Phosphorylation-dependent subfunctionalization of the calcium-dependent protein kinase CPK28

Calcium (Ca²⁺)-dependent protein kinases (CDPKs or CPKs) are a unique family of Ca²⁺ sensor/kinase-effector proteins with diverse functions in plants. In Arabidopsis thaliana, CPK28 contributes to immune homeostasis by promoting degradation of the key immune signaling receptor-like cytoplasmic kinase BOTRYTIS-INDUCED KINASE 1 (BIK1) and additionally functions in vegetative-to-reproductive stage transition. How CPK28 controls these seemingly disparate pathways is unknown. Here, we identify a single phosphorylation site in the kinase domain of CPK28 (Ser318) that is differentially required for its function in immune homeostasis and stem elongation. We show that CPK28 undergoes intermolecular autophosphorylation on Ser318 and can additionally be transphosphorylated on this residue by BIK1. Analysis of several other phosphorylation sites demonstrates that Ser318 phosphorylation is uniquely required to prime CPK28 for Ca²⁺ activation at physiological concentrations of Ca²⁺, possibly through stabilization of the Ca²⁺-bound active state as indicated by intrinsic fluorescence experiments. Together, our data indicate that phosphorylation of Ser318 is required for the activation of CPK28 at low intracellular [Ca²⁺] to prevent initiation of an immune response in the absence of infection. By comparison, phosphorylation of Ser318 is not required for stem elongation, indicating pathway-specific requirements for phosphorylation-based Ca²⁺-sensitivity priming. We additionally provide evidence for a conserved function for Ser318 phosphorylation in related group IV CDPKs, which holds promise for biotechnological applications by generating CDPK alleles that enhance resistance to microbial pathogens without consequences to yield.

Protein kinases represent one of the largest eukaryotic protein superfamilies. While roughly 500 protein kinases have been identified in humans (1), the genomes of Arabidopsis thaliana (hereafter, Arabidopsis) (2) and Oryza sativa (3) encode more than 1,000 and 1,500 protein kinases, respectively, including several families unique to plants. Among these protein kinases are the receptor-like kinases (RLKs), receptor-like cytoplasmic kinases (RLCKs), and calcium-dependent protein kinases (CDPKs or CPKs) that have emerged as key regulators of plant immunity (4–6). Despite encompassing only 2% of most eukaryotic genomes, protein kinases phosphorylate more than 40% of cellular proteins (7, 8), reflecting their diverse roles in coordinating intracellular signaling events. Reversible phosphorylation of serine (Ser), threonine (Thr), and tyrosine (Tyr) residues can serve an array of functions including changes in protein conformation and activation state (9, 10), protein stability and degradation (11, 12), subcellular localization (13–15), and interaction with protein substrates (16–18).Calcium (Ca²⁺) is a ubiquitous secondary messenger that acts cooperatively with protein phosphorylation to propagate intracellular signals. Spatial and temporal changes in intracellular Ca²⁺ levels occur in response to environmental and developmental cues (19–23). In plants, Ca²⁺ transients are decoded by four major groups of calcium sensor proteins, which possess one or more Ca²⁺-binding EF-hand motifs (24, 25): calmodulins (CaM), CaM-like proteins, calcineurin B–like proteins, CDPKs, and Ca²⁺/CaM-dependent protein kinases.At the intersection of phosphorylation cascades and Ca²⁺ signaling are CDPKs, a unique family of Ca²⁺ sensor/kinase-effector proteins. CDPKs have been identified in all land plants and green algae, as well as certain protozoan ciliates and apicomplexan parasites (26, 27). CDPKs have a conserved domain architecture, consisting of a canonical Ser/Thr protein kinase domain and an EF-hand containing Ca²⁺-binding CaM-like domain (CLD), linked together by an autoinhibitory junction (AIJ) and flanked by variable regions on both the amino (N) and carboxyl (C) termini (28, 29). As their name implies, most CDPKs require Ca²⁺ for their activation (30). Upon Ca²⁺ binding to all EF-hands in the CaM-like domain, a dramatic conformational change occurs, freeing the AIJ from the catalytic site of the kinase, rendering the enzyme active (31–33). CDPKs vary in their sensitivity to Ca²⁺ (30), presumably allowing proteins to perceive distinct stimuli through differences in Ca²⁺-binding affinity. For example, Arabidopsis CPK4 displays half maximal kinase activity in the presence of ∼3 μM free Ca²⁺ (30) while CPK5 only requires ∼100 nM (34). Importantly, CDPKs are signaling hubs with documented roles in multiple distinct pathways (4, 24, 35–38) and are therefore likely regulated beyond Ca²⁺ activation.Subfunctionalization is at least partially mediated by protein localization and interaction with pathway-specific binding partners, as is well documented for Arabidopsis CPK3 which functions in response to biotic and abiotic stimuli in distinct cellular compartments (39). Recent attention has been drawn to site-specific phosphorylation as a mechanism to regulate the activity of multifunctional kinases. For example, phosphorylation sites on the RLK BRASSINOSTEROID INSENSITIVE 1–ASSOCIATED KINASE 1 (BAK1) are differentially required for its function as a coreceptor with a subset of leucine-rich repeat –RLKs (40). Phosphoproteomic analyses indicate that CDPKs are differentially phosphorylated following exposure to distinct stimuli (41–48); however, the biochemical mechanisms by which site-specific phosphorylation regulates multifunctional CDPKs is still poorly understood.Arabidopsis CPK28 is a plasma membrane–localized protein kinase with dual roles in plant immune homeostasis (49–51) and phytohormone-mediated reproductive growth (52, 53). In vegetative plants, CPK28 serves as a negative regulator of immune signal amplitude by phosphorylating and activating two PLANT U-BOX–type E3 ubiquitin ligases, PUB25 and PUB26, which target the key immune RLCK BOTRYTIS-INDUCED KINASE 1 (BIK1) for proteasomal degradation (50). Owing to elevated levels of BIK1, CPK28 null plants (cpk28-1) have heightened immune responses and enhanced resistance to the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 (Pto DC3000) (51). Upon transition to the reproductive stage, cpk28-1 plants additionally present shorter leaf petioles, enhanced anthocyanin production, and a reduction in stem elongation (52, 53). The molecular basis for developmental phenotypes in the cpk28-1 knockout mutant, beyond hormonal imbalance (52, 53), are comparatively unknown.Our recent work demonstrated that autophosphorylation status dictates Ca²⁺-sensitivity of CPK28 peptide kinase activity in vitro (54). While dephosphorylated CPK28 is stimulated by the addition of 100 μM CaCl₂ compared to untreated protein, hyperphosphorylated CPK28 displayed similar levels of activity at basal Ca²⁺ concentrations (54). These results highlight the interesting possibility that phosphorylation status may control the activation of multifunctional kinases in distinct pathways by allowing CDPKs to respond to stimulus-specific Ca²⁺ signatures.In the present study, we identify a single autophosphorylation site, Ser318, that decouples the activity of CPK28 in immune signaling from its role in reproductive growth. We show that expression of a nonphosphorylatable Ser-to-Ala variant (CPK28^S318A) is unable to complement the immune phenotypes of cpk28-1 mutants but is able to complement defects in stem growth. Further, we uncover a functional role for phosphorylation of Ser318 in priming CPK28 for activation at low free [Ca²⁺]. Together, we demonstrate that site-specific phosphorylation can direct the activity of a multifunctional kinase in distinct pathways and provide evidence for a conserved mechanism in orthologous group IV CDPKs.     

Elementary mechanisms of calmodulin regulation of NaV1.5 producing divergent arrhythmogenic phenotypes

In cardiomyocytes, Na_V1.5 channels mediate initiation and fast propagation of action potentials. The Ca²⁺-binding protein calmodulin (CaM) serves as a de facto subunit of Na_V1.5. Genetic studies and atomic structures suggest that this interaction is pathophysiologically critical, as human mutations within the Na_V1.5 carboxy-terminus that disrupt CaM binding are linked to distinct forms of life-threatening arrhythmias, including long QT syndrome 3, a “gain-of-function” defect, and Brugada syndrome, a “loss-of-function” phenotype. Yet, how a common disruption in CaM binding engenders divergent effects on Na_V1.5 gating is not fully understood, though vital for elucidating arrhythmogenic mechanisms and for developing new therapies. Here, using extensive single-channel analysis, we find that the disruption of Ca²⁺-free CaM preassociation with Na_V1.5 exerts two disparate effects: 1) a decrease in the peak open probability and 2) an increase in persistent Na_V openings. Mechanistically, these effects arise from a CaM-dependent switch in the Na_V inactivation mechanism. Specifically, CaM-bound channels preferentially inactivate from the open state, while those devoid of CaM exhibit enhanced closed-state inactivation. Further enriching this scheme, for certain mutant Na_V1.5, local Ca²⁺ fluctuations elicit a rapid recruitment of CaM that reverses the increase in persistent Na current, a factor that may promote beat-to-beat variability in late Na current. In all, these findings identify the elementary mechanism of CaM regulation of Na_V1.5 and, in so doing, unravel a noncanonical role for CaM in tuning ion channel gating. Furthermore, our results furnish an in-depth molecular framework for understanding complex arrhythmogenic phenotypes of Na_V1.5 channelopathies.

Voltage-gated sodium channels (Na_V) are responsible for the initiation and spatial propagation of action potentials (AP) in excitable cells (1, 2). Na_V channels undergo rapid activation that underlie the AP upstroke while ensuing inactivation permits AP repolarization. The Na_V1.5 channel constitutes the predominant isoform in cardiomyocytes, whose pore-forming α-subunit is encoded by the SCN5A gene. Na_V1.5 dysfunction underlies diverse forms of cardiac disease including cardiomyopathies, arrhythmias, and sudden death (3–6). Human mutations in Na_V1.5 are associated with two forms of inherited arrhythmias–congenital long QT syndrome 3 (LQTS3) and Brugada syndrome (BrS) (7). LQTS3 stems from delayed or incomplete inactivation of Na_V1.5 that causes persistent Na influx that prolongs AP repolarization—a “gain-of-function” phenotype (7–9). BrS predisposes patients to sudden death and is associated with a reduction in the peak Na current that may slow cardiac conduction or cause region-specific repolarization differences—a “loss-of-function” phenotype (10, 11). Genetic studies have identified an expanding array of mutations in multiple Na_V1.5 domains, including the channel carboxy-terminus (CT) that is a hotspot for mutations linked to both LQTS3 and BrS (12, 13). This domain interacts with the Ca²⁺-binding protein calmodulin (CaM), suggesting that altered CaM regulation of Na_V1.5 may be a common pathophysiological mechanism (12, 14–16). More broadly, human mutations in the homologous regions of neuronal Na_V1.1 (17, 18), Na_V1.2 (19, 20), and Na_V1.6 (21) as well as skeletal muscle Na_V1.4 (22) are linked to varied clinical phenotypes including epilepsy, autism spectrum disorder, neurodevelopmental delay, and myotonia (23). Taken together, a common Na_V mechanistic deficit—defective CaM regulation—may underlie these diverse diseases.CaM regulation of Na_V channels is complex, isoform specific, and mediated by multiple interfaces within the channel (14–16). The Na_V CT consists of a dual vestigial EF hand segment and a canonical CaM-binding “IQ” (isoleucine–glutamine) domain (24, 25) (Fig. 1A). The IQ domain of nearly all Na_V channels binds to both Ca²⁺-free CaM (apoCaM) and Ca²⁺/CaM, similar to Ca_V channels (26–31). As CaM is typically a Ca²⁺-dependent regulator, much attention has been focused on elucidating Ca²⁺-dependent changes in Na_V gating. For skeletal muscle Na_V1.4, transient elevation in cytosolic Ca²⁺ causes a dynamic reduction in the peak current, a process reminiscent of Ca²⁺/CaM-dependent inactivation of Ca_V channels (32). Cardiac Na_V1.5 by comparison exhibits no dynamic effect of Ca²⁺ on the peak current (32–34). Instead, sustained Ca²⁺ elevation has been shown to elicit a depolarizing shift in Na_V1.5 steady-state inactivation (SSI or h_∞), although the magnitude and the presence of a shift have been debated (32, 35). Additional CaM-binding sites have been identified in the channel amino terminus domain (36) and the III-IV linker near the isoleucine, phenylalanine, and methionine (IFM) motif that is well recognized for its role in fast inactivation (35, 37). However, recent cryogenic electron microscopy structures, biochemical, and functional analyses suggest that both the III-IV linker and the Domain IV voltage-sensing domain might instead interact with the channel CT in a state-dependent manner (38–43).Open in a separate windowFig. 1.Absence of dynamic Ca²⁺/CaM effects on WT Na_V1.5 SSI. (A, Left) Structure of Na_V1.5 transmembrane domain (6UZ3) (70) juxtaposed with that of Na_V1.5 CT–apoCaM complex (4OVN) (28). (Right) Arrhythmia-linked CT mutations highlighted in Na_V1.5 CT–apoCaM structure (LQTS3, blue; BrS, magenta; mixed syndrome, purple). (B) Dynamic Ca²⁺-dependent changes in Na_V1.5 SSI probed using Ca²⁺ photouncaging. Na currents specifying h_∞ at ∼100 nM (Left) and ∼4 μM Ca²⁺ step (Right). (C) Population data for Na_V1.5 SSI under low (black, Left) versus high (red, Right) intracellular Ca²⁺ reveal no differences (P = 0.55, paired t test). Dots and bars are mean ± SEM (n = 8 cells). (D) FRET two-hybrid analysis of Cerulean-tagged apoCaM interaction with various Venus-tagged Na_V1.5 CT (WT, black; IQ/AA, red; S[1904]L, blue). Each dot is FRET efficiency measured from a single cell. Solid line fits show 1:1 binding isotherm.Beyond Ca²⁺-dependent effects, the loss of apoCaM binding to the Na_V1.5 IQ domain increases persistent current (34, 44), suggesting that apoCaM itself may be pathophysiologically relevant. Indeed, Na_V1.5 mutations in the apoCaM-binding interface are associated with LQTS3 and atrial fibrillation (7), as well as a loss-of-function BrS phenotype and a mixed-syndrome phenotype whereby some patients present with BrS while others with LQTS3 (Fig. 1A) (13, 45). How alterations in CaM binding paradoxically elicits both gain-of-function and loss-of-function effects is not fully understood, though important to delineate pathophysiological mechanisms and for personalized therapies.Here, using single- and multichannel recordings, we show that apoCaM binding elicits two distinct effects on Na_V1.5 gating: 1) an increase in the peak channel open probability (P_O/peak) and 2) a reduction in the normalized persistent channel open probability (R_persist), consistent with previous studies (34, 44). The two effects may explain how mixed-syndrome mutations in the Na_V1.5 CT produce either BrS or LQTS3 phenotypes. On one hand, the loss of apoCaM association may diminish P_O/peak and induce BrS by shunting cardiac AP. On the other hand, increased R_persist may prevent normal AP repolarization, resulting in LQTS3. Analysis of elementary mechanisms suggests that these changes relate to a switch in the state dependence of channel inactivation. Furthermore, dynamic changes in Ca²⁺ can inhibit persistent current for certain mutant Na_V1.5 owing to enhanced Ca²⁺/CaM binding that occurs over the timescale of a cardiac AP. This effect may result in beat-to-beat variability in persistent Na current for some mutations. In all, these findings explain how a common deficit in CaM binding can contribute to distinct arrhythmogenic mechanisms.     

Synaptic plasticity rules with physiological calcium levels

Spike-timing–dependent plasticity (STDP) is considered as a primary mechanism underlying formation of new memories during learning. Despite the growing interest in activity-dependent plasticity, it is still unclear whether synaptic plasticity rules inferred from in vitro experiments are correct in physiological conditions. The abnormally high calcium concentration used in in vitro studies of STDP suggests that in vivo plasticity rules may differ significantly from in vitro experiments, especially since STDP depends strongly on calcium for induction. We therefore studied here the influence of extracellular calcium on synaptic plasticity. Using a combination of experimental (patch-clamp recording and Ca²⁺ imaging at CA3-CA1 synapses) and theoretical approaches, we show here that the classic STDP rule in which pairs of single pre- and postsynaptic action potentials induce synaptic modifications is not valid in the physiological Ca²⁺ range. Rather, we found that these pairs of single stimuli are unable to induce any synaptic modification in 1.3 and 1.5 mM calcium and lead to depression in 1.8 mM. Plasticity can only be recovered when bursts of postsynaptic spikes are used, or when neurons fire at sufficiently high frequency. In conclusion, the STDP rule is profoundly altered in physiological Ca²⁺, but specific activity regimes restore a classical STDP profile.

Spike-timing–dependent plasticity (STDP) is a form of synaptic modification thought to constitute a mechanism underlying formation of new memories. The polarity of synaptic changes is controlled by the relative timing between pre- and postsynaptic activity and depends on intracellular Ca²⁺ signaling (review in refs. 1 and 2). In hippocampal and neocortical pyramidal neurons, timing-dependent long-term synaptic potentiation (t-LTP) is induced when synaptic activity is followed by one or more backpropagating action potentials in the postsynaptic cell (3–8). It involves postsynaptic Ca²⁺ influx through N-methyl-d-aspartate (NMDA) receptors that in turn activates protein kinases (3, 6, 8, 9). Timing-dependent long-term synaptic depression (t-LTD) is expressed when synaptic activity is repeatedly preceded by one or more backpropagating action potentials (4–7, 10). It depends on NMDA receptor activation, postsynaptic metabotropic glutamate receptors (mGluR), voltage-dependent calcium channels, protein phosphatases, cannabinoid receptor CB1, and astrocytic signaling (6, 10–16). Calcium therefore represents potentially a key factor in the induction of STDP. The intracellular Ca²⁺ dependence of STDP suggests that extracellular Ca²⁺ might play a critical role in shaping STDP. Yet, most if not all in vitro STDP studies (6–10, 17–19) used nonphysiological external Ca²⁺ concentrations ranging between 2 and 3 mM because elevated calcium is known to stabilize recording of synaptic transmission and to avoid intrinsic bursting that could obscure induction of STDP with single pre- and postsynaptic spikes (20, 21). In contrast, the physiological Ca²⁺ concentration is typically around 1.3 mM, with small (0.1–0.3 mM) variations between awake, sleep, and anesthesia, and with age, but in all cases concentrations are below 1.8 mM in rodent hippocampus (22–24).Calcium-based models of synaptic plasticity (25, 26) where Ca²⁺ transients result from backpropagating action potentials and excitatory postsynaptic potentials (EPSPs) predict that the sign, shape, and magnitude of STDP strongly depend on the amplitudes of calcium transients triggered by pre- and postsynaptic spikes and therefore on external Ca²⁺ concentration (26) (Fig. 1). These modeling studies suggest the possibility that plasticity rules at physiological concentrations might be very different from the ones inferred from currently available data. Several scenarios are possible: In the mildest one, high Ca²⁺ concentrations used in experimental studies would lead to an overestimate of the in vivo levels of plasticity; in the most extreme one, a complete lack of plasticity could be observed in physiological Ca²⁺. In addition, recent work shows that synaptic plasticity rules at a cerebellar synapse are profoundly altered in physiological calcium (27, 28). We therefore set out to determine STDP rules in physiological Ca²⁺ at the CA3-CA1 synapse of the hippocampus in vitro.Open in a separate windowFig. 1.Prediction of a calcium-based model of spike-timing–dependent plasticity. Cartoon showing qualitatively calcium transients induced by pairing a presynaptic spike with a postsynaptic spike with a delay ∆t, for three extracellular calcium concentrations (high on top, low on the bottom). Synaptic changes depend on two plasticity thresholds, one for LTP (blue) and one for LTD (red). The resulting ''STDP curves'' (change in synaptic stength ∆w as a function of ∆t) are shown on the right. At high extracellular calcium, the calcium transient exceeds LTP threshold in a range of positive ∆ts, and the STDP curves has a LTP window surrounded by two LTD windows. Decreasing extracellular calcium leads to a decrease in the amplitude of the calcium transient, which no longer cross the LTP threshold, resulting in a STDP curve with only LTD. Finally, a further reduction in extracellular calcium leads to no threshold crossing, and consequently no synaptic changes.We show here that the classical STDP rule (t-LTD for post-before-pre pairings, t-LTP for pre-before-post pairings) is obtained solely with a high external Ca²⁺ concentration (≥ 2.5 mM), whereas no plasticity could be induced for concentrations lower than 1.5 mM external Ca²⁺, and only t-LTD could be induced by positive or negative time delays in 1.8 mM external Ca²⁺. t-LTP could be restored only when bursts of three or four postsynaptic spikes were used instead of single spikes, or when the pairing frequency was increased from 0.33 to 5 or 10 Hz. We used two variants of a Ca²⁺-based plasticity model (26) in which both t-LTD and t-LTP depend on transient changes in postsynaptic Ca²⁺ (Fig. 1) to fit the data. We found that the nonlinearity of transient Ca²⁺ changes conferred by NMDA receptor activation is critical to quantitatively account for the entire experimental dataset. Our results indicate that the STDP rule is profoundly altered in physiological Ca²⁺, but that a classical STDP profile can be restored under specific activity regimes.     

Rapid Ca2+ channel accumulation contributes to cAMP-mediated increase in transmission at hippocampal mossy fiber synapses

The cyclic adenosine monophosphate (cAMP)-dependent potentiation of neurotransmitter release is important for higher brain functions such as learning and memory. To reveal the underlying mechanisms, we applied paired pre- and postsynaptic recordings from hippocampal mossy fiber-CA3 synapses. Ca²⁺ uncaging experiments did not reveal changes in the intracellular Ca²⁺ sensitivity for transmitter release by cAMP, but suggested an increase in the local Ca²⁺ concentration at the release site, which was much lower than that of other synapses before potentiation. Total internal reflection fluorescence (TIRF) microscopy indicated a clear increase in the local Ca²⁺ concentration at the release site within 5 to 10 min, suggesting that the increase in local Ca²⁺ is explained by the simple mechanism of rapid Ca²⁺ channel accumulation. Consistently, two-dimensional time-gated stimulated emission depletion microscopy (gSTED) microscopy showed an increase in the P/Q-type Ca²⁺ channel cluster size near the release sites. Taken together, this study suggests a potential mechanism for the cAMP-dependent increase in transmission at hippocampal mossy fiber-CA3 synapses, namely an accumulation of active zone Ca²⁺ channels.

Communication between neurons is largely mediated by chemical synapses. Synaptic strengths are not fixed, but change dynamically in the short and longer term in an activity-dependent manner (short- and long-term plasticity, 1–3). Moreover, neuromodulators act on presynaptic terminals to modulate synaptic strength. Such activity-dependent or modulatory changes are often mediated by the activation of second messengers, such as protein kinase A and C (2). Second messenger systems, particularly the cyclic adenosine monophosphate (cAMP)/PKA-dependent system, are important for higher brain functions, including learning and memory in Aplysia (3), flies (4, 5), and the mammalian brain (6). Despite its functional importance, the cellular and molecular mechanisms of cAMP-dependent modulation are still poorly understood regardless of whether Aplysia synapses and Drosophila neuromuscular junctions have been investigated (2, 7). Mammalian central synapses are no exception here, also reflecting technical difficulties due to the generally small size of the presynaptic terminals in the mammalian brain.Hippocampal mossy fiber-CA3 (MF-CA3) synapses are characterized by exceptionally large presynaptic terminals (hippocampal mossy fiber bouton, hMFB), which allow for the direct analysis of the cellular mechanisms of synaptic transmission and plasticity by using patch-clamp recordings (8–10). Thus, hMFBs provide a suitable model of cortical synapses in the mammalian brain. Moreover, these synapses are functionally important for brain function such as pattern separation (11). Mossy fiber synapses are known to exhibit unique presynaptic forms of short- and long-term synaptic potentiation and depression, which share the cAMP/PKA-dependent induction mechanism (12–15). In addition, the cAMP-dependent plasticity pathway is important for presynaptic modulation by dopamine and noradrenaline (16–18), which modulates hippocampal network activity and behavior. However, its underlying cellular mechanisms remain largely unclear. Enhancement of the molecular priming and docking of synaptic vesicles at mossy fiber synapses has been suggested by previous studies using genetics and electron microscopy (19–21). In particular, RIM1, an active zone scaffold protein, is crucial for cAMP-dependent long-term potentiation (LTP) (19) and is phosphorylated by PKA, although a corresponding phosphorylation mutant of RIM1 was found to have no effect on long-term potentiation (22, but see ref. 23). Other studies on hMFBs have implicated a role in positional priming, i.e., changes in the spatial coupling between Ca²⁺ channels and the release machinery (24). However, there is a lack of the direct visualization or manipulation of this regulation.In order to measure the intracellular Ca²⁺ sensitivity of transmitter release directly and examine the mechanisms of cAMP-dependent modulation quantitatively, we here carried out Ca²⁺ uncaging experiments at hippocampal mossy fiber synapses. Unexpectedly, our results failed to show changes in Ca²⁺ sensitivity, but instead uncovered an increase in local Ca²⁺ concentrations at the release sites. Furthermore, by live imaging of local Ca²⁺ using total internal reflection fluorescence (TIRF) microscopy as well as superresolution time gated STED (gSTED) microscopy, we provided evidence that rather rapid Ca²⁺ channel accumulation may underlie cAMP-induced potentiation instead of release machinery modulations. This study thus provides a potential mechanism of presynaptic modulation at central synapses.     

Neuromodulator release in neurons requires two functionally redundant calcium sensors

Neuropeptides and neurotrophic factors secreted from dense core vesicles (DCVs) control many brain functions, but the calcium sensors that trigger their secretion remain unknown. Here, we show that in mouse hippocampal neurons, DCV fusion is strongly and equally reduced in synaptotagmin-1 (Syt1)- or Syt7-deficient neurons, but combined Syt1/Syt7 deficiency did not reduce fusion further. Cross-rescue, expression of Syt1 in Syt7-deficient neurons, or vice versa, completely restored fusion. Hence, both sensors are rate limiting, operating in a single pathway. Overexpression of either sensor in wild-type neurons confirmed this and increased fusion. Syt1 traveled with DCVs and was present on fusing DCVs, but Syt7 supported fusion largely from other locations. Finally, the duration of single DCV fusion events was reduced in Syt1-deficient but not Syt7-deficient neurons. In conclusion, two functionally redundant calcium sensors drive neuromodulator secretion in an expression-dependent manner. In addition, Syt1 has a unique role in regulating fusion pore duration.

To date, over 100 genes encoding neuropeptides and neurotrophic factors, together referred to as neuromodulators, are identified, and most neurons express neuromodulators and neuromodulator receptors (1). Neuromodulators travel through neurons in dense core vesicles (DCVs) and, upon secretion, regulate neuronal excitability, synaptic plasticity, and neurite outgrowth (2–4). Dysregulation of DCV secretion is linked to many brain disorders (5–7). However, the molecular mechanisms that regulate neuromodulator secretion remain largely elusive.Neuromodulator secretion, like neurotransmitter secretion from synaptic vesicles (SVs), is tightly controlled by Ca²⁺. The Ca²⁺ sensors that regulate secretion have been described for other secretory pathways but not for DCV exocytosis in neurons. Synaptotagmin (Syt) and Doc2a/b are good candidate sensors due to their interaction with SNARE complexes, phospholipids, and Ca²⁺ (8–11). The Syt family consists of 17 paralogs (12, 13). Eight show Ca²⁺-dependent lipid binding: Syt1 to 3, Syt5 to 7, and Syt9 and 10 (14, 15). Syt1 mediates synchronous SV fusion (8), consistent with its low Ca²⁺-dependent lipid affinity (15, 16) and fast Ca²⁺/membrane dissociation kinetics (16, 17). Syt1 is also required for the fast fusion in chromaffin cells (18) and fast striatal dopamine release (19). Synaptotagmin-7 (Syt7), in contrast, drives asynchronous SV fusion (20), in line with its a higher Ca²⁺ affinity (15) and slower dissociation kinetics (16). Syt7 is also a major calcium sensor for neuroendocrine secretion (21) and secretion in pancreatic cells (22–24). Other sensors include Syt4, which negatively regulates brain-derived neurothropic factor (25) and oxytocin release (26), in line with its Ca²⁺ independency. Syt9 regulates hormone secretion in the anterior pituitary (27) and, together with Syt1, secretion from PC12 cells (28, 29). Syt10 controls growth factor secretion (30). However, Syt9 and Syt10 expression is highly restricted in the brain (31–33). Hence, the calcium sensors for neuronal DCV fusion remain largely elusive. Because DCVs are generally not located close to Ca²⁺ channels (34), we hypothesized that DCV fusion is triggered by high-affinity Ca²⁺ sensors. Because of their important roles in vesicle secretion, their Ca²⁺ binding ability, and their high expression levels in the brain (20, 31, 35–38), we addressed the roles of Doc2a/b, Syt1, and Syt7 in neuronal DCV fusion.In this study, we used primary Doc2a/b-, Syt1-, and Syt7-null (knockout, KO) neurons expressing DCV fusion reporters (34, 39–41) with single-vesicle resolution. We show that both Syt1 and Syt7, but not Doc2a/b, are required for ∼60 to 90% of DCV fusion events. Deficiency of both Syt1 and Syt7 did not produce an additive effect, suggesting they function in the same pathway. Syt1 overexpression (Syt1-OE) rescued DCV fusion in Syt7-null neurons, and vice versa, indicating that the two proteins compensate for each other in DCV secretion. Moreover, overexpression of Syt1 or Syt7 in wild-type (WT) neurons increased DCV fusion, suggesting they are both rate limiting for DCV secretion. We conclude that DCV fusion requires two calcium sensors, Syt1 and Syt7, that act in a single/serial pathway and that both sensors regulate fusion in a rate-limiting and dose-dependent manner.     

Stac protein regulates release of neuropeptides

Neuropeptides are important for regulating numerous neural functions and behaviors. Release of neuropeptides requires long-lasting, high levels of cytosolic Ca²⁺. However, the molecular regulation of neuropeptide release remains to be clarified. Recently, Stac3 was identified as a key regulator of L-type Ca²⁺ channels (CaChs) and excitation–contraction coupling in vertebrate skeletal muscles. There is a small family of stac genes in vertebrates with other members expressed by subsets of neurons in the central nervous system. The function of neural Stac proteins, however, is poorly understood. Drosophila melanogaster contain a single stac gene, Dstac, which is expressed by muscles and a subset of neurons, including neuropeptide-expressing motor neurons. Here, genetic manipulations, coupled with immunolabeling, Ca²⁺ imaging, electrophysiology, and behavioral analysis, revealed that Dstac regulates L-type CaChs (Dmca1D) in Drosophila motor neurons and this, in turn, controls the release of neuropeptides.

Neuropeptides are required for a myriad of brain functions, such as regulation of complex social behaviors, including emotional behavior (1). The activities of many neuropeptides within the brain are now well-established, but, despite the important role neuropeptides play, our understanding of the mechanisms by which neuropeptides are released by neurons and how release is regulated is not as advanced as that of classical neurotransmitters. A greater understanding of neuropeptide release could help untangle the mechanisms of complex behaviors as well as reveal therapeutic targets that could modulate aberrant behaviors.Release of neuropeptides, which are packaged in dense core vesicles (DCVs) rather than in small synaptic vesicles that contain neurotransmitters, often involves activation of L-type Ca²⁺ channels (CaChs) and Ca²⁺-induced Ca²⁺ release (CICR) (2). Much of what is known about DCV exocytosis is from the study of nonneural cells. For example, in adrenal chromaffin cells, the exocytosis of DCVs containing catecholamines and/or peptide hormones involves CICR either via the ryanodine receptor (RyR) or inositol trisphosphate receptor (iP₃R) (3, 4) initiated by an influx of Ca²⁺ through voltage-gated CaChs, such as L-type CaChs. In neurons, the release of neuropeptides is more complex. Neuropeptides can be released from dendrites, cell bodies, axons, and presynaptic terminals (5–7), and release can involve a kiss-and-run mechanism (8). DCVs are not tightly clustered as are small synaptic vesicles, and DCVs are generally not associated with presynaptic specializations for release of neurotransmitters, such as active zones (9). Furthermore, the DCVs within the central nervous system (CNS) are not as numerous nor as large as they are in chromaffin cells and neurohypophyseal terminals. In many neurons, the release of neuropeptides requires bursts of action potentials (10, 11). Based primarily upon pharmacological experiments, it appears that an influx of Ca²⁺ via L-type CaChs is necessary for release of neuropeptides from some neurons (8, 12–18), with some cases also involving Ca²⁺ release from internal stores while others not. This suggests the possibility that, in neurons, neuropeptide release may be initiated by an influx of Ca²⁺ via L-type CaCh.Drosophila provide an ideal system to study neuropeptide release due to the vast array of molecular and genetic tools available for manipulating them. Numerous neuropeptides are expressed by the Drosophila nervous system, including proctolin by motor neurons (19, 20). Furthermore, larval motor neurons fire bursts of high frequency action potentials (21, 22) so motor boutons are likely to have the long-lasting increases in Ca²⁺ transients in motor boutons that are thought to be required for the release of neuropeptides. Motor boutons contain a network of endoplasmic reticulum (ER) (23), and there is a RyR-dependent release of Ca²⁺ from the ER presumably via CICR, which is required for sustained release of neuropeptides at the neuromuscular junction (NMJ) of third instar larvae (24). Elegant dynamic examination of DCVs in boutons with fluorescence recovery after photobleaching showed that they are immobile in the resting state, but they move randomly and release neuropeptides for several minutes following activity-dependent Ca²⁺ increases (25). Furthermore, simultaneous photobleaching and imaging of DCVs suggest that DCVs release neuropeptides via multiple rounds of kiss-and-run events (26). Thus, in Drosophila, neuropeptide release at the NMJ appears to involve CICR from the ER.One way to regulate the release of neuropeptides is to control changes in cytoplasmic Ca²⁺ levels. In mammals, stac3 is a member of a small family of genes, along with stac1 and stac2, which are expressed by subsets of neurons (27–29). Stac3 was identified as a regulator of L-type CaChs and excitation–contraction (EC) coupling in zebrafish skeletal muscles (30, 31). Stac3 also regulates EC coupling in murine skeletal muscles (32) and is causal for the congenital Native American myopathy (30). EC coupling is the process that transduces changes in muscle membrane voltage to initiate release of Ca²⁺ from the sarcoplasmic reticulum (SR) and contraction. In vertebrate skeletal muscles, EC coupling is mediated by the L-type CaCh DHPR, which is in the transverse tubule membrane (t-tubules) and is the voltage sensor for EC coupling, and the RyR, which is the Ca²⁺ release channel in the SR (33–36). In zebrafish, Stac3 regulates EC coupling by colocalizing with DHPR and RyR, and by regulating DHPR stability and functionality, including the response to voltage of DHPRs, but not trafficking of DHPRs (31, 37).The in vivo function of the stac1 and stac2 genes expressed by neurons is, however, unknown. Recently, a stac-like gene, Dstac, was identified in Drosophila (38). There is a single stac gene in Drosophila, and it is expressed both by muscles and a subset of neurons, including in the lateral ventral neurons (LN_Vs) that express the neuropeptide, pigment-dispersing factor (PDF), in the brain. Previously, genetic manipulation of PDF demonstrated the necessity of PDF for circadian rhythm (39). Interestingly, knocking down Dstac selectively in the PDF neurons disrupted circadian rhythm, suggesting the hypothesis that Dstac regulates the release of neuropeptides such as PDF. Since Stac3 regulates the L-type CaCh in vertebrate skeletal muscle, Dstac might control neuropeptide release via regulation of the single L-type CaCh in Drosophila neurons (Dmca1D) (40). We tested this hypothesis by examining the role of Dstac for neuropeptide release by the more accessible presynaptic boutons of motor neurons at the NMJs of third instar larvae.     

Allosteric modulation of alternatively spliced Ca2+-activated Cl− channels TMEM16A by PI(4,5)P2 and CaMKII

Transmembrane 16A (TMEM16A, anoctamin1), 1 of 10 TMEM16 family proteins, is a Cl⁻ channel activated by intracellular Ca²⁺ and membrane voltage. This channel is also regulated by the membrane phospholipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂]. We find that two splice variants of TMEM16A show different sensitivity to endogenous PI(4,5)P₂ degradation, where TMEM16A(ac) displays higher channel activity and more current inhibition by PI(4,5)P₂ depletion than TMEM16A(a). These two channel isoforms differ in the alternative splicing of the c-segment (exon 13). The current amplitude and PI(4,5)P₂ sensitivity of both TMEM16A(ac) and (a) are significantly strengthened by decreased free cytosolic ATP and by conditions that decrease phosphorylation by Ca²⁺/calmodulin-dependent protein kinase II (CaMKII). Noise analysis suggests that the augmentation of currents is due to a rise of single-channel current (i), but not of channel number (N) or open probability (P_O). Mutagenesis points to arginine 486 in the first intracellular loop as a putative binding site for PI(4,5)P₂, and to serine 673 in the third intracellular loop as a site for regulatory channel phosphorylation that modulates the action of PI(4,5)P₂. In silico simulation suggests how phosphorylation of S673 allosterically and differently changes the structure of the distant PI(4,5)P₂-binding site between channel splice variants with and without the c-segment exon. In sum, our study reveals the following: differential regulation of alternatively spliced TMEM16A(ac) and (a) by plasma membrane PI(4,5)P₂, modification of these effects by channel phosphorylation, identification of the molecular sites, and mechanistic explanation by in silico simulation.

TMEM16A (anoctamin1) plays a wide range of physiological roles in diverse cell types, including contraction of smooth muscle and gastrointestinal motility, secretion of Cl⁻ in epithelial cells, detection of noxious heat in nociceptive neurons, modulation of neuronal excitability, and regulation of cell volume (1). TMEM16A channels, from a family of 10 anoctamin proteins (TMEM16A–K), continuously monitor the concentration of intracellular Ca²⁺ and function as Ca²⁺-activated Cl⁻ channels (2–4). Several splice variants of TMEM16A generated by combinatorial inclusion or exclusion of four exon segments, a, b, c, and d (5–7), display unique electrophysiological properties in tissues. Segments a and b lie in the N terminus, and segments c and d lie in the first intracellular loop of TMEM16A. Among the four segments, it is known that b and c help regulate the cytosolic Ca²⁺ sensitivity and voltage dependence of channel gating. For example, inclusion of the b-segment results in decreased channel sensitivity to intracellular Ca²⁺ rise, whereas skipping of the c-segment reduces channel activity and also impairs Ca²⁺ sensitivity (5, 8, 9). In addition to inclusion or skipping of each segment, calmodulin (10–13), phosphorylation (14–16), protons (17–19), and lipids (20–27) also impact on the gating of TMEM16A channels.Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂] is a key signaling phospholipid in the inner leaflet of the plasma membrane. It acts as a cofactor that regulates many types of ion channels and receptors (28–30), and thus depletion of membrane PI(4,5)P₂ by the activation of either phospholipase C (PLC) or phosphoinositide 5-phosphatases leads to decreases or increases in gating activity of ion channels. Of the TMEM16 family, TMEM16A, TMEM16B, and TMEM16F are ion channels best known to be modulated by PI(4,5)P₂ (21–27, 31). Several studies showed that PI(4,5)P₂ is required for sustained TMEM16A channel activity and stabilizes the Ca²⁺-bound open state of the channels (23, 24, 32). Further work located a PI(4,5)P₂ regulatory region and demonstrated how PI(4,5)P₂ interacts with TMEM16A to regulate channel gating by performing computational simulation. Le et al. (25) proposed that channel activation and desensitization are mediated by two distinct structural modules; one is a PI(4,5)P₂-binding module formed by putative PI(4,5)P₂-binding residues of TMs 3–5 located near the cytoplasmic membrane interface and another is a Ca²⁺-binding module of TMs 6–8 involved in the primary opening of the channel pore by Ca²⁺. Yu et al. (26) identified three key binding sites involved in TMEM16A–PI(4,5)P₂ interaction. When PI(4,5)P₂ interacts with these binding residues, which form networks with each other, it affects TMEM16A channel gating as a result of the conformational change of TM6.In our study, using exogenous lipid phosphatase tools and mutagenesis, we found that PI(4,5)P₂ differentially regulates channel activity depending on the TMEM16A splice variant. In addition, we found that the presence or absence of intracellular ATP is a key determinant of the PI(4,5)P₂ sensitivity of TMEM16A. Through structural analysis partly based on a recent cryogenic electron microscopy (cryo-EM) structure of TMEM16A, we also confirmed that phosphorylation of serine 673 by CaMKII allosterically regulates the structure of a PI(4,5)P₂ interaction site in the RDR domain of TMEM16A(ac) near to transmembrane segment 3 (TM3). Together, our data reveal a molecular mechanism of TMEM16A channel regulation by PI(4,5)P₂, demonstrating that PI(4,5)P₂-dependent TMEM16A channel activation can be allosterically modulated by phosphorylation and alternative splicing.     

Impaired TRPV4-eNOS signaling in trabecular meshwork elevates intraocular pressure in glaucoma

Primary Open Angle Glaucoma (POAG) is the most common form of glaucoma that leads to irreversible vision loss. Dysfunction of trabecular meshwork (TM) tissue, a major regulator of aqueous humor (AH) outflow resistance, is associated with intraocular pressure (IOP) elevation in POAG. However, the underlying pathological mechanisms of TM dysfunction in POAG remain elusive. In this regard, transient receptor potential vanilloid 4 (TRPV4) cation channels are known to be important Ca²⁺ entry pathways in multiple cell types. Here, we provide direct evidence supporting Ca²⁺ entry through TRPV4 channels in human TM cells and show that TRPV4 channels in TM cells can be activated by increased fluid flow/shear stress. TM-specific TRPV4 channel knockout in mice elevated IOP, supporting a crucial role for TRPV4 channels in IOP regulation. Pharmacological activation of TRPV4 channels in mouse eyes also improved AH outflow facility and lowered IOP. Importantly, TRPV4 channels activated endothelial nitric oxide synthase (eNOS) in TM cells, and loss of eNOS abrogated TRPV4-induced lowering of IOP. Remarkably, TRPV4-eNOS signaling was significantly more pronounced in TM cells compared to Schlemm’s canal cells. Furthermore, glaucomatous human TM cells show impaired activity of TRPV4 channels and disrupted TRPV4-eNOS signaling. Flow/shear stress activation of TRPV4 channels and subsequent NO release were also impaired in glaucomatous primary human TM cells. Together, our studies demonstrate a central role for TRPV4-eNOS signaling in IOP regulation. Our results also provide evidence that impaired TRPV4 channel activity in TM cells contributes to TM dysfunction and elevated IOP in glaucoma.

Glaucoma is a heterogenic group of multifactorial neurodegenerative diseases characterized by progressive optic neuropathy. It is the leading cause of irreversible vision loss with more than 70 million people affected worldwide (1), and the prevalence is estimated to increase to 111.6 million by the year 2040 (2). Primary open angle glaucoma (POAG) is the most common form of glaucoma, accounting for ∼70% of all cases (1). POAG is characterized by progressive loss of retinal ganglion cell axons that leads to an irreversible loss of vision (1, 3). Elevated intraocular pressure (IOP) is a major, and the only treatable, risk factor associated with POAG (4). The trabecular meshwork (TM), a molecular sieve-like structure, maintains homeostatic control over IOP by constantly adjusting the resistance to aqueous humor (AH) outflow. In POAG, there is increased resistance to AH outflow, elevating IOP (5). This increase in AH outflow resistance is associated with dysfunction of the TM (6–8).The TM has an intrinsic ability to sense the AH flow and regulate outflow facility to maintain IOP homeostasis (6), although the precise flow-sensing mechanisms in TM cells are unclear. In this regard, transient receptor potential vanilloid 4 (TRPV4) cation channels have emerged as a major flow-activated Ca²⁺ entry pathway in multiple cell types (9–12). Upon activation, TRPV4 channels allow localized Ca²⁺ influx (termed as TRPV4 sparklets), which influences a variety of cellular homeostatic processes (13, 14). TRPV4 sparklets are spatially restricted signals with a spatial spread (maximum width at half maximal amplitude) of ∼11 microns (13). Treatment with a selective TRPV4 channel activator GSK1016790A (GSK101) lowered IOP in rats and mice (15). Furthermore, baseline IOP was higher in global TRPV4^−/− mice compared to their wild-type (WT) littermates (15). However, the exact cell type responsible for these IOP-lowering effects is not known. Previous studies have shown that TRPV4 channel protein is expressed in TM cells and tissues (15, 16). The physiological roles of TRPV4 channels in TM cells (TRPV4_TM) and downstream signaling mechanisms remain unknown. TM constitutively expresses Ca²⁺-sensitive endothelial nitric oxide synthase (eNOS) (17), a known regulator of outflow facility and IOP (18–22). In vascular endothelial cells, TRPV4 channels are important regulators of eNOS activity (23–26). We, therefore, hypothesized that TRPV4_TM-eNOS signaling promotes outflow facility and reduces IOP.Glaucoma-associated pathological changes are known to impair physiological function of TM (8). One of the hallmarks of the glaucomatous TM is its inability to maintain normal IOP and AH outflow resistance (6). Here, we postulated that impaired TRPV4_TM-eNOS signaling contributes to TM dysfunction and elevated IOP in glaucoma. In this report, our studies in human TM cells and TM tissue showed shear stress–mediated activation of TRPV4-eNOS signaling. Moreover, reduced AH outflow and elevated IOPs were observed in TM-specific TRPV4^−/− (TRPV4_TM^−/−) mice and eNOS^−/− mice. Importantly, TRPV4_TM activity and shear stress–mediated activation of TRPV4_TM-eNOS signaling are compromised in human glaucomatous TM cells. Our results provide direct evidence for a physiological role of TRPV4_TM-eNOS signaling and indicate that impaired TRPV4_TM-eNOS signaling may underlie TM dysfunction and IOP dysregulation in glaucoma.     

Regulation of longevity by depolarization-induced activation of PLC-β–IP3R signaling in neurons

Mitochondrial ATP production is a well-known regulator of neuronal excitability. The reciprocal influence of plasma-membrane potential on ATP production, however, remains poorly understood. Here, we describe a mechanism by which depolarized neurons elevate the somatic ATP/ADP ratio in Drosophila glutamatergic neurons. We show that depolarization increased phospholipase-Cβ (PLC-β) activity by promoting the association of the enzyme with its phosphoinositide substrate. Augmented PLC-β activity led to greater release of endoplasmic reticulum Ca²⁺ via the inositol trisphosphate receptor (IP₃R), increased mitochondrial Ca²⁺ uptake, and promoted ATP synthesis. Perturbations that decoupled membrane potential from this mode of ATP synthesis led to untrammeled PLC-β–IP₃R activation and a dramatic shortening of Drosophila lifespan. Upon investigating the underlying mechanisms, we found that increased sequestration of Ca²⁺ into endolysosomes was an intermediary in the regulation of lifespan by IP₃Rs. Manipulations that either lowered PLC-β/IP₃R abundance or attenuated endolysosomal Ca²⁺ overload restored animal longevity. Collectively, our findings demonstrate that depolarization-dependent regulation of PLC-β–IP₃R signaling is required for modulation of the ATP/ADP ratio in healthy glutamatergic neurons, whereas hyperactivation of this axis in chronically depolarized glutamatergic neurons shortens animal lifespan by promoting endolysosomal Ca²⁺ overload.

Spatially circumscribed ATP production at nerve termini is predicated on local mitochondria that are energized when voltage-gated Ca²⁺ channels provide the [Ca²⁺] elevations needed to overcome the low sensitivity of the mitochondrial Ca²⁺ uniporter (MCU) (1–3). In neuronal soma, however, bulk cytosolic [Ca²⁺] is not elevated to levels needed for mitochondrial sequestration. Rather, mitochondrial Ca²⁺ uptake in the somatodendritic compartment occurs at specialized points of contact between mitochondria and endoplasmic reticulum (ER) where Ca²⁺ released by IP₃Rs is transferred into the mitochondrial matrix (4). Approximately 75 to 90% of the somatic ATP synthesized following interorganellar transfer of Ca²⁺ is consumed by Na⁺/K⁺ ATPases, which help establish resting membrane potential and permit repolarization during activity (5, 6). Therefore, defects in neuronal ATP synthesis result in loss of membrane potential and hyperexcitability (6).Whether excitability of the somatic plasma membrane (PM) exerts reciprocal influence on mitochondrial [Ca²⁺] and ATP production remains poorly understood. In an attempt to fill some of the gaps in knowledge, we examined the effects of PM potential on mitochondrial ATP production and Ca²⁺ homeostasis in Drosophila neurons. Owing to recent reports of neuronal hyperexcitability being a driver of diminished longevity in organisms ranging from Caenorhabditis elegans to humans (7–9), we hoped our studies would inform insights into the regulation of aging and lifespan. Moreover, since neuronal hyperexcitability, Ca²⁺ dyshomeostasis, and bioenergetic dysfunction characterize neurodegenerative diseases (6, 10, 11), uncovering actionable molecular targets that bridge these perturbations may bear therapeutic value. Our findings reveal a previously unknown mechanism by which excitability regulates bioenergetics and Ca²⁺ signaling and points to the utility of this signaling circuit in the regulation of longevity.     

A unique C2 domain at the C terminus of Munc13 promotes synaptic vesicle priming

Neurotransmitter release during synaptic transmission comprises a tightly orchestrated sequence of molecular events, and Munc13-1 is a cornerstone of the fusion machinery. A forward genetic screen for defects in neurotransmitter release in Caenorhabditis elegans identified a mutation in the Munc13-1 ortholog UNC-13 that eliminated its unique and deeply conserved C-terminal module (referred to as HC2M) containing a Ca²⁺-insensitive C2 domain flanked by membrane-binding helices. The HC2M module could be functionally replaced in vivo by protein domains that localize to synaptic vesicles but not to the plasma membrane. HC2M is broadly conserved in other Unc13 family members and is required for efficient synaptic vesicle priming. We propose that the HC2M domain evolved as a vesicle/endosome adaptor and acquired synaptic vesicle specificity in the Unc13ABC protein family.

Chemical synaptic transmission is the primary mode of cellular communication within the nervous system. The presynaptic piece of this process encompasses a remarkable set of sequential and highly regulated interactions between a host of proteins, synaptic vesicles (SV), the plasma membrane, and calcium ions (Ca²⁺). Fusion of neurotransmitter-containing vesicles with the presynaptic plasma membrane is driven by the assembly of the neuronal SNAREs SNAP-25 and Syntaxin 1 on the plasma membrane and Synaptobrevin-2/VAMP2 on the SV. The assembly process and its coupling to intracellular Ca²⁺ are choreographed by a deeply conserved group of proteins including Munc13, Munc18, Synaptotagmin 1, and Complexin (1–4). Together with the SNAREs, these proteins form the core of the fusion apparatus across all metazoan nervous systems (5–7).First identified in a landmark genetic screen for nervous system mutants in the nematode Caenorhabditis elegans, UNC-13 is the founding member of the highly conserved metazoan Unc13 secretory protein family that includes Unc13ABC in humans (Munc13-1/2/3 in mice) (8–10). Munc13-1/UNC-13 localizes to the presynaptic active zone and is implicated in numerous presynaptic functions including initiation of release site assembly, SV docking and priming, Ca²⁺- and lipid-dependent forms of short-term synaptic plasticity, opening and positioning Syntaxin 1 for SNARE assembly, and protecting SNARE complexes from disassembly by NSF/alpha-SNAP (3, 11–13). Loss of Munc13-1 orthologs in the nervous system almost entirely eliminates all forms of chemical synaptic transmission, establishing the Unc13 family as essential to this process (14–16). All UNC-13 orthologs contain a large Syntaxin-binding MUN domain flanked by a Ca²⁺- and lipid-binding C1-C2 module and an additional C2 domain on its C terminus referred to as C2C (5, 10, 17).The C-terminal end of UNC-13 is the least understood domain within the Unc13 protein family in terms of both structure and mechanism (18, 19). Recent work on the MUN and C2C domains of Munc13-1 both in vitro and in cultured hippocampal synapses supports the notion that the MUN-C2C region attaches Munc13-1 to SVs as a means of preparing SVs for fusion (20, 21), but several questions remain unresolved. Is the SV interaction mediated by direct membrane binding? Does the C2C domain itself bind to SVs or does the MUN domain serve this role? Does either domain provide cargo specificity as part of the priming process? Interestingly, the C-terminal end of the MUN domain of CAPS, another Unc13 family member, can bind dense-core vesicles (DCVs) although it lacks a C-terminal C2 domain (22). Moreover, the MUN domain without the C2C domain has also been demonstrated to bind liposomes through an interaction with Synaptobrevin 2 (23). These observations bring up several possibilities for interactions with the C terminus of Munc13 including direct MUN–membrane interactions, C2C–membrane interactions, or protein–protein interactions involving either or both domains. Other Unc13 family members possessing a MUN domain with a C-terminal C2 domain such as Unc13D/Munc13-4 and BAIAP3 have been proposed to tether specific cargo such as endosomes, secretory granules, and large DCVs (24, 25). How Unc13 proteins select among different cargos remains largely unanswered (24, 26, 27).Through behavioral, electrophysiological, biochemical, and genetic approaches, we uncover a deeply conserved C-terminal membrane-binding domain within Munc13-1/UNC-13 termed the Munc13 C-terminal (MCT) domain. This region, together with C2C and a neighboring N-terminal helix fold together into a stable membrane-binding protein domain in vitro, and loss of any part of this module in vivo impairs SV priming and nervous system function. Moreover, the C-terminal domain can be replaced by foreign domains that bind SVs but not the plasma membrane, demonstrating a role in SV interactions at the synapse. Phylogenetic protein sequence comparisons suggest that the ancestral Unc13/BAIAP3 homolog possessed a similar C-terminal domain prior to the emergence of metazoa, and subsequently, the UNC-13ABC subfamily domain evolved as an SV adaptor that plays a critical role in neurotransmission in all animals.     

Cellular pathways of calcium transport and concentration toward mineral formation in sea urchin larvae

Sea urchin larvae have an endoskeleton consisting of two calcitic spicules. The primary mesenchyme cells (PMCs) are the cells that are responsible for spicule formation. PMCs endocytose sea water from the larval internal body cavity into a network of vacuoles and vesicles, where calcium ions are concentrated until they precipitate in the form of amorphous calcium carbonate (ACC). The mineral is subsequently transferred to the syncytium, where the spicule forms. Using cryo-soft X-ray microscopy we imaged intracellular calcium-containing particles in the PMCs and acquired Ca-L_2,3 X-ray absorption near-edge spectra of these Ca-rich particles. Using the prepeak/main peak (L₂′/ L₂) intensity ratio, which reflects the atomic order in the first Ca coordination shell, we determined the state of the calcium ions in each particle. The concentration of Ca in each of the particles was also determined by the integrated area in the main Ca absorption peak. We observed about 700 Ca-rich particles with order parameters, L₂′/ L₂, ranging from solution to hydrated and anhydrous ACC, and with concentrations ranging between 1 and 15 M. We conclude that in each cell the calcium ions exist in a continuum of states. This implies that most, but not all, water is expelled from the particles. This cellular process of calcium concentration may represent a widespread pathway in mineralizing organisms.

Calcium ions play a critical role in many cellular processes. Calcium ions are messengers for a wide range of cellular activities, including fertilization, cell differentiation, secretion, muscle contraction, and programmed cell death (1, 2). Therefore, the concentration of Ca²⁺ in the cytosol is highly regulated at around 100 to 200 nM during resting stages (3–5). Ca²⁺ homeostasis is mediated by Ca-binding proteins and different organelles in the cell, including the endoplasmic reticulum (ER) and the mitochondria that serve as significant Ca²⁺ stores and as signal generators (6–8).Calcium ions are an important component of many biominerals such as bones, teeth, shells, and spines (9). In comparison to Ca²⁺ signaling, which requires small amounts of Ca²⁺, biomineralization processes require massive sequestering and transport of ions from the environment and/or from the food to the site of mineralization. The sequestered ions can reach the mineralization site as solutes but can also concentrate intracellularly inside vesicles, where they precipitate to form highly disordered mineral phases (10–15). In the latter case, specialized cells take up the ions through ion pumps, ion channels, or by endocytosis of extracellular fluid and process the calcium ions until export to the final mineralization location (16–19).In this study, we evaluate the contents of calcium-containing vesicles in primary mesenchyme cells (PMCs), which are involved in the formation of the calcitic skeleton of the sea urchin larvae. In this way, we obtain insights into how calcium ions, extracted from the environment, are concentrated and stored for spicule formation.Paracentrotus lividus sea urchin embryos form an endoskeleton consisting of two calcitic spicules within 72 h after fertilization (20, 21). The source of the calcium ions is the surrounding sea water, whereas the carbonate ions are thought to originate from both sea water and metabolic processes in the embryo (22, 23). Sea water enters the embryonic body cavity (blastocoel) through the permeable ectoderm cell layer of the embryo (24). Endocytosis of sea water and blastocoel fluid into PMCs as well as endothelial and epithelial cells (25, 26) was tracked by labeling sea water with calcein, a fluorescent calcium-binding and membrane-impermeable dye (17, 26–28). The endocytosed fluid in the PMCs was observed to form a network of vacuoles and vesicles (26). Beniash et al. observed electron-dense granules of sizes 0.5 to 1.5 µm in PMCs, which are composed of amorphous calcium carbonate (ACC) (10). Intracellular vesicles of similar size were observed Vidavsky et al., using cryo-scanning electron microscopy (SEM) and air-SEM, containing calcium carbonate deposits composed of nanoparticles 20 to 30 nm in size (25). Ca deposits within the same range of sizes were also observed in the rough ER of PMCs (29).The intracellularly produced ACC is subsequently exported to the growing spicule, where it partially transforms into calcite through secondary nucleation (30–33). The location and distribution of ACC and calcite in the spicule were studied by using extended X-ray absorption fine structure, X-ray absorption near-edge spectroscopy (XANES), and photoelectron emission microscopy (PEEM) (30, 34, 35). Three distinct mineral phases were identified in the spicule: hydrated ACC (ACC*H₂O), anhydrous ACC, and crystalline calcite (34). According to theoretical simulations of Rez and Blackwell (36), the two different amorphous phases arise from different levels of ordering of the oxygen coordination polyhedron around calcium. The coordination polyhedron becomes more ordered as the transformation to the crystalline phase progresses. The phase information contained in the XANES spectra is exploited here to characterize the mineral phases in the intracellular vesicles.Cryo-soft X-ray transmission microscopy (cryo-SXM) is an attractive technique for tomography and spectromicroscopy of biological samples in the hydrated state (37–39). Imaging is performed in the “water window” interval of X-ray energies, namely between the carbon (C) K-edge (284 eV) and the oxygen (O) K-edge (543 eV). As a result, in the “water window” C is highly absorbing, whereas O, and thus H₂O, is almost transparent. Subsequently, carbon-rich moieties such as lipid bodies, proteins, and membranes appear dark in transmission, whereas the water rich cytosol appears lighter (40). The Ca L_2,3-absorption edge between ∼346 and 356 eV also resides in the “water window” (41). Therefore, imaging across this absorption edge enables the characterization of Ca-rich moieties in whole, hydrated cells. Each pixel of the same field of view, imaged as the energy is varied across the Ca L_2,3-edge, can be assigned an individual Ca L_2,3-edge XANES spectrum. This technique was applied to the calcifying coccolithophorid alga (42, 43). In this study, we use cryo-SXM and XANES to locate and characterize both the phases and the concentrations of Ca-rich bodies in sea urchin larval cells.     

Secretagogin marks amygdaloid PKCδ interneurons and modulates NMDA receptor availability

The perception of and response to danger is critical for an individual’s survival and is encoded by subcortical neurocircuits. The amygdaloid complex is the primary neuronal site that initiates bodily reactions upon external threat with local-circuit interneurons scaling output to effector pathways. Here, we categorize central amygdala neurons that express secretagogin (Scgn), a Ca²⁺-sensor protein, as a subset of protein kinase Cδ (PKCδ)⁺ interneurons, likely “off cells.” Chemogenetic inactivation of Scgn⁺/PKCδ⁺ cells augmented conditioned response to perceived danger in vivo. While Ca²⁺-sensor proteins are typically implicated in shaping neurotransmitter release presynaptically, Scgn instead localized to postsynaptic compartments. Characterizing its role in the postsynapse, we found that Scgn regulates the cell-surface availability of NMDA receptor 2B subunits (GluN2B) with its genetic deletion leading to reduced cell membrane delivery of GluN2B, at least in vitro. Conclusively, we describe a select cell population, which gates danger avoidance behavior with secretagogin being both a selective marker and regulatory protein in their excitatory postsynaptic machinery.

The amygdala is a brain structure critical for the acquisition and relay of threatening stimuli to execute behavioral responses to danger (1, 2). Much of our understanding about this process is based on the concept of internuclear lateral-to-medial information flow within the amygdaloid complex, with the lateral (LA) and central (CeA) amygdaloid nuclei being the main input and output, respectively (3). During the acquisition of experience-induced danger responses, unconditioned threat and context-specific environmental cues are linked in the basolateral amygdaloid nucleus, anterior part (BLA) (4). In turn, the CeA serves as a relay and processes information via its local recurrent inhibitory circuits before feeding those toward effector structures (5–7), including the prefrontal cortex (8). Instead of being a passive relay station, the CeA is likely to participate in the learning of danger responses (9–13): Anatomical and physiological evidence converge to indicate that output neurons in the medial part of the CeA (CeM) are under inhibitory control, which originates in its lateral subregion (CeL) (5, 12, 14, 15). In accord with this principle, neuronal subsets specific for the different CeA subnuclei were identified and causally related to the regulation of danger avoidance behavior (10). In the CeL, PKCδ-positive⁽⁺⁾ (“fear-off”) neurons project onto and inhibit CeM output neurons that trigger danger-induced behavior, such as freezing (10). In turn, “fear-on” CeL neurons marked by the expression of the neuropeptide somatostatin (SOM⁺) modulate afferent and efferent signals locally within the CeL or via inhibitory connections between CeL and CeM, probably involving neuropeptide-Y Y2 receptors (16). Additionally, these CeL neurons form mutual inhibitory connections with corticotropin releasing hormone (CRH)⁺ neurons, which determine the balance between conditioned flight and fright (freezing) behaviors (17). While fear-on neurons suppress fear-off neurons, both cell types receive direct excitatory input from the LA (18). Based on these wiring principles, a series of studies have charted the cellular constituents of threat-responsive CeA circuits, with cell type-specific genetic manipulations allowing for inferences to be made toward fear-related disorders (9). Nevertheless, molecular mechanisms specific to distinct interneuron subclasses amenable to gating behavioral responses remain less well explored.Ca²⁺ plays critical roles in determining the physiology of synaptic neurotransmission. Upon synaptic activity, Ca²⁺ entry activates Ca²⁺-sensor proteins to trigger cell state- and context-specific intracellular signaling events by recruiting partner proteins in signalosome complexes (19). Secretagogin (Scgn) is one such Ca²⁺-sensor protein whose expression is activity dependent (20) and specific to a hitherto undefined GABA interneuron subclass in the CeL (21). However, the role of Scgn in the amygdala remains unknown.Here, we hypothesized that Scgn could modulate excitatory neurotransmission locally to scale behavioral responses in danger. By combining classical neuroanatomy, neurochemistry, electrophysiology, and behavioral genetics, we found that Scgn marks a subpopulation of PKCδ⁺ CeL interneurons whose inhibition triggers freezing in a typical behavioral paradigm for the assessment of danger responses in rodents, termed “fear conditioning” (15, 22). Ultrastructural analysis showed Scgn enrichment in the subsynaptic region of dendrites apposing excitatory afferents, which was biochemically confirmed by synaptic fractionation and Western blotting. Postsynaptic localization is unexpected because the bulk of studies on Scgn implicates this Ca²⁺-sensor protein in presynaptic neurotransmitter release. By reanalyzing our open-source proteomics data, we identified the 2B subunit of the NMDA receptor (GluN2B) as a stable member of the Scgn signalosome, confirmed a putative protein–protein interaction by immunoprecipitation, and thus suggest a role for Scgn in shaping GluN2B surface availability by using fluorescence recovery after photobleaching (FRAP) combined with gene silencing in vitro. These data suggest that Scgn⁺ CeL neurons are central to gate danger responses with Scgn contributing to the assembly of the excitatory postsynaptic machinery at the cell membrane.     

Tropospheric carbonyl sulfide mass balance based on direct measurements of sulfur isotopes

Robust estimates for the rates and trends in terrestrial gross primary production (GPP; plant CO₂ uptake) are needed. Carbonyl sulfide (COS) is the major long-lived sulfur-bearing gas in the atmosphere and a promising proxy for GPP. Large uncertainties in estimating the relative magnitude of the COS sources and sinks limit this approach. Sulfur isotope measurements (³⁴S/³²S; δ³⁴S) have been suggested as a useful tool to constrain COS sources. Yet such measurements are currently scarce for the atmosphere and absent for the marine source and the plant sink, which are two main fluxes. Here we present sulfur isotopes measurements of marine and atmospheric COS, and of plant-uptake fractionation experiments. These measurements resulted in a complete data-based tropospheric COS isotopic mass balance, which allows improved partition of the sources. We found an isotopic (δ³⁴S ± SE) value of 13.9 ± 0.1‰ for the troposphere, with an isotopic seasonal cycle driven by plant uptake. This seasonality agrees with a fractionation of −1.9 ± 0.3‰ which we measured in plant-chamber experiments. Air samples with strong anthropogenic influence indicated an anthropogenic COS isotopic value of 8 ± 1‰. Samples of seawater-equilibrated-air indicate that the marine COS source has an isotopic value of 14.7 ± 1‰. Using our data-based mass balance, we constrained the relative contribution of the two main tropospheric COS sources resulting in 40 ± 17% for the anthropogenic source and 60 ± 20% for the oceanic source. This constraint is important for a better understanding of the global COS budget and its improved use for GPP determination.

The Earth system is going through rapid changes as the climate warms and CO₂ level rises. This rise in CO₂ is mitigated by plant uptake; hence, it is important to estimate global and regional photosynthesis rates and trends (1). Yet, robust tools for investigating these processes at a large scale are scarce (2). Recent studies suggest that carbonyl sulfide (COS) could provide an improved constraint on terrestrial photosynthesis (gross primary production, GPP) (2–12). COS is the major long-lived sulfur-bearing gas in the atmosphere and the main supplier of sulfur to the stratospheric sulfate aerosol layer (13), which exerts a cooling effect on the Earth’s surface and regulates stratospheric ozone chemistry (14).During terrestrial photosynthesis, COS diffuses into leaf stomata and is consumed by photosynthetic enzymes in a similar manner to CO₂ (3–5). Contrary to CO₂, COS undergoes rapid and irreversible hydrolysis mainly by the enzyme carbonic-anhydrase (6, 7). Thus, COS can be used as a proxy for the one-way flux of CO₂ removal from the atmosphere by terrestrial photosynthesis (2, 8–11). However, the large uncertainties in estimating the COS sources weaken this approach (10–12, 15). Tropospheric COS has two main sources: the oceans and anthropogenic emissions, and one main sink–terrestrial plant uptake (8, 10–13). Smaller sources include biomass burning, soil emissions, wetlands, volcanoes, and smaller sinks include OH destruction, stratospheric destruction, and soil uptake (12). The largest source of COS to the atmosphere is the ocean, both as direct COS emission, and as indirect carbon disulfide (CS₂) and dimethylsulfide (DMS) emissions that are rapidly oxidized to COS (10, 16–20). Recent studies suggest oceanic COS emissions are in the range of 200–4,000 GgS/y (19–22). The second major COS source is the anthropogenic source, which is dominated by indirect emissions derived from CS₂ oxidation, mainly from the use of CS₂ as an industrial solvent. Direct emissions of COS are mainly derived from coal and fuel combustion (17, 23, 24). Recent studies suggest that anthropogenic emissions are in the range of 150–585 GgS/y (23, 24). The terrestrial plant uptake is estimated to be in the range of 400–1,360 GgS/y (11). Measurements of sulfur isotope ratios (δ³⁴S) in COS may be used to track COS sources and thus reduce the uncertainties in their flux estimations (15, 25–27). However, the isotopic mass balance approach works best if the COS end members are directly measured and have a significantly different isotopic signature. Previous δ³⁴S measurements of atmospheric COS are scarce and there have been no direct measurements of two important components: the δ³⁴S of oceanic COS emissions, and the isotopic fractionation of COS during plant uptake (15, 25–27). In contrast to previous studies that used assessments for these isotopic values, our aim was to directly measure the isotopic values of these missing components, and to determine the tropospheric COS δ³⁴S variability over space and time.     

Structures of the junctophilin/voltage-gated calcium channel interface reveal hot spot for cardiomyopathy mutations

Junctophilins (JPH) are a class of proteins found at junctions between the plasma membrane and the endoplasmic or sarcoplasmic reticulum, allowing for communications between proteins embedded in different membranes. JPHs have been proposed to interact with lipids as well as several ion channels, allowing for specialized communication between them. The JPH3 isoform is the target for repeats that cause Huntington’s disease-like 2, whereas JPH2 is a hot spot for mutations linked to cardiomyopathy. Here we present crystal structures of two JPH isoforms, which resemble a twisted skeleton with ribs formed by membrane occupation recognition nexus repeats, and a backbone built by a long α-helix. We captured the structure of a complex between JPH2 and a C-terminal binding site in the L-type calcium channel (Ca_V1.1) and show that this interaction is required for clustering of these channels and for robust muscle excitation–contraction coupling. Over 80 sequence variants linked to cardiomyopathy are found in different structurally important regions of JPH2, most of which affect stabilizing interactions. A subset directly affects the interaction with the L-type calcium channel. In parallel, sequence variants in the L-type calcium channel, linked to cardiac arrhythmia, also affect critical interactions.

Junctions between the plasma membrane and the endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) are found in multiple cell types and allow for specialized communication between proteins embedded in these different membranes. Junctophilins (JPH) are key to enabling the formation of such junctions, by virtue of a C-terminal transmembrane helix, located in the ER or SR membrane, and an N-terminal domain containing MORN (membrane occupation recognition nexus) motifs, thought to interact with phospholipids in the plasma membrane (1, 2). As such, JPHs play critical roles in diverse signaling processes, often allowing functional or mechanical cross-talk between ion channels embedded in different membranes.Four isoforms (JPH1–JPH4) are encoded in the human genome, with JPH1 primarily expressed in skeletal muscle, JPH2 in skeletal, cardiac, and smooth muscle, and JPH3/4 mostly found in the brain (3) and in sensory neurons (4). Additionally, JPHs have been found in multiple cell types, including pancreatic β cells (5) and T cells (6). In muscle tissue, JPHs allow for the communication between L-type voltage-gated calcium channels (Ca_V), located in the transverse-tubule (T-tubule) membrane, and ryanodine receptors (RyRs) in the SR membrane. In cardiac myocytes, a depolarization of the plasma membrane activates the Ca_V1.2 isoform, and the influx of Ca²⁺ then triggers opening of RyR2, in a process known as Ca²⁺-induced Ca²⁺ release (7). In skeletal muscle, direct mechanical coupling is thought to occur between the corresponding Ca_V1.1 and RyR1 isoforms, although any direct contacts between these two proteins remain to be elucidated (8, 9). In both scenarios, the coupling requires proximity between the T-tubule and SR membranes, for which JPHs are critical. JPHs have also been suggested to directly bind both Ca_Vs and RyRs, as well as other ion channels including Ca²⁺-activated potassium channels (10–12) and KCNQ1 (13).The importance of JPHs is underscored by the various disorders associated with them. JPH1 has been identified as a modifier of Charcot-Marie-Tooth disease (14). JPH2 is a hot spot for mutations linked to hypertrophic cardiomyopathy (HCM) (15–17), and repeats in JPH3 have been found to cause Huntington disease-like 2 (HDL-2) (18). In addition, cardiac stress results in activation of calpain, which cleaves JPH2 and drives heart failure progression (19–24). Cleaved fragments of JPH2 have also been shown to migrate to the nucleus, where, depending on the fragment type, they either attenuate (22) or promote (21) cellular remodeling. To date, no high-resolution structural information is available for any JPH isoform, hampering insights into its basic functions and disease mechanisms.In this study, we describe high-resolution structures of two JPH isoforms, reveal how JPHs bind the C-terminal region of Ca_V1.1 via a groove formed by the MORN repeats, how this association is important for normal excitation–contraction coupling, and how disease-associated mutations may affect this process.     

Nitric oxide resets kisspeptin-excited GnRH neurons via PIP2 replenishment

Fertility relies upon pulsatile release of gonadotropin-releasing hormone (GnRH) that drives pulsatile luteinizing hormone secretion. Kisspeptin (KP) neurons in the arcuate nucleus are at the center of the GnRH pulse generation and the steroid feedback control of GnRH secretion. However, KP evokes a long-lasting response in GnRH neurons that is hard to reconcile with periodic GnRH activity required to drive GnRH pulses. Using calcium imaging, we show that 1) the tetrodotoxin-insensitive calcium response evoked by KP relies upon the ongoing activity of canonical transient receptor potential channels maintaining voltage-gated calcium channels in an activated state, 2) the duration of the calcium response is determined by the rate of resynthesis of phosphatidylinositol 4,5-bisphosphate (PIP₂), and 3) nitric oxide terminates the calcium response by facilitating the resynthesis of PIP₂ via the canonical pathway guanylyl cyclase/3′,5′-cyclic guanosine monophosphate/protein kinase G. In addition, our data indicate that exposure to nitric oxide after KP facilitates the calcium response to a subsequent KP application. This effect was replicated using electrophysiology on GnRH neurons in acute brain slices. The interplay between KP and nitric oxide signaling provides a mechanism for modulation of the refractory period of GnRH neurons after KP exposure and places nitric oxide as an important component for tonic GnRH neuronal pulses.

Gonadotropin-releasing hormone (GnRH)-secreting neurons integrate multiple physiological and environmental cues and translate them into GnRH secretory patterns, to drive gonadotrophs, which in turn control the gonads. In both sexes, tonic GnRH pulses regulate gametogenesis and steroidogenesis via luteinizing hormone (LH) and follicle-stimulating pulses. Females require a GnRH surge to trigger LH surge and ovulation [reviewed in (1, 2)]. However, disruption of the normal pulsatile GnRH secretion impairs fertility in both sexes (3). Although insufficient for optimal reproductive health (4), the direct action of kisspeptins (KP) on GnRH neurons, via the KP receptor Kiss1r, is required for fertility (4–6). KP neurons play a critical regulatory role in the hypothalamic–pituitary–gonadal axis, including puberty onset (7–9), the preovulatory GnRH surge (5, 9, 10), and tonic GnRH pulses (6, 11). In rodents, two KP neuronal subpopulations exist with distinct functions: the anteroventral periventricular nucleus (AVPV) subpopulation, linked to puberty onset [reviewed in (12)] and preovulatory surge [reviewed in (13)], and the arcuate nucleus (ARC) subpopulation, also known as Kisspeptin-Neurokinin B-Dynorphin (KNDy) neurons, linked to tonic pulses (reviewed in ref. 14).Exogenous KP at the GnRH cell body evokes a long-lasting increase in intracellular calcium levels ([Ca²⁺]_i) (15), often leading to the summation of individual oscillations into [Ca²⁺]_i plateaus (15, 16). This observation is in agreement with an increase in electrical activity where GnRH neurons in acute slices go from burst firing to tonic firing after KP application (17–19). One could argue that this prolonged response is an artifact of the exogenously applied KP. However, endogenously released KP by AVPV stimulation also evokes a long-lasting increase in firing rate (20). Under normal conditions, [Ca²⁺]_i oscillations are driven by bursts of action potentials (AP) (21, 22). Yet, AP are not necessary for the KP-evoked [Ca²⁺]_i response to occur, as it is driven by multiple effectors including transient receptor potential-canonical channels (TRPC), voltage-gated calcium channels (VGCC), and inositol 1,4,5-trisphosphate receptors (InsP3R) (15, 16, 19, 23). Thus, the versatility of Kiss1r signaling pathway underlies the functionality of KP projections along GnRH neuron processes (24), with KP locally applied on nerve terminals also evoking a long-lasting increase in [Ca²⁺]_i (16).While the long-lasting KP response is suitable to trigger the preovulatory surge, it seems incompatible with the KNDy model for tonic pulses. GnRH and LH pulses occur every ∼20 min in ovariectomized mice (25). Indeed, the KNDy model provides on-/off- signals for KNDy neurons, neurokinin B and dynorphin respectively, and an on-signal for GnRH neurons, KP. However, this model lacks an off-signal for GnRH neurons. KNDy neurons trigger GnRH/LH pulses via Kiss1r (26), but the “extinction” of KNDy neurons by dynorphin is not obligatory for the termination of GnRH/LH pulses in rodents (27, 28), and dynorphin is lacking in human KP-neurokinin neurons (29).In fact, how the response in firing and [Ca²⁺]_i to KP relate to GnRH secretion is unknown. In acute coronal brain slices, the KP-evoked increase in firing at the cell body cannot be repeated (19, 30). In contrast, in acute sagittal brain slices, using fast scan cyclic voltammetry, the KP-evoked GnRH secretion from cells in the preoptic area (POA) and fibers in the median eminence (ME) can be triggered repeatedly (31). The difference in repeatability at the cell body is puzzling. One explanation could be a technical consequence of brain slicing or conventional patch clamp. Another explanation could be a dissociation between firing and [Ca²⁺]_i during the second KP application. In fact, the common feature of the KP-evoked increase in [Ca²⁺]_i and GnRH secretion, at the GnRH neuron cell body and nerve terminal, is that it is AP-independent (15, 16, 31). The current study uses calcium imaging and electrophysiology to address the mechanisms that allow 1) [Ca²⁺]_i in GnRH neurons to return to baseline after KP stimulation, and 2) GnRH neurons to respond to a second KP stimulation (i.e., repeatability) and shows nitric oxide as an important component for tonic GnRH neuronal pulses.