 (63%) RyR2R4496Cϩ/Ϫ myocytes (Pϭ0.001). Superfusion with isoproterenol (30 nmol/L) induced small DADs (45%) and no triggered activity in WT myocytes, whereas it elicited DADs in 87% and triggered activity in 60% of RyR2R4496Cϩ/Ϫ myocytes (Pϭ0.001). DADs and triggered activity were abolished by ryanodine (10 mol/L) but not by K201 (1 mol/L or 10 mol/L). In vivo administration of K201 failed to prevent induction of polymorphic ventricular tachycardia (VT) in RyR2R4496Cϩ/Ϫ mice. Measurement of the FKBP12.6/RyR2 ratio in the heavy sarcoplasmic reticulum membrane showed normal RyR2-FKBP12.6 interaction both in WT and RyR2R4496Cϩ/Ϫ either before and after treatment with caffeine and epinephrine. We suggest that (1) triggered activity is the likely arrhythmogenic mechanism of CPVT; (2) K201 fails to prevent DADs in RyR2R4496Cϩ/Ϫ myocytes and ventricular arrhythmias in RyR2R4496Cϩ/Ϫ mice; and (3) RyR2-FKBP12.6 interaction in RyR2 R4496Cϩ/Ϫ is identical to that of WT both before and after epinephrine and caffeine, thus suggesting that it is unlikely that the R4496C mutation interferes with the RyR2/FKBP12.6 complex. Key Words: cardiac electrophysiology Ⅲ ryanodine receptor Ⅲ sudden death Ⅲ transgenic mice Ⅲ ventricular tachycardia C atecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmogenic disease characterized by adrenergically mediated bidirectional or polymorphic ventricular tachycardia leading to syncope and/or sudden cardiac death in individuals without structural heart disease. 1,2 In 2001, we reported that the autosomal dominant form of CPVT is caused by mutations in the ryanodine receptor gene (RyR2). 3 Based on the evidence that the morphology of ventricular tachycardia observed in CPVT resembles that of digitalis induced ventricular tachycardia (VT), it had been suggested that arrhythmogenesis in CPVT could be mediated by delayed afterdepolarizations (DADs) and triggered activity. Although the discovery that CPVT is caused by mutations in the ryanodine receptor has substantiated this hypothesis, up to now no conclusive demonstration that DADs cause CPVT is available.Furthermore, although several authors have characterized in vitro the functional consequences of RyR2 mutations, 4 -6 the molecular and electrophysiological derangements leading to arrhythmias in CPVT patients are still uncl...

ResearchFusion with a fertilizing spermatozoon induces the mammalian oocyte to undergo a remarkable series of oscillations in cytosolic Ca 2+ , leading to oocyte activation and development of the embryo. The exact molecular mechanism for generating Ca 2+ oscillations has not been established. A sperm-specific zeta isoform of phospholipase C (PLCζ) has been identified in mice. Mouse PLCζ triggers Ca 2+ oscillations in mouse oocytes and exhibits properties synonymous with the 'sperm factor' that has been proposed to diffuse into the oocyte after gamete fusion. The present study isolated the PLCζ homologue from human and cynomolgus monkey testes. Comparison with mouse and monkey PLCζ protein sequences indicates a shorter X-Y linker region in human PLCζ and predicts a distinctly different isoelectric point. Microinjection of complementary RNA for both human and cynomolgus monkey PLCζ elicits Ca 2+ oscillations in mouse oocytes equivalent to those seen during fertilization in mice. Moreover, human PLCζ elicits mouse egg activation and early embryonic development up to the blastocyst stage, and exhibits greater potency than PLCζ from monkeys and mice. These results are consistent with the proposal that sperm PLCζ is the molecular trigger for egg activation during fertilization and that the role and activity of PLCζ is highly conserved across mammalian species.

Reproduction 124, 611-623). PLC may represent the physiological stimulus for egg activation and development at mammalian fertilization. PLC is the smallest known mammalian PLC isozyme, comprising two EF hand domains, a C2 domain, and the catalytic X and Y core domains. To gain insight into PLC structure-function, we assessed the ability of PLC and a series of domain-deletion constructs to cause phosphatidylinositol 4,5-bisphosphate hydrolysis in vitro and also to generate cytoplasmic Ca 2؉ changes in intact mouse eggs. PLC and the closely related PLC␦1 had similar K m values for phosphatidylinositol 4,5-bisphosphate, but PLC was around 100 times more sensitive to Ca 2؉ than was PLC␦1. Notably, specific phosphatidylinositol 4,5-bisphosphate hydrolysis activity was retained in PLC constructs that had either EF hand domains or the C2 domain removed, or both. In contrast, Ca 2؉ sensitivity was greatly reduced when either one, or both, of the EF hand domains were absent, and the Hill coefficient was reduced upon deletion of the C2 domain. Microinjection into intact mouse eggs revealed that all domain-deletion constructs were ineffective at initiating Ca 2؉ oscillations. These data suggest that the exquisite Ca 2؉ -dependent features of PLC regulation are essential for it to generate inositol 1,4,5-trisphosphate and Ca 2؉ oscillations in intact mouse eggs.

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At fertilization in mammals, the sperm induces a characteristic series of Ca2+ oscillations in the egg which serve as the essential trigger for egg activation and early development of the embryo. It is not known how the sperm initiates this fundamental process, however, nor has any pathway linking sperm-egg membrane-receptor binding with intracellular Ca2+ release been demonstrated. Microinjection of sperm extracts into mammalian eggs elicits Ca2+ oscillations identical to those occurring at fertilization, which suggests that sperm may introduce a Ca2+ oscillation-inducing factor into the egg on gamete membrane fusion. Here we identify a soluble sperm protein that exhibits Ca2+ oscillation-inducing ('oscillogen') activity in eggs. Sperm oscillogen exists as an oligomer with a subunit of M(r) 33K and a specific intracellular localization at the equatorial segment of the sperm head. Cloning of the 33K oscillogen complementary DNA indicates similarity with a hexose phosphate isomerase found in prokaryotes. This sperm-derived oscillogen, termed oscillin, may represent the physiological trigger for development in mammals.

We have demonstrated recently (Mitchell, K. J., Pinton, P., Varadi, A., Tacchetti, C., Ainscow, E. K., Pozzan, T., Rizzuto, R., and Rutter, G. A. (2001