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.     

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.     

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.     

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.     

Heme-binding protein CYB5D1 is a radial spoke component required for coordinated ciliary beating

Coordinated beating is crucial for the function of multiple cilia. However, the molecular mechanism is poorly understood. Here, we characterize a conserved ciliary protein CYB5D1 with a heme-binding domain and a cordon-bleu ubiquitin-like domain. Mutation or knockdown of Cyb5d1 in zebrafish impaired coordinated ciliary beating in the otic vesicle and olfactory epithelium. Similarly, the two flagella of an insertional mutant of the CYB5D1 ortholog in Chlamydomonas (Crcyb5d1) showed an uncoordinated pattern due to a defect in the cis-flagellum. Biochemical analyses revealed that CrCYB5D1 is a radial spoke stalk protein that binds heme only under oxidizing conditions. Lack of CrCYB5D1 resulted in a reductive shift in flagellar redox state and slowing down of the phototactic response. Treatment of Crcyb5d1 with oxidants restored coordinated flagellar beating. Taken together, these data suggest that CrCYB5D1 may integrate environmental and intraciliary signals and regulate the redox state of cilia, which is crucial for the coordinated beating of multiple cilia.

Cilia and flagella are highly conserved organelles that project from the surface of most eukaryotic cells and perform sensory, secretory, and motile functions (1, 2). The single motile flagellum of spermatozoa propels cell body movement, whereas the two flagella of the green alga Chlamydomonas reinhardtii mediate oriented swimming. Coordinated beating of multiple cilia on epithelial surfaces, for example, in the trachea, oviduct, and brain ventricles of vertebrates, is crucial for mucus clearance in the airway, ovum transport in the oviduct, and cerebrospinal fluid circulation in the brain ventricles (3). Impairment of coordinated beating of these multiple cilia in humans can result in ciliopathies such as chronic respiratory problems, infertility, and hydrocephalus (4).The beating of individual cilia/flagella depends on axonemal dyneins, which are AAA⁺ enzymes that convert ATP (adenosine triphosphate) hydrolysis into mechanical force, resulting in the sliding of doublet microtubules (5). The activity of dyneins is spatiotemporally controlled by various regulatory pathways including changes in Ca²⁺, phosphorylation, and redox state (5). Recent Cryo-EM (cryo-electron microscopy) studies have revealed that these dyneins are generally in a force-balanced state in straight axonemal regions, and that the initiation and formation of a ciliary bend by microtubule sliding may depend on an inhibitory signal that is propagated from the base-to-tip to disrupt the force-balanced state (6). To achieve coordinated beating, the activity of multiple cilia must be synchronized by both intrinsic and extrinsic factors including hydrodynamic coupling, axonemal mechanical feedback, connective structures in the basal body region, and Ca²⁺ signaling (7–10). The hydrodynamic hypothesis is supported by the observation that when two sperm or two micropipette-held somatic cells of Volvox carteri were placed in close proximity under hydrodynamic flow, the beating of two flagella was spontaneously synchronized (8, 10). However, multiple cilia can still maintain coordinated beating when fluid flow disappears (11), suggesting that other mechanisms are also involved.The mechanical feedback system was proposed based on the study of light chain 1 (LCI) from Chlamydomonas. LC1 contains six leucine-rich repeats that form an elongated barrel (12) and binds to the microtubule-binding domain of the γ-HC (γ heavy chain) specifically (13). LC1 mutant proteins expressed in wild type Chlamydomonas resulted in uncoordinated flagellar beating (14). Knockdowns of LC1 in planaria also led to the loss of metachronal synchrony in the ventral cilia (7). As a conformational switch, LC1 may sense changes in axonemal curvature imposed by hydrodynamic flow and thereby modulate the activity of outer dynein arm to coordinate flagellar beating. This model is also supported by recent structural data on the LC1–γ-HC microtubule-binding domain complex (15).Coordinated beating of algal flagella is also achieved by basal coupling through filamentary connections between basal bodies (10). These connectors control basal body alignment and cilia orientation in multiciliated cells and may also participate in coordinated beating by integrating the chemical or mechanical signals among cilia (9). However, the nature of these signals remains unknown. Ca²⁺ is a common second messenger in cilia and may also participate in coordinated beating. Knock-down of the components of the calmodulin-associated complex, such as CaM-IP2, CaM-IP3, PF6, and PCDP1, resulted in uncoordinated flagellar beating in Chlamydomonas (16–18). However, the coordination defect may be a secondary effect since motility-related structures such as the central pair and radial spokes exhibit structural alterations in these mutants. The two flagella of Chlamydomonas respond differently to changes of Ca²⁺ concentration at the low μM range, which alters flagellar coordination and results in phototactic turning (19, 20). Therefore, Ca²⁺ functions in modulating flagellar coordination during these processes.Recent studies have shown that redox state also participates in regulating ciliary/flagellar beating in Chlamydomonas and mammals. The beat frequency of flagella was reduced when Chlamydomonas cells were treated with oxidizing agents (21). Redox poise also regulates the sign of phototaxis in Chlamydomonas (22). When multiple cilia on airway epithelial cells were exposed to oxidants, the mucociliary transport was inhibited (23). In addition, several redox-sensitive components have been identified in flagellar motility-related complexes, such as LC3, LC5, γ-HC, and outer dynein arm-docking complex protein 3 (DC3) in Chlamydomonas (24–26). Disruption of human TXNDC3 and TXNDC6, which contain modules orthologous to the thioredoxin-like LC3 and LC5 proteins in Chlamydomonas, results in respiratory disease (27, 28). Taken together, these findings suggest that redox plays a vital role in coordinating ciliary beating; however, the molecular mechanisms and pathways that mediate the redox response are unknown.Here, we identify a highly conserved ciliary protein cytochrome b5-like/heme-binding domain (CYB5D1), which is required for the coordinated beating of multiple cilia in zebrafish and Chlamydomonas. Furthermore, our results indicate that this heme-binding protein may function in a redox signaling pathway to coordinate ciliary beating.     

Reconstitution of β-adrenergic regulation of CaV1.2: Rad-dependent and Rad-independent protein kinase A mechanisms

L-type voltage-gated Ca_V1.2 channels crucially regulate cardiac muscle contraction. Activation of β-adrenergic receptors (β-AR) augments contraction via protein kinase A (PKA)–induced increase of calcium influx through Ca_V1.2 channels. To date, the full β-AR cascade has never been heterologously reconstituted. A recent study identified Rad, a Ca_V1.2 inhibitory protein, as essential for PKA regulation of Ca_V1.2. We corroborated this finding and reconstituted the complete pathway with agonist activation of β1-AR or β2-AR in Xenopus oocytes. We found, and distinguished between, two distinct pathways of PKA modulation of Ca_V1.2: Rad dependent (∼80% of total) and Rad independent. The reconstituted system reproduces the known features of β-AR regulation in cardiomyocytes and reveals several aspects: the differential regulation of posttranslationally modified Ca_V1.2 variants and the distinct features of β1-AR versus β2-AR activity. This system allows for the addressing of central unresolved issues in the β-AR–Ca_V1.2 cascade and will facilitate the development of therapies for catecholamine-induced cardiac pathologies.

Cardiac excitation–contraction coupling crucially depends on the L-type voltage-dependent Ca²⁺ channel, Ca_V1.2. Influx of extracellular Ca²⁺ via Ca_V1.2 triggers Ca²⁺ release from the sarcoplasmic reticulum via the Ca²⁺ release channel (1). Activation of the sympathetic nervous system increases heart rate, relaxation rate and contraction force. The latter is largely due to increased Ca²⁺ influx via Ca_V1.2 (2, 3). Pathological prolonged sympathetic activation progressively impairs cardiac function, causing heart failure, partly due to misregulation of Ca_V1.2 (4, 5).Cardiac Ca_V1.2 is a heterotrimer comprising the pore-forming subunit α_1C (∼240 kDa), the intracellular Ca_Vβ₂ (∼68 kDa) and the extracellular α2δ (∼170 kDa) (Fig. 1A) (6, 7). The N and C termini (NT, CT respectively) of α_1C are cytosolic and vary among Ca_V1.2 isoforms. Further, most of the cardiac α_1C protein is posttranslationally cleaved at the CT, around amino acid (a.a.) 1800, to produce the truncated ∼210-kDa α_1C protein and the ∼35-kDa cleaved distal CT (dCT); however, the full-length protein is also present (8–11).Open in a separate windowFig. 1.cAMP regulation of Ca_V1.2 is enhanced by coexpression of Rad. (A) Ca_V1.2 and Rad. α_1C and α2δ subunits are shown schematically, with structures of β_2b (38) and Rad (74). The truncation in α_1CΔ1821 was at a.a. 1,821 (red cross mark) similar to naturally truncated cardiac α_1C, ∼a.a. 1800 (9). Ca_Vβ binds to the cytosolic loop I, L1, that connects repeat domains I and II. Rad exerts inhibitory action on the channel, in part through an interaction with Ca_Vβ. (B) Rad reduces the Ba²⁺ current of Ca_V1.2-α_1CΔ1821 (α_1CΔ1821, β_2b and α2δ; 1.5 ng RNA of each subunit) in a dose-dependent manner. Pearson correlation, r = −0.82, P = 0.023. Each point represents mean ± SEM from 7 to 10 oocytes recorded during 1 d. The linear regression line was drawn for nonzero doses of Rad. (C) Rad enhances the cAMP-induced increase in I_Ba. Diary plots of the time course of change in I_Ba (normalized to initial I_Ba) are shown before and after intracellular injection of cAMP in representative cells. No Rad: Upper; with Rad: Lower. (Insets) Currents at +20 mV before (black trace) and 10 min after cAMP injection (red trace). (D) “before–after” plots of cAMP-induced changes in I_Ba in individual cells injected Rad RNA while varying Rad:β_2b RNA ratio (by weight, wt/wt). Empty symbols–before cAMP; red-filled–after cAMP. n = 3 experiments; statistics: paired t test. (E) cAMP-induced increase in I_Ba at different Rad/β_2b RNA levels (summary of data from D). Each symbol represents fold increase in I_Ba induced by cAMP injection in one cell. Here and in the following figures, box plots show 25 to 75 percentiles, whiskers show the 5/95 percentiles, and black and red horizontal lines within the boxes are the median and mean, respectively. At all Rad:β_2b RNA ratios except 1:20, the cAMP-induced increase in I_Ba was significantly greater than without Rad (Kruskal–Wallis test; H = 36.1, 6 degrees of freedom, P < 0.001). (F) Summary of cAMP effects in 10 experiments without and with Rad at 1:2 and 1:1 Rad:β_2b RNA ratios (pooled). Number of cells: within the bars. Statistics: Mann–Whitney U test; U = 19.0, P < 0.001.The sympathetic nervous system activates cardiac β-adrenergic receptors (β-AR), primarily β1-AR (which is coupled to G_s, is globally distributed in cardiomyocytes, and mediates most of the β-AR-enhancement of contraction and Ca_V1.2 activity) and β2-AR, which can couple to both G_s and G_i (12). The cascade of adrenergic modulation of Ca_V1.2 comprises agonist binding to β-ARs, activation of G_s and adenylyl cyclase, elevated intracellular cAMP levels, and activation of protein kinase A (PKA) by cAMP-induced dissociation of its catalytic subunit (PKA-CS) from the regulatory subunit. However, the final step, how PKA-CS enhances Ca_V1.2 activity, remained enigmatic. A long-standing paradigm was a direct phosphorylation by PKA-CS of α_1C and/or Ca_Vβ subunits (3, 13–16). However, numerous studies critically challenged this theory. In particular, mutated Ca_V1.2 channels in genetically engineered mice lacking putative PKA phosphorylation sites on α_1C and/or β_2b, were still up-regulated by PKA (9, 17–21) (reviewed in refs. 6 and 22).One significant obstacle in deciphering the mechanism of PKA regulation of Ca_V1.2 was a recurrent lack of success in reconstituting the regulation in heterologous systems, which proved challenging and controversial (23). Studies in heterologous cellular models, including Xenopus oocytes, demonstrated that cAMP failed to up-regulate Ca_V1.2 containing the full-length α_1C, Ca_V1.2-α_1C (24–26). However, robust β-AR–induced up-regulation of Ca²⁺ currents was observed in oocytes injected with total heart RNA (27, 28), suggesting the necessity of an auxiliary protein, the “missing link” (24, 25). Interestingly, partial regulation was observed with dCT-truncated α_1C (16, 29). Intracellular injection of cAMP or PKA-CS in Xenopus oocytes caused a modest (30 to 40%) up-regulation of Ca_V1.2, containing a dCT-truncated α_1C, Ca_V1.2-α_1CΔ1821 (29). This regulation required the presence of the initial segment of the long-NT of α_1C but did not involve Ca_Vβ subunit. We proposed that this mechanism might account for part of the adrenergic regulation of Ca_V1.2 in the heart (29). Normally adrenergic stimulation in cardiomyocytes increases the Ca²⁺ current two- to threefold; thus, a major part of the regulation has remained unexplained.Recently, Liu et al. identified Rad as the “missing link” in PKA regulation of Ca_V1.2 (20). Rad is a member of the Ras-related GTP-binding protein subfamily (RGK) that inhibit high voltage-gated calcium channels Ca_V1 and Ca_V2 (30). Rad tonically inhibits Ca_V1.2, largely via an interaction with Ca_Vβ (31, 32). Ablation of Rad in murine heart was shown to increase basal Ca_V1.2 activity and rendered the channel insensitive to β-AR regulation, probably through a “ceiling” effect (33, 34). Liu et al. (20) reconstituted a major part of the Ca_V1.2 regulation cascade, initiated by forskolin-activated adenylyl cyclase in mammalian cells, ultimately attaining an approximately twofold increase in Ca²⁺ current. The regulation required phosphorylation of Rad, the presence of Ca_Vβ, and the interaction of Ca_Vβ with the cytosolic loop I of α_1C, suggesting that PKA phosphorylation of Rad reduces its interaction with Ca_Vβ and relieves the tonic inhibition of Ca_V1.2 (20, 35).Importantly, the complete adrenergic cascade, starting with β-AR activation, has not yet been heterologously reconstituted for Ca_V1.2. Also, the relation between the Rad-dependent regulation and the regulation reported in our previous study (29) is not clear. Here, we utilized the Xenopus oocyte heterologous expression system and successfully reconstituted the entire β-AR cascade. We demonstrate two distinct pathways of PKA modulation of Ca_V1.2 (Rad dependent and Rad independent) and characterize the roles of NT and CT of α_1C, β_2b, and Rad in the adrenergic modulation of cardiac Ca_V1.2 channels. Reproducing the complete β-AR cascade in a heterologous expression system will promote the identification and characterization of intracellular proteins that regulate the cascade, eventually assisting efforts to develop therapies to treat heart failure and other catecholamine-induced cardiac pathologies.     

INAUGURAL ARTICLE by a Recently Elected Academy Member:The effect of parathyroid hormone on osteogenesis is mediated partly by osteolectin

We previously described a new osteogenic growth factor, osteolectin/Clec11a, which is required for the maintenance of skeletal bone mass during adulthood. Osteolectin binds to Integrin α11 (Itga11), promoting Wnt pathway activation and osteogenic differentiation by leptin receptor⁺ (LepR⁺) stromal cells in the bone marrow. Parathyroid hormone (PTH) and sclerostin inhibitor (SOSTi) are bone anabolic agents that are administered to patients with osteoporosis. Here we tested whether osteolectin mediates the effects of PTH or SOSTi on bone formation. We discovered that PTH promoted Osteolectin expression by bone marrow stromal cells within hours of administration and that PTH treatment increased serum osteolectin levels in mice and humans. Osteolectin deficiency in mice attenuated Wnt pathway activation by PTH in bone marrow stromal cells and reduced the osteogenic response to PTH in vitro and in vivo. In contrast, SOSTi did not affect serum osteolectin levels and osteolectin was not required for SOSTi-induced bone formation. Combined administration of osteolectin and PTH, but not osteolectin and SOSTi, additively increased bone volume. PTH thus promotes osteolectin expression and osteolectin mediates part of the effect of PTH on bone formation.

The maintenance and repair of the skeleton require the generation of new bone cells throughout adult life. Osteoblasts are relatively short-lived cells that are constantly regenerated, partly by skeletal stem cells within the bone marrow (1). The main source of new osteoblasts in adult bone marrow is leptin receptor-expressing (LepR⁺) stromal cells (2–4). These cells include the multipotent skeletal stem cells that give rise to the fibroblast colony-forming cells (CFU-Fs) in the bone marrow (2), as well as restricted osteogenic progenitors (5) and adipocyte progenitors (6–8). LepR⁺ cells are a major source of osteoblasts for fracture repair (2) and growth factors for hematopoietic stem cell maintenance (9–11).One growth factor synthesized by LepR⁺ cells, as well as osteoblasts and osteocytes, is osteolectin/Clec11a, a secreted glycoprotein of the C-type lectin domain superfamily (5, 12, 13). Osteolectin is an osteogenic factor that promotes the maintenance of the adult skeleton by promoting the differentiation of LepR⁺ cells into osteoblasts. Osteolectin acts by binding to integrin α11β1, which is selectively expressed by LepR⁺ cells and osteoblasts, activating the Wnt pathway (12). Deficiency for either Osteolectin or Itga11 (the gene that encodes integrin α11) reduces osteogenesis during adulthood and causes early-onset osteoporosis in mice (12, 13). Recombinant osteolectin promotes osteogenic differentiation by bone marrow stromal cells in culture and daily injection of mice with osteolectin systemically promotes bone formation.Osteoporosis is a progressive condition characterized by reduced bone mass and increased fracture risk (14). Several factors contribute to osteoporosis development, including aging, estrogen insufficiency, mechanical unloading, and prolonged glucocorticoid use (14). Existing therapies include antiresorptive agents that slow bone loss, such as bisphosphonates (15, 16) and estrogens (17), and anabolic agents that increase bone formation, such as parathyroid hormone (PTH) (18), PTH-related protein (19), and sclerostin inhibitor (SOSTi) (20). While these therapies increase bone mass and reduce fracture risk, they are not a cure.PTH promotes both anabolic and catabolic bone remodeling (21–24). PTH is synthesized by the parathyroid gland and regulates serum calcium levels, partly by regulating bone formation and bone resorption (23–25). PTH1R is a PTH receptor (26, 27) that is strongly expressed by LepR⁺ bone marrow stromal cells (8, 28–30). Recombinant human PTH (Teriparatide; amino acids 1 to 34) and synthetic PTH-related protein (Abaloparatide) are approved by the US Food and Drug Administration (FDA) for the treatment of osteoporosis (19, 31). Daily (intermittent) administration of PTH increases bone mass by promoting the differentiation of osteoblast progenitors, inhibiting osteoblast and osteocyte apoptosis, and reducing sclerostin levels (32–35). PTH promotes osteoblast differentiation by activating Wnt and BMP signaling in bone marrow stromal cells (28, 36, 37), although the mechanisms by which it regulates Wnt pathway activation are complex and uncertain (38).Sclerostin is a secreted glycoprotein that inhibits Wnt pathway activation by binding to LRP5/6, a widely expressed Wnt receptor (7, 8), reducing bone formation (39, 40). Sclerostin is secreted by osteocytes (8, 41), negatively regulating bone formation by inhibiting the differentiation of osteoblasts (41, 42). SOSTi (Romosozumab) is a humanized monoclonal antibody that binds sclerostin, preventing binding to LRP5/6 and increasing Wnt pathway activation and bone formation (43). It is FDA-approved for the treatment of osteoporosis (20, 44) and has activity in rodents in addition to humans (45, 46).The discovery that osteolectin is a bone-forming growth factor raises the question of whether it mediates the effects of PTH or SOSTi on osteogenesis.     

Structure and activity of SLAC1 channels for stomatal signaling in leaves

Stomata in leaves regulate gas exchange between the plant and its atmosphere. Various environmental stimuli elicit abscisic acid (ABA); ABA leads to phosphoactivation of slow anion channel 1 (SLAC1); SLAC1 activity reduces turgor pressure in aperture-defining guard cells; and stomatal closure ensues. We used electrophysiology for functional characterizations of Arabidopsis thaliana SLAC1 (AtSLAC1) and cryoelectron microscopy (cryo-EM) for structural analysis of Brachypodium distachyon SLAC1 (BdSLAC1), at 2.97-Å resolution. We identified 14 phosphorylation sites in AtSLAC1 and showed nearly 330-fold channel-activity enhancement with 4 to 6 of these phosphorylated. Seven SLAC1-conserved arginines are poised in BdSLAC1 for regulatory interaction with the N-terminal extension. This BdSLAC1 structure has its pores closed, in a basal state, spring loaded by phenylalanyl residues in high-energy conformations. SLAC1 phosphorylation fine-tunes an equilibrium between basal and activated SLAC1 trimers, thereby controlling the degree of stomatal opening.

The stomatal pore, formed by a pair of specialized guard cells in plant leaves, serves as the gateway for water transpiration and atmospheric CO₂ influx for photosynthesis (1, 2). These pores need to be tightly controlled, as inadequate CO₂ intake is harmful and excessive water loss is devastating for plants (3). The guard cells respond to a wide range of environmental stimuli, including high CO₂ levels, O₃, low air humidity, and drought, and transduce these signals into appropriate turgor pressure changes that can adjust stomatal pore aperture (4, 5). It is well established that turgor pressure of guard cells is regulated by ion transport across the membrane, notably anions and potassium ions (6–8).The phytohormone abscisic acid (ABA) plays a central role in controlling stomatal closure via activation of a complex signaling pathway that is mediated by receptors, kinase/phosphatases, and ion channels (5, 9). It has been known that the activation of a slow anion current in guard cells is a key event leading to stomatal closure (10–13). The corresponding gene, slow anion channel 1 (SLAC1), was discovered in genetic screens for O₃ or CO₂ sensitivity in Arabidopsis; when this particular channel was mutated out, the mutants showed impaired response to stimuli (14–16).Arabidopsis thaliana SLAC1 (AtSLAC1) contains 556 amino acid (aa) residues, with a conserved transmembrane domain (TMD) between hydrophilic tails at both the N and C termini (15). Within the ABA signaling pathway, SLAC1 channel activity is controlled by a protein kinase-phosphatase pair (OST1/ABI1) (17–19). In the absence of ABA, OST1 kinase is bound and inhibited by ABI1 phosphatase (20–22). Under drought condition, ABA is accumulated into guard cells and perceived by the PYR1 receptor (23, 24). This resulting hormone–receptor complex then binds to phosphatase ABI1 to form a ternary hormone–receptor–phosphatase complex (25–28), which activates OST1 kinase, and leads to phosphorylation of SLAC1 (17, 18). Subsequently, SLAC1 releases anions (Cl⁻ and NO₃⁻) from guard cells, leading to membrane depolarization. Depolarization in turn activates potassium channels to release K⁺ via K⁺ channels and drives water out of the cell, decreasing guard cell turgor to close down the stomatal pore (8).It has been shown that SLAC1 activation is due to phosphorylation at its N terminus (17, 18, 29–31). The phosphorylation of S120 by OST1 is essential but not sufficient for SLAC1 activation monitored in Xenopus oocytes (18). S59 has been shown to be the target for both OST1 and other kinases (29, 31). In another phosphoproteomic analysis, using the fragment SLAC1¹⁻¹⁸⁶ as substrate, S86 and S113 were also shown to be phosphorylated by OST1 (32). OST1 also phosphorylates SLAC1 at the C terminus (17), with T513 identified as a potential phosphorylation site for SLAC1 activation (31). Although multiple phosphorylation sites have been proposed, the underlying mechanism for SLAC1 activation remains elusive.Our previous study on a SLAC1 bacterial homolog from Haemophilus influenzae (HiTehA) revealed a superfamily of trimeric anion channels (33) and found each protomer gated by a highly conserved phenylalanine. We observed an unusual high-energy rotameric conformation for this gating phenylalanine (F262 in HiTehA), and further mutational study on the corresponding site in AtSLAC1 (F450) suggested a unique mechanism for channel activation (33). Nevertheless, since HiTehA shares a mere 19% in sequence identity with AtSLAC1 and lacks the substantial N and C termini that are essential for channel activation, questions remain about SLAC1 structure and its regulation by kinase phosphorylation (7).Here we describe the cryoelectron microscopy (cryo-EM) structure of SLAC1 from Brachypodium distachyon (BdSLAC1) at 2.97-Å resolution, and we further characterize the channel activity on Arabidopsis SLAC1. BdSLAC1 shares 63% overall sequence identity with AtSLAC1, with 73% for the pore-forming TMD and 48% for the regulatory N and C termini. As for the bacterial homolog, each BdSLAC1 TMD comprises five tandemly repeated helical hairpins that surround a transmembrane pore in each protomer of the trimeric assembly. The SLAC1 pores are electropositive, consistent with its function as an anion channel. The regulatory N-terminal domain (190 aa) and C-terminal domain (65 aa) are flexibly associated and not discernible in the structure.We employed a SLAC1::OST1 fusion design for examining SLAC1 phosphorylation, whereby we systematically characterized critical sites responsible for channel activation. Inspired by the structure, we also conducted mutagenesis on the highly conserved phenylalanine residues related to channel gating. Altogether, our structural and functional analyses provide insights into SLAC1 gating and activity modulation. These findings allow us to propose a mechanism for finely tuned SLAC1 control of the stomatal pore in response to environmental stimuli.     

Shear stress activates mitochondrial oxidative phosphorylation by reducing plasma membrane cholesterol in vascular endothelial cells

Vascular endothelial cells (ECs) sense and respond to hemodynamic shear stress, which is critical for circulatory homeostasis and the pathophysiology of vascular diseases. The mechanisms of shear stress mechanotransduction, however, remain elusive. We previously demonstrated a direct role of mitochondria in the purinergic signaling of shear stress: shear stress increases mitochondrial adenosine triphosphate (ATP) production, triggering ATP release and Ca²⁺ signaling via EC purinoceptors. Here, we showed that shear stress rapidly decreases cholesterol in the plasma membrane, thereby activating mitochondrial ATP production. Imaging using domain 4 mutant-derived cholesterol biosensors showed that the application of shear stress to cultured ECs markedly decreased cholesterol levels in both the outer and inner plasma membrane bilayers. Flow cytometry showed that the cholesterol levels in the outer bilayer decreased rapidly after the onset of shear stress, reached a minimum (around 60% of the control level) at 10 min, and plateaued thereafter. After the shear stress ceased, the decreased cholesterol levels returned to those seen in the control. A biochemical analysis showed that shear stress caused both the efflux and the internalization of plasma membrane cholesterol. ATP biosensor imaging demonstrated that shear stress significantly increased mitochondrial ATP production. Similarly, the treatment of cells with methyl-β-cyclodextrin (MβCD), a membrane cholesterol-depleting agent, increased mitochondrial ATP production. The addition of cholesterol to cells inhibited the increasing effects of both shear stress and MβCD on mitochondrial ATP production in a dose-dependent manner. These findings indicate that plasma membrane cholesterol dynamics are closely coupled to mitochondrial oxidative phosphorylation in ECs.

Vascular endothelial cells (ECs) recognize shear stress, a biomechanical force generated by flowing blood, and transduce it into intracellular biochemical signals, thereby causing responses such as changes in cell morphology, function, and gene expression (1). These EC responses play crucial roles in maintaining the homeostasis of the circulatory system, and their impairments cause various vascular diseases such as hypertension, aneurysm, and atherosclerosis (2–4). To date, numerous studies have elucidated the mechanism of EC mechanotransduction and have revealed a unique feature: shear stress activates multiple signal transduction pathways through a variety of membrane molecules, including ion channels, receptors, and adhesion proteins, almost simultaneously (5). Recently, it has become apparent that the plasma membrane itself plays an important role in EC mechanotransduction. EC plasma membranes rapidly respond to shear stress by altering their physical properties, such as fluidity, viscosity, and lipid order (6, 7). We previously demonstrated that shear stress decreases the lipid order of not only EC plasma membranes but also, artificial lipid bilayer membranes, thereby causing a transition from the liquid-ordered state to the liquid-disordered state, along with an increase in membrane fluidity (8). These changes in membrane physical properties were linked to downstream signaling pathways, such as the activation of mitogen-activated protein kinase (9) and the phosphorylation of vascular endothelial growth factor receptors (VEGFRs) (10). This mechanism, in which changes in membrane physical properties occur initially and are then followed by the activation of membrane molecules, may characterize the EC mechanotransduction described above (11).Ca²⁺ signaling is known to play a critical role in EC mechanotransduction (12). ECs rapidly release intrinsic adenosine triphosphate (ATP) in response to shear stress (13, 14), and this ATP activates purinoceptors, such as ligand-gated channel P2X and G protein-coupled P2Y receptors, located in the plasma membranes; these purinoceptors are responsible for extracellular Ca²⁺ influx and Ca²⁺ release from the endoplasmic reticulum (15–17). The increase in cytoplasmic Ca²⁺ activates a variety of EC functions. In P2X4-knockout mice, ECs exhibited neither Ca²⁺ signaling nor the production of a potent vasodilator, nitric oxide, in response to shear stress, thereby impairing the blood flow-dependent vasodilator response and vascular remodeling and resulting in hypertension (4). Our recent study revealed that mitochondria play an important role in shear stress-induced ATP release and Ca²⁺ signaling in ECs (18). Upon shear stress stimulation, the ECs rapidly augmented their mitochondrial ATP production, triggering ATP release and subsequent Ca²⁺ signaling. However, the means by which shear stress acting on the plasma membrane affects mitochondrial oxidative phosphorylation in ECs remains unknown.Cholesterol plays a dominant role in determining the mechanical properties of plasma membranes by affecting the membrane lipid order, fluidity, bending modulus, thickness, stiffness, and bilayer pressure profile; in this manner, cholesterol modulates the conformation and function of membrane proteins (19). Plasma membrane cholesterol is involved in not only the control of various cell functions, such as signaling, adhesion, motility, and remodeling of the cytoskeleton (20), but also EC responses to biomechanical forces (10, 21–23). For instance, the depletion of EC plasma membrane cholesterol inhibits the shear stress-induced activation of extracellular signal-regulated kinase (22), whereas the addition of cholesterol to EC plasma membranes suppresses the shear stress-induced phosphorylation of VEGFRs (10). However, the roles of plasma membrane cholesterol in EC mechanotransduction have not been fully elucidated, partly because of a lack of information concerning how plasma membrane cholesterol behaves in ECs under shear stress.In the last decade, domain 4 (D4) of perfringolysin O (PFO), a cholesterol-binding toxin, has been widely used as a cholesterol-specific molecular sensor for measuring and imaging cholesterol in cellular membranes (24). When D4 and its mutants labeled with fluorophores are added to the extracellular medium, cholesterol in the outer leaflet of the plasma membrane can be visualized in living cells; when these sensors are introduced into the cells by gene transfer or microinjection, cholesterol in the inner leaflets of the membrane can be visualized. A recent study showed that these D4-based cholesterol sensors did not lyse the cells or cross the plasma membrane, allowing trans-bilayer asymmetries in plasma membrane cholesterol to be identified in various mammalian cells (25).In the present study, we applied controlled levels of shear stress to cultured human pulmonary aortic endothelial cells (HPAECs) and examined changes in the amounts and distribution of plasma membrane cholesterol using fluorescence imaging and flow cytometry with D4 mutant-based cholesterol biosensors. We also used biochemical measurements to analyze changes in the amounts of cholesterol in whole cells and isolated plasma membranes. Furthermore, to determine whether changes in plasma membrane cholesterol affect mitochondrial oxidative phosphorylation, we examined changes in mitochondrial ATP concentrations using real-time imaging with a fluorescence resonance energy transfer (FRET)-based ATP biosensor (26).     

Goblet cell LRRC26 regulates BK channel activation and protects against colitis in mice

Goblet cells (GCs) are specialized cells of the intestinal epithelium contributing critically to mucosal homeostasis. One of the functions of GCs is to produce and secrete MUC2, the mucin that forms the scaffold of the intestinal mucus layer coating the epithelium and separates the luminal pathogens and commensal microbiota from the host tissues. Although a variety of ion channels and transporters are thought to impact on MUC2 secretion, the specific cellular mechanisms that regulate GC function remain incompletely understood. Previously, we demonstrated that leucine-rich repeat-containing protein 26 (LRRC26), a known regulatory subunit of the Ca²⁺-and voltage-activated K⁺ channel (BK channel), localizes specifically to secretory cells within the intestinal tract. Here, utilizing a mouse model in which MUC2 is fluorescently tagged, thereby allowing visualization of single GCs in intact colonic crypts, we show that murine colonic GCs have functional LRRC26-associated BK channels. In the absence of LRRC26, BK channels are present in GCs, but are not activated at physiological conditions. In contrast, all tested MUC2⁻ cells completely lacked BK channels. Moreover, LRRC26-associated BK channels underlie the BK channel contribution to the resting transepithelial current across mouse distal colonic mucosa. Genetic ablation of either LRRC26 or BK pore-forming α-subunit in mice results in a dramatically enhanced susceptibility to colitis induced by dextran sodium sulfate. These results demonstrate that normal potassium flux through LRRC26-associated BK channels in GCs has protective effects against colitis in mice.

The colonic epithelium is composed of a single layer of heterogeneous cells, covered by mucus, that separate the luminal contents from host tissues. Acting both in concert and individually, the diverse cells comprising the epithelial layer play the functions of protection (1), sensation (2, 3), transport of substances (4, 5), and repair (6). Colonic epithelial cells belong to three lineages: Absorptive enterocytes, enteroendocrine cells, and goblet cells (GCs). The colonic epithelium is morphologically organized into repeating units called crypts of Lieberkühn, where stem cells located at the base of the crypts divide and successively differentiate into the mature lineages as they migrate toward the crypt surface (7). Many of the key specialized functions of epithelial cells are, in part, defined by proteins involved in ion transport, located either on their luminal or basolateral membrane. Thus, among different gastrointestinal epithelial cells, ion channels, carriers, exchangers, and pumps work in concert to define a variety of essential functions: 1) Solute and electrolyte absorption and secretion in absorptive enterocytes (reviewed in refs. 5 and 8); 2) environment sensation and serotonin secretion by enteroendocrine cells (2, 9); and 3) mucus secretion by GCs and subsequent mucus maturation into the protective layer covering the epithelial surface (10–12). Despite this progress, ionic transport in GCs and its implications in GC physiology is a topic that remains poorly understood. Here, we address the role of the Ca²⁺- and voltage-activated K⁺ channel (BK channel) in GCs.GCs play two primary roles: One related to the maintenance of the mucosal barrier (reviewed in refs. 1 and 13) and one related with the mucosal immune homeostasis (reviewed in refs. 14 and 15). The role of GCs in barrier maintenance consists in generation of the mucus layer lining the intestinal lumen. One way GCs carry out this role is by secreting MUC2, the gel-forming mucin that forms the scaffold of the mucus layer separating luminal pathogens and commensal microbiota from the epithelial surface (11, 12, 15, 16). This separation is critical, as has been demonstrated in both animal models and humans: Mouse models with deficient mucus layer generation develop spontaneous colitis (16, 17), whereas a more penetrable mucus layer has been observed in patients with ulcerative colitis (UC), a form of human inflammatory bowel disease (IBD) (18, 19). The constant replenishment of the mucus layer involves MUC2 exocytosis from GCs, and subsequent maturation (hydration and expansion) of the secreted MUC2 to form the gel-like mucus coating the epithelium (15). Both exocytosis and maturation of MUC2 are highly dependent on anion and K⁺ transport (10–12, 20). It has been proposed that mucin exocytosis in colon requires activities of the Na⁺/K⁺/2Cl⁻ cotransporter (NKCC1) (20, 21), and also anion and K⁺ channels whose identities are still unclear (20). It is also not clearly known whether specific ionic conductances are intrinsic to GCs or are located in the surrounding absorptive enterocytes. Although several types of K⁺ channels—including K_Ca3.1, Kv7.1, and BK channels—have been found in colonic epithelial cells (22–27), to what extent any of those K⁺ channels are specifically associated with GCs or critical to their function remains unclear. To date, most functional studies about colonic K⁺ channels have focused on their roles in electrolyte and fluid secretion/absorption of the whole colon, whereas the cellular events relating K⁺ channels to specific roles in GC function are still poorly understood.Among colonic epithelial K⁺ channels, the BK channel (also known as K_Ca1.1), the Ca²⁺- and voltage-activated K⁺ channel of high conductance, has been proposed to be the main component of colonic K⁺ secretion into the lumen (28–30). BK channels are homotetramers of the pore-forming BKα subunit, but can also contain tissue-specific regulatory subunits that critically define the functional properties of the channel (31). BK channels composed exclusively of the pore-forming BKα subunit are unlikely to be activated at the physiological conditions of epithelial cells and, as a consequence, the molecular properties of colonic BK channels that would allow them to contribute to colonic ion transport remain unclear. Recently, we established that the leucine-rich repeat-containing protein 26 (LRRC26), a BK regulatory γ-subunit, is specifically expressed in secretory epithelial cells, including GCs of the gastrointestinal tract (32). When LRRC26 is present in a BK channel complex, the resulting channel activates near normal resting physiological conditions, even in the absence of any elevation of intracellular Ca²⁺ (33).In the present study, we have specifically probed the role of BK channels in cells of the colonic epithelium and examined the impact of deletions of either the BKα subunit or LRRC26 on colonic function. Here, through recordings from identified GCs in intact colonic crypts, we show that LRRC26-associated BK channels contribute the major K⁺ current at low intracellular Ca²⁺ (∼250 nM) in mouse colonic GCs. Furthermore, the LRRC26-containing BK channels are activated near −40 mV, even in the absence of intracellular Ca²⁺. In contrast, in identified GCs from Lrrc26^−/− mice, BK current is present, but it is only activated at membrane potentials unlikely to ever occur physiologically. Surprisingly, all colonic epithelial MUC2⁻ cells sampled completely lack functional BK channels. To establish that the LRRC26-containing BK channels contribute to normal K⁺ fluxes in intact colon tissue, we show that the transepithelial current across distal colon at rest has a component dependent on LRRC26-associated BK channels, which is absent when either BKα or LRRC26 is genetically deleted. Moreover, the genetic ablation of either LRRC26 or BK channel results in a dramatically enhanced susceptibility to colitis induced by dextran sodium sulfate (DSS). Overall, our results suggest that normal potassium flux through LRRC26-associated BK channels in GCs has a protective role against development of colitis.     

A multiplier peroxiporin signal transduction pathway powers piscine spermatozoa

The primary task of a spermatozoon is to deliver its nuclear payload to the egg to form the next-generation zygote. With polyandry repeatedly evolving in the animal kingdom, however, sperm competition has become widespread, with the highest known intensities occurring in fish. Yet, the molecular controls regulating spermatozoon swimming performance in these organisms are largely unknown. Here, we show that the kinematic properties of postactivated piscine spermatozoa are regulated through a conserved trafficking mechanism whereby a peroxiporin ortholog of mammalian aquaporin-8 (Aqp8bb) is inserted into the inner mitochondrial membrane to facilitate H₂O₂ efflux in order to maintain ATP production. In teleosts from more ancestral lineages, such as the zebrafish (Danio rerio) and the Atlantic salmon (Salmo salar), in which spermatozoa are activated in freshwater, an intracellular Ca²⁺-signaling directly regulates this mechanism through monophosphorylation of the Aqp8bb N terminus. In contrast, in more recently evolved marine teleosts, such the gilthead seabream (Sparus aurata), in which spermatozoa activation occurs in seawater, a cross-talk between Ca²⁺- and oxidative stress-activated pathways generate a multiplier regulation of channel trafficking via dual N-terminal phosphorylation. These findings reveal that teleost spermatozoa evolved increasingly sophisticated detoxification pathways to maintain swimming performance under a high osmotic stress, and provide insight into molecular traits that are advantageous for postcopulatory sexual selection.

For many dioecious animals, spermatozoon velocity, progressivity, and duration of motility are vital determinants of reproductive success and are thus major selection criteria for sperm evolution (1–6). Maximizing such kinematic properties contributes to spermatozoon vigor (7); however, due to the limitations in sperm ATP stores, which provide the chemical energy for flagellar contractions, a trade-off between swimming fast and for extended periods typically exists (8). Optimal combinations of traits that improve spermatozoon vigor are nevertheless important in polyandrous vertebrates facing sperm competition, which represents a powerful form of postcopulatory sexual selection (9–15). Since the phenomenon of sperm competition was first recognized (16), investigators have sought to understand the underlying mechanisms that could explain advantageous trait selection (17). To date, however, most research has focused on the physical and morphological properties involved in sperm competition, and very little is known concerning the molecular and genetic mechanisms underpinning spermatozoon performance (15, 18, 19).One positively selected morphological change in respect of spermatozoon velocity and longevity in vertebrates as diverse as fishes, birds, and mammals, has been the increase in the spermatozoon midpiece size and the number or scale of mitochondria therein (20–22). Such changes have logically been associated with increased mitochondrial production of ATP for improved flagellar motility. However, the biochemical reactions that lead to increased ATP synthesis also generate elevated levels of hydrogen peroxide (H₂O₂), a reactive oxygen species (ROS) that inhibits mitochondrial function and suppresses flagellar motility (23–27). With osmotic stress of the exposed ejaculate generating additional ROS (26, 28), it has been unclear how sperm evolved molecular mechanisms that surmount such signaling conflicts.A solution to this apparent paradox was recently discovered in the spermatozoa of a marine teleost, in which a water channel protein, now termed Aqp8bb (a peroxiporin ortholog of mammalian aquaporin-8) (29), is rapidly (<1 s) trafficked to the inner mitochondrial membrane upon activation in seawater (SW) to facilitate H₂O₂ efflux and the maintenance of ATP production and flagellar motility (26). The importance of Aqp8bb, which mainly functions as a peroxiporin in these germ cells, was demonstrated through immunological inhibitory experiments, which highlighted the channel-trafficking mechanism as a critical regulator of the spermatozoon velocity and motility (26). To date, however, the signal transduction pathways that regulate peroxiporin trafficking in vertebrate spermatozoa remain completely unknown.Among externally fertilizing vertebrates, the highest known intensity of sperm competition occurs in true bony fishes (teleosts) (6), and we therefore focused our investigations on these model organims. In contrast to amniotic vertebrates, in which ejaculates become gelatinous when emitted (30), the ejaculates of freshwater (FW) and marine teleosts are not only rapidly diluted, but respectively face tremendous and opposing osmotic stresses, which in most species activate sperm motility (31–33). To understand the significance of such harsh environments for peroxiporin signal transduction pathway evolution, we selected model species from ancient and modern lineages of teleosts, including the FW ostariphysan zebrafish (Danio rerio), the FW protacanthopterygian Atlantic salmon (Salmo salar), and the modern marine acanthomorph gilthead seabream (Sparus aurata). Using a combination of pharmacological, molecular, and physiological approaches, we uncover the evolution of increasingly sophisticated peroxiporin signal transduction pathways powering their spermatozoa. The findings provide insight into the underlying hierarchy of systemic molecular traits that regulate the velocity, progressivity and duration of spermatozoon motility.     

PIEZO ion channel is required for root mechanotransduction in Arabidopsis thaliana

Plant roots adapt to the mechanical constraints of the soil to grow and absorb water and nutrients. As in animal species, mechanosensitive ion channels in plants are proposed to transduce external mechanical forces into biological signals. However, the identity of these plant root ion channels remains unknown. Here, we show that Arabidopsis thaliana PIEZO1 (PZO1) has preserved the function of its animal relatives and acts as an ion channel. We present evidence that plant PIEZO1 is expressed in the columella and lateral root cap cells of the root tip, which are known to experience robust mechanical strain during root growth. Deleting PZO1 from the whole plant significantly reduced the ability of its roots to penetrate denser barriers compared to wild-type plants. pzo1 mutant root tips exhibited diminished calcium transients in response to mechanical stimulation, supporting a role of PZO1 in root mechanotransduction. Finally, a chimeric PZO1 channel that includes the C-terminal half of PZO1 containing the putative pore region was functional and mechanosensitive when expressed in naive mammalian cells. Collectively, our data suggest that Arabidopsis PIEZO1 plays an important role in root mechanotransduction and establish PIEZOs as physiologically relevant mechanosensitive ion channels across animal and plant kingdoms.

Plants extend roots within the soil to access water and nutrients as well as provide stability for the aerial parts of the plant. Underground barriers caused by drought and/or heterogeneous soil components can exert mechanical resistance that alters root extension and penetration (1–3). The root cap at the very tip of the primary root is a dynamic organ that contains different classes of stem cells that divide asymmetrically and is essential for growth through harder media and soils (4). Bending or poking root tips elicits a transient Ca²⁺ influx with short latency that is blocked by lanthanides, including Gd³⁺, a nonselective inhibitor of mechanically activated (MA) cation channels (5–7). However, the molecular identity of putative ion channels underlying this response is unknown. Only a few mechanosensitive ion channels have been described in plants (8). MSL8 plays a mechanosensory role in pollen (9), MSL10 is involved in cell swelling (8, 10), OSCA1 has mainly been characterized for its role in osmosensation (11), and OSCA1.3 regulates stomatal closure during immune signaling (12). It has been proposed that MCA1, expressed in the elongation zone but not the root cap, is a stretch-activated calcium permeable ion channel involved in soil penetration; however, evidence for its being a bona fide ion channel capable of detecting mechanical force is lacking (13–15). Recently, it has been shown that mechanosensitive Ca²⁺ channel activity is dependent on the developmental regulator DEK1; however, whether DEK1 is a pore-forming ion channel has not yet been addressed (16, 17). The genome of Arabidopsis thaliana encodes an ortholog of the mammalian mechanosensitive ion channels PIEZO1 and PIEZO2 (18). Given that PIEZOs play prominent roles in multiple aspects of animal mechanosensation and physiology (19–22), we investigated the role of A. thaliana PIEZO1 (PZO1) in plant mechanosensation. A recent study reported that PZO1 regulated virus translocation within the plant, but its specific role in mechanotransduction was not addressed (23). Here we use genetic tools, electrophysiological methods, and calcium imaging to investigate the role of PZO1 in root mechanosensation.     

Stress-dependent activation of myosin in the heart requires thin filament activation and thick filament mechanosensing

Myosin-based regulation in the heart muscle modulates the number of myosin motors available for interaction with calcium-regulated thin filaments, but the signaling pathways mediating the stronger contraction triggered by stretch between heartbeats or by phosphorylation of the myosin regulatory light chain (RLC) remain unclear. Here, we used RLC probes in demembranated cardiac trabeculae to investigate the molecular structural basis of these regulatory pathways. We show that in relaxed trabeculae at near-physiological temperature and filament lattice spacing, the RLC-lobe orientations are consistent with a subset of myosin motors being folded onto the filament surface in the interacting-heads motif seen in isolated filaments. The folded conformation of myosin is disrupted by cooling relaxed trabeculae, similar to the effect induced by maximal calcium activation. Stretch or increased RLC phosphorylation in the physiological range have almost no effect on RLC conformation at a calcium concentration corresponding to that between beats. These results indicate that in near-physiological conditions, the folded myosin motors are not directly switched on by RLC phosphorylation or by the titin-based passive tension at longer sarcomere lengths in the absence of thin filament activation. However, at the higher calcium concentrations that activate the thin filaments, stretch produces a delayed activation of folded myosin motors and force increase that is potentiated by RLC phosphorylation. We conclude that the increased contractility of the heart induced by RLC phosphorylation and stretch can be explained by a calcium-dependent interfilament signaling pathway involving both thin filament sensitization and thick filament mechanosensing.

The contraction of cardiac muscle is generated by reciprocal sliding of actin-containing thin filaments and myosin-containing thick filaments in the sarcomere driven by myosin motors. The interaction of the myosin motors with the overlapping thin filament is primarily controlled by calcium-induced structural changes in the thin filament linked to the intracellular calcium transient (1). Calcium ions released in the cytoplasm following an action potential bind to troponin, triggering the movement of tropomyosin around the filament, which uncovers actin sites to which the motors can bind and power contraction (2). However, some of the myosin motors may not be available for actin binding, as they are folded onto the thick filament surface in relaxing conditions (3). Thick filament–based regulatory mechanisms control the release of the myosin motors from the folded conformation and contribute to the regulation of contractility of striated muscle (3–6).Electron microscopy (EM) studies on isolated thick filaments from vertebrate heart muscle showed that the myosin motors in the region of the filament that contains myosin-binding protein C (MyBP-C), the C-zone, are sequestered in helical tracks on the thick filament surface and are folded back onto their tails in an asymmetric conformation called the interacting-heads motif, or IHM (7, 8), originally identified in two-dimensional crystals of dephosphorylated smooth muscle myosin (9). The IHM has also been associated with a biochemical state of myosin with a very low adenosine triphosphate (ATP)-ase rate, called the “super-relaxed” state (10), which is considered to be an OFF state of myosin. A recent X-ray diffraction study of cardiac muscle (11) extended that concept and suggested that in diastole, the resting phase of the cardiac cycle, three distinct motor conformations coexist in the thick filament in roughly equal numbers: folded helical, folded nonhelical, and disordered. The folded helical motors are likely to correspond to the IHM conformation and are confined to the C-zone. All the folded motors would be unavailable for actin binding and therefore OFF, but the disordered motors would constitute a population of constitutively ON motors that are immediately available for actin binding upon activation of the thin filament.Stress sensing in the thick filament can control the release of the myosin motors from the folded states and might be responsible for modulating the strength of contraction of cardiac muscle in response to changes in the afterload (i.e., the arterial pressure) (12). Moreover, the transitions between these motor conformations, together with the calcium-induced structural changes in the thin filament, control the speed of contraction and relaxation (11). According to this mechanosensing paradigm of thick filament regulation, the constitutively ON motors play a fundamental role in the activation of cardiac muscle, as the force generated by these motors immediately after the electrical stimulus triggers a positive mechanosensing feedback loop that controls the number of active motors and the dynamics of contraction. Destabilization of the folded conformations by mutations in myosin and other thick filament proteins can alter the equilibrium between these motor conformations, leading to a hypercontractile phenotype in some hypertrophic cardiomyopathies (HCM) (13, 14). Pharmacological therapies targeting thick filament proteins to treat HCM (15–17) have been aimed at reversing the destabilization of the folded states caused by these mutations.The contractility of the heart is also controlled by phosphorylation of the myosin regulatory light chain (RLC) (18) and by β-adrenergic signaling pathways mediated by phosphorylation of MyBP-C in the thick filament (19) as well as troponin in the thin filament, which are also likely to alter the equilibrium between regulatory conformations of the motors. RLC phosphorylation is essential for the normal function of the heart. The pattern of contraction of the heart may depend on a spatial gradient of RLC phosphorylation across the ventricle wall (20), and a decrease in the level of RLC phosphorylation is associated with heart failure (21, 22). RLC phosphorylation potentiates the contractility of vertebrate and invertebrate striated muscle, an effect that is generally thought to be mediated by disrupting the folded helical conformation of the myosin motors on the thick filament and increasing the number of motors available for interaction with actin during contraction at a given [Ca²⁺] (23, 24). Disordering of the myosin motors on the thick filament by RLC phosphorylation has been shown in in vitro studies on isolated thick filaments (23) and is the main mechanism of thick filament activation in intact striated muscle of tarantula during contraction (25). However, the effect of RLC phosphorylation on the structure of the cardiac thick filament in diastole is unclear. More generally, the large changes in force and the speed of contraction and relaxation of cardiac muscle produced by β-adrenergic agonists are not associated with significant changes in the diastolic structure of the thick filament (26), so they are not simply mediated by increasing the number of ON motors in diastole. Similarly, length-dependent activation (LDA), the cellular correlate of the Frank–Starling law of the heart and a key autoregulatory mechanism that adjusts the cardiac output in response to different extents of diastolic filling (27, 28), seems not to be simply mediated by a stretch-induced change in the structure of the thick filament in diastole (11, 26). Conflicting results have been reported (29), however, and mathematical models have suggested that activation of myosin motors induced by the passive tension transmitted to the thick filament by titin might contribute to LDA in cardiac muscle (30, 31).Here, we investigated the in situ conformation of the myosin motors and its dependence on temperature, RLC phosphorylation, [Ca²⁺], and sarcomere length (SL) in demembranated cardiac trabeculae from rat hearts using the polarized fluorescence from probes on the N- and C-lobes of the RLC (18, 32). We show that, at the low [Ca²⁺] values that maintain the relaxed state and at near-physiological temperature and lattice spacing, the RLC-lobe orientations are consistent with about one-third of the myosin motors being in the folded helical conformation corresponding to the IHM, likely stabilized by MyBP-C in the C-zone of the filament. At the low [Ca²⁺] values that maintain the relaxed state, the folded conformation of the myosin motors is disrupted by cooling but not by RLC phosphorylation or stretch. However, stretching cardiac muscle at higher [Ca²⁺] that partially activates the thin filament triggers a stress-dependent activation of the thick filament and a force increase that is potentiated by RLC phosphorylation. This increase in contractility, induced by RLC phosphorylation and stretch, can be explained by an interfilament signaling pathway that links the stress-dependent activation of the thick filament to the activation state of the thin filament.     

Zn2+ influx activates ERK and Akt signaling pathways

Zinc (Zn²⁺) is an essential metal in biology, and its bioavailability is highly regulated. Many cell types exhibit fluctuations in Zn²⁺ that appear to play an important role in cellular function. However, the detailed molecular mechanisms by which Zn²⁺ dynamics influence cell physiology remain enigmatic. Here, we use a combination of fluorescent biosensors and cell perturbations to define how changes in intracellular Zn²⁺ impact kinase signaling pathways. By simultaneously monitoring Zn²⁺ dynamics and kinase activity in individual cells, we quantify changes in labile Zn²⁺ and directly correlate changes in Zn²⁺ with ERK and Akt activity. Under our experimental conditions, Zn²⁺ fluctuations are not toxic and do not activate stress-dependent kinase signaling. We demonstrate that while Zn²⁺ can nonspecifically inhibit phosphatases leading to sustained kinase activation, ERK and Akt are predominantly activated via upstream signaling and through a common node via Ras. We provide a framework for quantification of Zn²⁺ fluctuations and correlate these fluctuations with signaling events in single cells to shed light on the role that Zn²⁺ dynamics play in healthy cell signaling.

Zinc (Zn²⁺) is an essential metal in biology, with approximately 10% of the proteins encoded by the human genome predicted to bind Zn²⁺ (1). All cells maintain and regulate a small pool of labile Zn²⁺ that can be exchanged among Zn²⁺-binding proteins and Zn²⁺ biosensors. The concentration of labile Zn²⁺ in the cytosol, measured in the hundreds of picomolar range (2–5), falls within the affinity range of many Zn²⁺ binding proteins, suggesting that under normal conditions many of these proteins will bind Zn²⁺ and function properly. However, some Zn²⁺ binders may need higher Zn²⁺ concentrations in order to function (6). Furthermore, there is growing evidence that mammalian cells experience fluctuations in available Zn²⁺, and these dynamics have been shown to be important for cell physiology (7–11).In addition to serving as an important biological cofactor (12), there are increasing examples that Zn²⁺ also plays a role in biological signaling. Crosstalk has been observed between Zn²⁺ dynamics and calcium signaling where increases in cytosolic Zn²⁺ lead to decrease in endoplasmic reticulum (ER) calcium, and conversely, increases in cytosolic calcium change Zn²⁺ homeostasis in the ER³. Zn²⁺ sequestration has been shown to block cell cycle progression in both meiotic oocytes (13) and mitotic cells (14–16). At a molecular level, picomolar concentrations of Zn²⁺ potentiate the response of the ryanodine receptor in cardiomyocytes (17). Zn²⁺ has also been implicated in metabotropic signaling via the G protein–coupled receptor 39 (GPR39 (18)), direct modulation of protein kinase C activity (19), and activation of MAPK kinase signaling pathways in neurons (20), cardiomyocytes (21), and mast cells (7). While the above studies demonstrate that Zn²⁺ fluctuations influence cellular processes, in many cases the molecular details of how Zn²⁺ interacts with canonical signaling pathways, second messengers, or serves as a signal itself are unclear. This is especially true for the MAPK pathway.MAPK signaling plays a role in cell proliferation, differentiation, and development and is one of the most well-studied signaling pathways (22). A connection between MAPK signaling and Zn²⁺ was first reported in 1996 when it was observed that addition of 300 μM ZnCl₂ to 3T3 fibroblasts led to increased phosphorylation of ERK1/2 kinases in the MAPK pathway (23). Early studies used epithelial cell lines to study the connection between Zn²⁺ and ERK signaling (23, 24). More recently, Zn²⁺ elevation has been demonstrated to increase ERK phosphorylation in dissociated neurons and transformed HT22 cells, where ERK signaling has been linked to synaptic plasticity and memory consolidation (20, 25, 26). The mechanism of ERK activation by Zn²⁺ remains enigmatic. The leading hypothesis has been that Zn²⁺ inhibits protein phosphatases, leading to sustained ERK activation. This idea is supported by the observation that ERK-directed phosphatase PP2A activity is reduced when Zn²⁺ is added to cell lysates (20, 25). Furthermore, it has been demonstrated that certain phosphatases are inhibited by nano- and picomolar concentrations of Zn²⁺ in vitro, although these phosphatases are not known to directly interact with ERK1/2 (27, 28). However, it is unclear how these bulk in vitro analyses relate to the role of Zn²⁺ fluctuations in living cells.The connection between Zn²⁺ and modulation of the MAPK pathway is even more perplexing when examining how Zn²⁺ influences Ras activity, which acts upstream of Raf-MEK-ERK. Two studies that involved acute perturbation of Zn²⁺ by adding high concentrations of Zn²⁺ concluded that Zn²⁺ promotes Ras activation (29, 30). On the other hand, two genetic screens in Caenorhabditis elegans suggested that Zn²⁺ inhibits Ras activity (31, 32). While these studies involved different model systems (cell lines versus C. elegans) and different means of altering Zn²⁺ (acute elevation versus chronic manipulation), it is important to note that there is a lack of consensus on how Zn²⁺ influences the Ras-Raf-MEK-ERK pathway.In this work we set out to dissect the connection between Zn²⁺ and ERK in an effort to elucidate the mechanism of activation. Using a combination of kinase translocation reporters and a Förster resonance energy transfer (FRET)-sensor for Zn²⁺, we quantified the changes in intracellular Zn²⁺ in response to subtle extracellular perturbations and correlated them directly with changes in kinase activity at the single cell level. We found that while elevated Zn²⁺ broadly inhibits phosphatase activity to some extent in vitro, in live cells, Zn²⁺ primarily activates ERK via upstream signaling, suggesting that ERK phosphatase inhibition can’t fully account for the Zn²⁺-induced increase in ERK activity. Finally, we demonstrate that our Zn²⁺ conditions activate Ras and Akt signaling along with ERK but that few other kinases are activated, including stress-response kinases JNK, p38, and p53. We therefore propose a mechanism of action where Zn²⁺ activates ERK and Akt pathways upstream of Ras, while the specific Zn²⁺-protein interaction remains elusive.