Time course and calcium dependence of transmitter release at a single ribbon synapse

At the first synapse in the auditory pathway, the receptor potential of mechanosensory hair cells is converted into a firing pattern in auditory nerve fibers. For the accurate coding of timing and intensity of sound signals, transmitter release at this synapse must occur with the highest precision. To measure directly the transfer characteristics of the hair cell afferent synapse, we implemented simultaneous whole-cell recordings from mammalian inner hair cells (IHCs) and auditory nerve fiber terminals that typically receive input from a single ribbon synapse. During a 1-s IHC depolarization, the synaptic response depressed >90%, representing the main source for adaptation in the auditory nerve. Synaptic depression was slightly affected by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor desensitization; however, it was mostly caused by reduced vesicular release. When the transfer function between transmitter release and Ca(2+) influx was tested at constant open probability for Ca(2+) channels (potentials >0 mV), a super linear relation was found. This relation is presumed to result from the cooperative binding of three to four Ca(2+) ions at the Ca(2+) sensor. However, in the physiological range for receptor potentials (-50 to -30 mV), the relation between Ca(2+) influx and afferent activity was linear, assuring minimal distortion in the coding of sound intensity. Changes in Ca(2+) influx caused an increase in release probability, but not in the average size of multivesicular synaptic events. By varying Ca(2+) buffering in the IHC, we further investigate how Ca(2+) channel and Ca(2+) sensor at this synapse might relate.     

Podosomes are present in a postsynaptic apparatus and participate in its maturation

A critical step in synapse formation is the clustering of neurotransmitter receptors in the postsynaptic membrane, directly opposite the nerve terminal. At the neuromuscular junction, a widely studied model synapse, acetylcholine receptors (AChRs) initially aggregate to form an ovoid postsynaptic plaque. As the synapse matures, the plaque becomes perforated and is eventually transformed into a complex, branched structure. We found that this transformation also occurs in myotubes cultured in the absence of neurons, and used this system to seek machinery that orchestrates postsynaptic maturation. We show that perforations in the AChR aggregate bear structures resembling podosomes, dynamic actin-rich adhesive organelles involved in matrix remodeling in non-neuronal cells but not described in neural structures. The location and dynamics of synaptic podosomes are spatiotemporally correlated with changes in AChR aggregate topology, and pharmacological disruption of podosomes leads to rapid alterations in AChR organization. Our results indicate that synaptic podosomes play critical roles in maturation of the postsynaptic membrane.     

Asynchronous Ca2+ current conducted by voltage-gated Ca2+ (CaV)-2.1 and CaV2.2 channels and its implications for asynchronous neurotransmitter release

We have identified an asynchronously activated Ca(2+) current through voltage-gated Ca(2+) (Ca(V))-2.1 and Ca(V)2.2 channels, which conduct P/Q- and N-type Ca(2+) currents that initiate neurotransmitter release. In nonneuronal cells expressing Ca(V)2.1 or Ca(V)2.2 channels and in hippocampal neurons, prolonged Ca(2+) entry activates a Ca(2+) current, I(Async), which is observed on repolarization and decays slowly with a half-time of 150-300 ms. I(Async) is not observed after L-type Ca(2+) currents of similar size conducted by Ca(V)1.2 channels. I(Async) is Ca(2+)-selective, and it is unaffected by changes in Na(+), K(+), Cl(-), or H(+) or by inhibitors of a broad range of ion channels. During trains of repetitive depolarizations, I(Async) increases in a pulse-wise manner, providing Ca(2+) entry that persists between depolarizations. In single-cultured hippocampal neurons, trains of depolarizations evoke excitatory postsynaptic currents that show facilitation followed by depression accompanied by asynchronous postsynaptic currents that increase steadily during the train in parallel with I(Async). I(Async) is much larger for slowly inactivating Ca(V)2.1 channels containing β(2a)-subunits than for rapidly inactivating channels containing β(1b)-subunits. I(Async) requires global rises in intracellular Ca(2+), because it is blocked when Ca(2+) is chelated by 10 mM EGTA in the patch pipette. Neither mutations that prevent Ca(2+) binding to calmodulin nor mutations that prevent calmodulin regulation of Ca(V)2.1 block I(Async). The rise of I(Async) during trains of stimuli, its decay after repolarization, its dependence on global increases of Ca(2+), and its enhancement by β(2a)-subunits all resemble asynchronous release, suggesting that I(Async) is a Ca(2+) source for asynchronous neurotransmission.     

Reduced release probability prevents vesicle depletion and transmission failure at dynamin mutant synapses

Endocytic recycling of synaptic vesicles after exocytosis is critical for nervous system function. At synapses of cultured neurons that lack the two "neuronal" dynamins, dynamin 1 and 3, smaller excitatory postsynaptic currents are observed due to an impairment of the fission reaction of endocytosis that results in an accumulation of arrested clathrin-coated pits and a greatly reduced synaptic vesicle number. Surprisingly, despite a smaller readily releasable vesicle pool and fewer docked vesicles, a strong facilitation, which correlated with lower vesicle release probability, was observed upon action potential stimulation at such synapses. Furthermore, although network activity in mutant cultures was lower, Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) activity was unexpectedly increased, consistent with the previous report of an enhanced state of synapsin 1 phosphorylation at CaMKII-dependent sites in such neurons. These changes were partially reversed by overnight silencing of synaptic activity with tetrodotoxin, a treatment that allows progression of arrested endocytic pits to synaptic vesicles. Facilitation was also counteracted by CaMKII inhibition. These findings reveal a mechanism aimed at preventing synaptic transmission failure due to vesicle depletion when recycling vesicle traffic is backed up by a defect in dynamin-dependent endocytosis and provide new insight into the coupling between endocytosis and exocytosis.     

Rab3B protein is required for long-term depression of hippocampal inhibitory synapses and for normal reversal learning

Rab3B, similar to other Rab3 isoforms, is a synaptic vesicle protein that interacts with the Rab3-interacting molecule (RIM) isoforms RIM1α and RIM2α as effector proteins in a GTP-dependent manner. Previous studies showed that at excitatory synapses, Rab3A and RIM1α are essential for presynaptically expressed long-term potentiation (LTP), whereas at inhibitory synapses RIM1α is required for endocannabinoid-dependent long-term depression (referred to as "i-LTD"). However, it remained unknown whether i-LTD also involves a Rab3 isoform and whether i-LTD, similar to other forms of long-term plasticity, is important for learning and memory. Here we show that Rab3B is highly enriched in inhibitory synapses in the CA1 region of the hippocampus. Using electrophysiological recordings in acute slices, we demonstrate that knockout (KO) of Rab3B does not alter the strength or short-term plasticity of excitatory or inhibitory synapses but does impair i-LTD significantly without changing classical NMDA receptor-dependent LTP. Behaviorally, we found that Rab3B KO mice exhibit no detectable changes in all basic parameters tested, including the initial phase of learning and memory. However, Rab3B KO mice did display a selective enhancement in reversal learning, as measured using Morris water-maze and fear-conditioning assays. Our data support the notion that presynaptic forms of long-term plasticity at excitatory and inhibitory synapses generally are mediated by a common Rab3/RIM-dependent pathway, with various types of synapses using distinct Rab3 isoforms. Moreover, our results suggest that i-LTD contributes to learning and memory, presumably by stabilizing circuits established in previous learning processes.     

Opioid agonists inhibit excitatory neurotransmission in ganglia and at the neuromuscular junction in Guinea pig gallbladder

BACKGROUND & AIMS: Opiates administered therapeutically could have an inhibitory effect on the neuromuscular axis of the gallbladder, and thus contribute to biliary stasis and acalculous cholecystitis. METHODS: Intracellular recordings were made from gallbladder neurons and smooth muscle, and tension measurements were made from muscle strips. Opioid receptor-specific agonists tested: delta, DPDPE; kappa, U-50488H; and mu, DAMGO. RESULTS: Opioid agonists had no effect on gallbladder neurons or smooth muscle. Each of the opioid agonists potently suppressed the fast excitatory synaptic input to gallbladder neurons, in a concentration-dependent manner with half-maximal effective concentration values of about 1 pmol/L. Also, each agonist caused a concentration-dependent reduction in the amplitude of the neurogenic contractile response (half-maximal effective concentration values: DPDPE, 189 pmol/L; U-50488H, 472 pmol/L; and DAMGO, 112 pmol/L). These ganglionic and neuromuscular effects were attenuated by the highly selective opioid-receptor antagonist, naloxone. Opioid-receptor activation also inhibited the presynaptic facilitory effect of cholecystokinin in gallbladder ganglia. Immunohistochemistry with opioid receptor-specific antisera revealed immunostaining for all 3 receptor subtypes in nerve bundles and neuronal cell bodies within the gallbladder, whereas opiate-immunoreactive nerve fibers are sparse in the gallbladder. CONCLUSIONS: These results show that opiates can cause presynaptic inhibition of excitatory neurotransmission at 2 sites within the wall of the gallbladder: vagal preganglionic terminals in ganglia and neuromuscular nerve terminals. These findings support the concept that opiates can contribute to gallbladder stasis by inhibiting ganglionic activity and neurogenic contractions.     

Distinct kinetics of synaptic structural plasticity,memory formation,and memory decay in massed and spaced learning

Long-lasting memories are formed when the stimulus is temporally distributed (spacing effect). However, the synaptic mechanisms underlying this robust phenomenon and the precise time course of the synaptic modifications that occur during learning remain unclear. Here we examined the adaptation of horizontal optokinetic response in mice that underwent 1 h of massed and spaced training at varying intervals. Despite similar acquisition by all training protocols, 1 h of spacing produced the highest memory retention at 24 h, which lasted for 1 mo. The distinct kinetics of memory are strongly correlated with the reduction of floccular parallel fiber–Purkinje cell synapses but not with AMPA receptor (AMPAR) number and synapse size. After the spaced training, we observed 25%, 23%, and 12% reduction in AMPAR density, synapse size, and synapse number, respectively. Four hours after the spaced training, half of the synapses and Purkinje cell spines had been eliminated, whereas AMPAR density and synapse size were recovered in remaining synapses. Surprisingly, massed training also produced long-term memory and halving of synapses; however, this occurred slowly over days, and the memory lasted for only 1 wk. This distinct kinetics of structural plasticity may serve as a basis for unique temporal profiles in the formation and decay of memory with or without intervals.During learning, memories are formed in a specific population of neuronal circuits and are consolidated for persistence (1, 2). These memory processes are supported by discrete subcellular events such as reversible modifications in the efficacy of synaptic transmission (3–5) or persistent structural modifications in the size and number of synaptic connections (6–8). However, how these synaptic modifications relate to the dynamics of formation and decay of memories in behaving animals remains elusive. Memory formation and its persistence are also sensitive to the temporal features of stimulus presentation, as observed in the well-known “spacing effect.” Training trials that include resting intervals between them (spaced training) produce stronger and longer-lasting memories than do the same number of trials with no intervals (massed training) (9). The spacing effect has been observed in a variety of explicit and implicit memory tasks (10–13), and the molecular mechanisms supporting this phenomenon have been reported (14–18). Various intracellular signaling molecules such as CREB (19), mitogen-activated protein (MAP) kinase (20, 21), and PKA (22, 23) underlie the spacing effect and are implicated in the remodeling of neuronal structures (23). In vitro studies showed that spaced stimuli induced the protrusion of new filopodia (20) and the recruitment of new synapses (24) in hippocampal neurons. However, despite the existence of numerous behavioral and molecular studies, no conjoint study has elucidated the synaptic correlates that underpin the expression of the spacing effect during learning. Here we studied the temporal evolution and decay of memory and its correlation with synaptic modifications during learning with distinct temporal patterns of training.We used an adaptation of the horizontal optokinetic response (HOKR), which is a simple model of cerebellum-dependent motor learning. It is a compensatory eye movement for stabilization of the visual image on the retina during horizontal motion of the surroundings. A surrounding that oscillates horizontally at a given frequency causes retinal slips in naive animals and facilitates HOKR 1 h after training (HOKR adaptation) (25–27). The amount of adaptation can be quantitatively monitored, and the flocculus (Fl), which is a phylogenetically preserved cerebellar lobule, is involved in the adaptation of the HOKR (28, 29). These features render this paradigm as an experimental model, useful for investigating neural correlates and mechanisms involved in motor learning. In a previous study, we showed that the short-term adaptation of HOKR induced by 1-h training was accompanied by a rapid and transient reduction (28%) in the number of AMPA receptors (AMPARs) in parallel fiber (PF) to Purkinje cell (PC) synapses, whereas the long-term adaptation induced by repeated 1-h training over 5 d was accompanied by a slowly developing reduction (45%) of PF–PC synapses (30). Despite recent controversies on the role of long-term depression (LTD) and a postulated role of long-term potentiation in cerebellar motor learning (31–33), this study first showed that LTD as a form of reduced AMPARs in PF–PC synapses does occur in physiological learning.In the present study, we further examined how the spacing effect is correlated with the structural plasticity in PF–PC synapses. We showed that spaced training including 1-h intervals induced stable long-lasting memories within 4 h after the training, which was accompanied by a rapid and long-lasting (>1 mo) reduction of PF–PC synapses after a transient reduction in AMPAR density and shrinkage of PF–PC synapses and PC spines. One hour of massed training also induced a gradual reduction of the PF–PC synapses, which reached the same level as that observed for the spaced training 5 d later but recovered faster within 10 d. The time course corresponded well with the slower establishment and quicker decay of long-lasting memory induced by massed training. The tight correlation observed between the structural modifications and the kinetics of long-lasting memory pinpoints the distinct temporal regulation of synaptic connections as a mechanism underlying the spacing effect.     

Distinct roles of isoforms of the heme-liganded nuclear receptor E75, an insect ortholog of the vertebrate Rev-erb, in mosquito reproduction

Mosquitoes are adapted to using vertebrate blood as a nutrient source to promote egg development and as a consequence serve as disease vectors. Blood-meal activated reproductive events in female mosquitoes are hormonally and nutritionally controlled with an insect steroid hormone 20-hydroxyecdysone (20E) playing a central role. The nuclear receptor E75 is an essential factor in the 20E genetic hierarchy, however functions of its three isoforms - E75A, E75B, and E75C - in mosquito reproduction are unclear. By means of specific RNA interference depletion of E75 isoforms, we identified their distinct roles in regulating the level and timing of expression of key genes involved in vitellogenesis in the fat body (an insect analog of vertebrate liver and adipose tissue) of the mosquito Aedes aegypti. Heme is required in a high level of expression of 20E-controlled genes in the fat body, and this heme action depends on E75. Thus, in mosquitoes, heme is an important signaling molecule, serving as a sensor of the availability of a protein meal for egg development. Disruption of this signaling pathway could be explored in the design of mosquito control approaches.     

Pathogenic immune mechanisms at the neuromuscular synapse: the role of specific antibody‐binding epitopes in myasthenia gravis

Autoantibodies against three different postsynaptic antigens and one presynaptic antigen at the neuromuscular junction are known to cause myasthenic syndromes. The mechanisms by which these antibodies cause muscle weakness vary from antigenic modulation and complement‐mediated membrane damage to inhibition of endogenous ligand binding and blocking of essential protein–protein interactions. These mechanisms are related to the autoantibody titre, specific epitopes on the target proteins and IgG autoantibody subclass. We here review the role of specific autoantibody‐binding epitopes in myasthenia gravis, their possible relevance to the pathophysiology of the disease and potential implications of epitope mapping knowledge for new therapeutic strategies.     

Spontaneous transmitter release recruits postsynaptic mechanisms of long-term and intermediate-term facilitation in Aplysia

Whereas short-term (minutes) facilitation at Aplysia sensory–motor neuron synapses is presynaptic, long-term (days) facilitation involves synaptic growth, which requires both presynaptic and postsynaptic mechanisms. How are the postsynaptic mechanisms recruited, and when does that process begin? We have been investigating the possible role of spontaneous transmitter release from the presynaptic neuron. In the previous paper, we found that spontaneous release is critical for the induction of long-term facilitation, and this process begins during an intermediate-term stage of facilitation that is the first stage to involve postsynaptic as well as presynaptic mechanisms. We now report that increased spontaneous release during the short-term stage acts as an orthograde signal to recruit postsynaptic mechanisms of intermediate-term facilitation including increased IP3, Ca²⁺, and membrane insertion and recruitment of clusters of AMPA-like receptors, which may be first steps in synaptic growth during long-term facilitation. These results suggest that the different stages of facilitation involve a cascade of pre- and postsynaptic mechanisms, which is initiated by spontaneous release and may culminate in synaptic growth.     

Distinct roles of dopamine D2L and D2S receptor isoforms in the regulation of protein phosphorylation at presynaptic and postsynaptic sites

Dopamine D2 receptors are highly expressed in the dorsal striatum where they participate in the regulation of (i) tyrosine hydroxylase (TH), in nigrostriatal nerve terminals, and (ii) the dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32), in medium spiny neurons. Two isoforms of the D2 receptor are generated by differential splicing of the same gene and are referred to as short (D2S) and long (D2L) dopamine receptors. Here we have used wild-type mice, dopamine D2 receptor knockout mice (D2 KO mice; lacking both D2S and D2L receptors) and D2L receptor-selective knockout mice (D2L KO mice) to evaluate the involvement of each isoform in the regulation of the phosphorylation of TH and DARPP-32. Incubation of striatal slices from wild-type mice with quinpirole, a dopamine D2 receptor agonist, decreased the state of phosphorylation of TH at Ser-40 and its enzymatic activity. Both effects were abolished in D2 KO mice but were still present in D2L KO mice. In wild-type mice, quinpirole inhibits the increase in DARPP-32 phosphorylation at Thr-34 induced by SKF81297, a dopamine D1 receptor agonist. This effect is absent in D2 KO as well as D2L KO mice. The inability of quinpirole to regulate DARPP-32 phosphorylation in D2L KO mice cannot be attributed to decreased coupling of D2S receptors to G proteins, because quinpirole produces a similar stimulation of [(35)S]GTPgammaS binding in wild-type and D2L KO mice. These results demonstrate that D2S and D2L receptors participate in presynaptic and postsynaptic dopaminergic transmission, respectively.     

Direct and indirect roles for Nodal signaling in two axis conversions during asymmetric morphogenesis of the zebrafish heart

The Nodal signaling pathway plays a conserved role in determining left-sided identity in vertebrates with this early left-right (L/R) patterning influencing the asymmetric development and placement of visceral organs. We have studied the role of Nodal signaling in asymmetric cardiac morphogenesis in zebrafish and describe two distinct rotations occurring within the heart. The first is driven by an asymmetric migration of myocardial cells during cardiac jogging, resulting in the conversion of the L/R axis to the dorsal-ventral (D/V) axis of the linear heart. This first rotation is directly influenced by the laterality of asymmetric gene expression. The second rotation occurs before cardiac looping and positions the original left cells exposed to Nodal signaling back to the left of the wild-type (WT) heart by 48 hours postfertilization (hpf). The direction of this second rotation is determined by the laterality of cardiac jogging and is not directly influenced by asymmetric gene expression. Finally, we have identified a role for Nodal signaling in biasing the location of the inner ventricular and outer atrial curvature formations. These results suggest that Nodal signaling directs asymmetric cardiac morphogenesis through establishing and subsequently reinforcing laterality information over the course of cardiac development.     

Complexins facilitate neurotransmitter release at excitatory and inhibitory synapses in mammalian central nervous system

Complexins (Cplxs) are key regulators of synaptic exocytosis, but whether they act as facilitators or inhibitors is currently being disputed controversially. We show that genetic deletion of all Cplxs expressed in the mouse brain causes a reduction in Ca(2+)-triggered and spontaneous neurotransmitter release at both excitatory and inhibitory synapses. Our results demonstrate that at mammalian central nervous system synapses, Cplxs facilitate neurotransmitter release and do not simply act as inhibitory clamps of the synaptic vesicle fusion machinery.     

Expression and functional analysis of two isoforms of the human GM-CSF receptor α chain in myeloid development and leukaemia

The human granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR) α chain RNA is alternatively spliced to yield receptor isoforms. Two of these, α₁ and α₂, differ in their cytoplasmic domains. Because the GM-CSFR β chain (β_c) is shared with the receptors for interleukins 3 and 5 it is possible that the α chain confers specificity on the GM-CSF response and that the different isoforms might refine this response further. Studies have been directed at determination of the respective biological roles of the α₁ and α₂ isoforms. Expression of the isoforms was examined by RNase protection analysis in normal granulocytes and a variety of cell lines of haemopoietic origin, at different stages of differentiation and activation. Expression was also analysed in cells from patients with a variety of leukaemic subtypes. Results demonstrated that the relative abundance of the isoforms was similar in all cell populations examined. The human GM-CSFR α₁ or α₂ receptors were independently expressed in the murine factor-dependent cell line FDC-P1, so that the properties of the receptors could be compared. Cell lines that expressed either receptor could be converted to growth in response to human GM-CSF and assumed a more differentiated phenotype when compared with the parental cell line. However, the morphology, expression of cell surface antigens and dose–growth response characteristics did not differ significantly between cells that expressed either the α₁ or α₂ receptor. These studies demonstrate that the α₁ and α₂ subunits of the GM-CSF receptor are co-ordinately regulated in both normal and malignant haemopoiesis. Furthermore, each receptor is able to deliver both proliferative and differentiative signals to myeloid cells.     

Spontaneous transmitter release is critical for the induction of long-term and intermediate-term facilitation in Aplysia

Long-term plasticity can differ from short-term in recruiting the growth of new synaptic connections, a process that requires the participation of both the presynaptic and postsynaptic components of the synapse. How does information about synaptic plasticity spread from its site of origin to recruit the other component? The answer to this question is not known in most systems. We have investigated the possible role of spontaneous transmitter release as such a transsynaptic signal. Until recently, relatively little has been known about the functions of spontaneous release. In this paper, we report that spontaneous release is critical for the induction of a learning-related form of synaptic plasticity, long-term facilitation in Aplysia. In addition, we have found that this signaling is engaged quite early, during an intermediate-term stage that is the first stage to involve postsynaptic as well as presynaptic molecular mechanisms. In a companion paper, we show that spontaneous release from the presynaptic neuron acts as an orthograde signal to recruit the postsynaptic mechanisms of intermediate-term facilitation and initiates a cascade that can culminate in synaptic growth with additional stimulation during long-term facilitation. Spontaneous release could make a similar contribution to learning-related synaptic plasticity in mammals.     

Distinct temporal-spatial roles for rho kinase and myosin light chain kinase in epithelial purse-string wound closure

BACKGROUND & AIMS: Small epithelial wounds heal by purse-string contraction of an actomyosin ring that is regulated by myosin light chain (MLC) kinase (MLCK) and rho kinase (ROCK). These studies aimed to define the roles of these kinases in purse-string wound closure. METHODS: Oligocellular and single-cell wounds were created in intestinal epithelial monolayers. Fluorescence imaging and electrophysiologic data were collected during wound closure. Human biopsies were studied immunohistochemically. RESULTS: Live-cell imaging of enhanced green fluorescent protein-beta-actin defined rapid actin ring assembly within 2 minutes after wounding. This progressed to a circumferential ring within 8 minutes that subsequently contracted and closed the wound. We therefore divided this process into 2 phases: ring assembly and wound contraction. Activated rho and ROCK localized to the wound edge during ring assembly. Consistent with a primary role in the assembly phase, ROCK inhibition prevented actin ring assembly and wound closure. ROCK inhibition after ring assembly was complete had no effect. Recruitment and activation of MLCK occurred after ring assembly was complete and coincided with ring contraction. MLCK inhibition slowed and then stopped contraction but did not prevent ring assembly. MLCK inhibition also delayed barrier function recovery. Studies of human colonic biopsy specimens suggest that purse-string wound closure also occurs in vivo, because MLC phosphorylation was enhanced surrounding oligocellular wounds. CONCLUSIONS: These results suggest complementary roles for these kinases in purse-string closure of experimental and in vivo oligocellular epithelial wounds; rho and ROCK are critical for actin ring assembly, while the activity of MLCK drives contraction.     

Selenoprotein N is required for ryanodine receptor calcium release channel activity in human and zebrafish muscle

Mutations affecting the seemingly unrelated gene products, SepN1, a selenoprotein of unknown function, and RyR1, the major component of the ryanodine receptor intracellular calcium release channel, result in an overlapping spectrum of congenital myopathies. To identify the immediate developmental and molecular roles of SepN and RyR in vivo, loss-of-function effects were analyzed in the zebrafish embryo. These studies demonstrate the two proteins are required for the same cellular differentiation events and are needed for normal calcium fluxes in the embryo. SepN is physically associated with RyRs and functions as a modifier of the RyR channel. In the absence of SepN, ryanodine receptors from zebrafish embryos or human diseased muscle have altered biochemical properties and have lost their normal sensitivity to redox conditions, which likely accounts for why mutations affecting either factor lead to similar diseases.