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.     

Comparative structural analysis of human Nav1.1 and Nav1.5 reveals mutational hotspots for sodium channelopathies

Among the nine subtypes of human voltage-gated sodium (Na_v) channels, the brain and cardiac isoforms, Na_v1.1 and Na_v1.5, each carry more than 400 missense mutations respectively associated with epilepsy and cardiac disorders. High-resolution structures are required for structure–function relationship dissection of the disease variants. We report the cryo-EM structures of the full-length human Na_v1.1–β4 complex at 3.3 Å resolution here and the Na_v1.5-E1784K variant in the accompanying paper. Up to 341 and 261 disease-related missense mutations in Na_v1.1 and Na_v1.5, respectively, are resolved. Comparative structural analysis reveals several clusters of disease mutations that are common to both Na_v1.1 and Na_v1.5. Among these, the majority of mutations on the extracellular loops above the pore domain and the supporting segments for the selectivity filter may impair structural integrity, while those on the pore domain and the voltage-sensing domains mostly interfere with electromechanical coupling and fast inactivation. Our systematic structural delineation of these mutations provides important insight into their pathogenic mechanism, which will facilitate the development of precise therapeutic interventions against various sodium channelopathies.

The nine subtypes of human voltage-gated sodium (Na_v) channels are responsible for the initiation and transmission of electrical impulses in different tissues: Na_v1.1 to Na_v1.3 and Na_v1.6 mainly function in the central nervous system, Na_v1.7 to Na_v1.9 are mostly distributed in the peripheral nervous system, Na_v1.4 is specialized in skeletal muscle, and Na_v1.5 is the primary cardiac isoform (1–4). Abnormalities of these channels, hinging on their tissue specificity, are associated with a broad spectrum of channelopathies. To date, more than 1,000 disease mutations have been identified in the primary sequence of Na_v channels, among which Na_v1.1 and Na_v1.5 each host more than 400 missense mutations (5–8).Na_v1.1 is encoded by SCN1A, which may have the largest number of epilepsy-related mutations. Up to 900 SCN1A mutations, more than half of which result in truncations (9), have been identified in epilepsy syndromes with different severities. Nonsense and hundreds of missense mutations of SCN1A are found in 70 to 80% of patients with Dravet syndrome, which is also known as the severe myoclonic epilepsy of infancy (10–13) (SI Appendix, Table S1). Several dozen missense mutations are associated with generalized epilepsy with febrile seizures plus and intractable childhood epilepsy with generalized tonic-clonic seizures (10) (SI Appendix, Table S2). Although most of the Na_v1.1 disease mutations lead to loss of function to different degrees, some represent gain of function. In most cases, the pathogenic mechanism remains elusive.A brief summary of Na_v1.5 pathophysiology is presented in the companion paper (14). Mechanistic understanding of the sodium channelopathies entails high-resolution structures of human Na_v channels. In the past 4 y, we have reported the cryoelectron microscopy (cryo-EM) structures, at resolutions ranging between 2.6 and 4.0 Å, of Na_v channels from insect (Na_vPaS), electric eel (EeNa_v1.4), and finally human, including Na_v1.2, Na_v1.4, Na_v1.5, and Na_v1.7, in the presence of multiple modulators, such as β1 and β2 subunits, peptide toxins, and small-molecule toxins tetrodotoxin and saxitoxin (15–21). Structures of a truncated rat Na_v1.5 were recently reported (22). All structurally resolved eukaryotic Na_v channels except for Na_vPaS exhibit similar conformations of potentially inactivated state.Notwithstanding these advances, high-resolution structures of human Na_v1.1 and Na_v1.5 wild-type and representative disease variants are necessary to provide accurate templates to directly map the disease mutations and to facilitate drug discovery. Furthermore, as these two channels harbor 80% of all identified mutations related to sodium channelopathies, a comparative analysis of their structures may reveal potential mutational hotspots, offering invaluable insight into the function and disease mechanism of Na_v channels.Here we present the cryo-EM structure of human Na_v1.1 associated with a modulating auxiliary subunit β4. In the accompanying paper, we report the structure of human Na_v1.5 that carries a common disease variant E1784K. Comparative structural analyses have revealed several clusters of disease mutations that are common to both Na_v1.1 and Na_v1.5.     

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

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

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

β-Adrenergic control of sarcolemmal CaV1.2 abundance by small GTPase Rab proteins

The number and activity of Ca_v1.2 channels in the cardiomyocyte sarcolemma tunes the magnitude of Ca²⁺-induced Ca²⁺ release and myocardial contraction. β-Adrenergic receptor (βAR) activation stimulates sarcolemmal insertion of Ca_V1.2. This supplements the preexisting sarcolemmal Ca_V1.2 population, forming large “superclusters” wherein neighboring channels undergo enhanced cooperative-gating behavior, amplifying Ca²⁺ influx and myocardial contractility. Here, we determine this stimulated insertion is fueled by an internal reserve of early and recycling endosome-localized, presynthesized Ca_V1.2 channels. βAR-activation decreased Ca_V1.2/endosome colocalization in ventricular myocytes, as it triggered “emptying” of endosomal Ca_V1.2 cargo into the t-tubule sarcolemma. We examined the rapid dynamics of this stimulated insertion process with live-myocyte imaging of channel trafficking, and discovered that Ca_V1.2 are often inserted into the sarcolemma as preformed, multichannel clusters. Similarly, entire clusters were removed from the sarcolemma during endocytosis, while in other cases, a more incremental process suggested removal of individual channels. The amplitude of the stimulated insertion response was doubled by coexpression of constitutively active Rab4a, halved by coexpression of dominant-negative Rab11a, and abolished by coexpression of dominant-negative mutant Rab4a. In ventricular myocytes, βAR-stimulated recycling of Ca_V1.2 was diminished by both nocodazole and latrunculin-A, suggesting an essential role of the cytoskeleton in this process. Functionally, cytoskeletal disruptors prevented βAR-activated Ca²⁺ current augmentation. Moreover, βAR-regulation of Ca_V1.2 was abolished when recycling was halted by coapplication of nocodazole and latrunculin-A. These findings reveal that βAR-stimulation triggers an on-demand boost in sarcolemmal Ca_V1.2 abundance via targeted Rab4a- and Rab11a-dependent insertion of channels that is essential for βAR-regulation of cardiac Ca_V1.2.

The influx of Ca²⁺ through L-type Ca²⁺ channels (Ca_V1.2) is indispensable for cardiac excitation–contraction coupling (EC-coupling). These multimeric proteins consist of a pore-forming and voltage-sensing α_1c subunit, and auxiliary β- and α₂δ-subunits. In ventricular myocytes, Ca_V1.2 mainly localize to the t-tubule sarcolemma and open briefly, allowing a small amount of Ca²⁺ influx, in response to the wave of depolarization that travels through the conduction system of the heart from its point of origin, usually in the SA-node. This initial influx is amplified manifold though Ca²⁺-induced Ca²⁺ release (CICR) from juxtaposed type 2 ryanodine receptors (RyR2) on the junctional sarcoplasmic reticulum, a short ∼12 nm across the dyadic cleft. The synchronous opening of thousands of RyR2 generates a transient, global elevation in intracellular calcium concentration ([Ca²⁺]_i), resulting in contraction. Reducing Ca_V1.2 channel current (I_Ca) results in less CICR, smaller [Ca²⁺]_i transients, and less forceful contractions. Conversely, larger amplitude I_Ca elicits greater Ca²⁺ release from the sarcoplasmic reticulum, producing more forceful contractions. The level of Ca²⁺ influx through Ca_V1.2 channels therefore tunes EC-coupling.I_Ca is a product of the number of channels in the sarcolemma and their open probability (P_o). Consequently, there are two possible, nonmutually exclusive strategies that may be adopted to alter I_Ca and consequently the magnitude of EC-coupling: 1) Adjust Ca_V1.2 channel activity (P_o) and 2) modify sarcolemmal Ca_V1.2 channel expression (N). The first strategy of increasing channel P_o has long been associated with β-adrenergic receptor (βAR)-mediated signaling in the heart (1–3). During acute physical or emotional stress, norepinephrine spills from sympathetic varicosities onto cardiomyocytes, activating βARs. The ensuing G_αs/adenylyl cyclase/cAMP/PKA signaling cascade culminates in PKA phosphorylation of several effector proteins, including Ca_V1.2 [or an element of their interactome (4)], enhancing their activity to generate this positive inotropic response.As to the second strategy to increase I_Ca, there remains a paucity of information regarding the mechanisms regulating Ca_V1.2 channel abundance in the cardiomyocyte sarcolemma. Classic secretory transport literature suggests that Ca_V1.2 channels are trafficked from the endoplasmic reticulum to the trans-Golgi-network and onward to their dyadic position in the sarcolemma. Underscoring the importance of faithful Ca_V1.2 channel trafficking, altered Ca_V1.2 channel density has been reported in both failing (5) and aging (6) ventricular myocytes, and impaired anterograde trafficking of Ca_V1.2 channels to the t-tubules of human ventricular myocytes has been linked to dilated cardiomyopathy (7). Yet, despite the importance of tight homeostatic control of Ca_V1.2 channel trafficking to prevent Ca²⁺ dysregulation, the molecular steps defining Ca_V1.2 channel sorting and insertion remain poorly understood. Therefore, elucidation of the trafficking pathways that regulate Ca_V1.2 channel abundance is critical for our understanding of the pathophysiology of heart failure and myocardial aging, and could potentially reveal new therapeutic or rejuvenation targets. Along that vein, in the treatment of cystic fibrosis, multiple drugs are in various stages of use or development to improve trafficking to, or to amplify or stabilize, CFTR channels at the apical membrane of airway epithelial cells (8).There exist no measurements of Ca_V1.2 channel lifetimes in cardiomyocytes, but pulse-chase experiments in immortalized cell lines support a lifetime of plasma membrane (PM)-localized Ca_V1.2 of ∼3 h (9), while total cellular Ca_V1.2 lifetime is >20 h (10). This disparity suggests membrane-Ca_V1.2 turns over much more dynamically than the total cellular channel content and implies ongoing local control by endosomal trafficking. Disturbance of the equilibrium between channel insertion/recycling and internalization would be predicted to lead to alterations in sarcolemmal Ca_V1.2 channel abundance. Trafficking of vesicular cargo through the endosomal pathway is regulated by Rab-GTPases, a >60-member family within the larger Ras superfamily of small GTPases (11, 12). Rab5 is involved in endocytosis and control of vesicular cargo influx into early endosomes (EEs; also called sorting endosomes), while Rab4 controls efflux of cargo out of EEs and fast recycling (t_1/2 ∼1 to 2 min) back to the PM (13). Rab11, expressed on recycling endosomes (RE; also called the endocytic recycling compartment or ERC), regulates slow recycling (t_1/2 ∼12 min) of cargo from this compartment back to the PM (13). In cortical neurons and pancreatic β-cells, activity-dependent Ca_V1.2 channel internalization has been postulated to play important roles in Ca²⁺ homeostasis, with implications for homeostatic synaptic plasticity and insulin production, respectively (11, 14). In mouse neonatal cardiomyocytes, Rab11b has been reported to limit Ca_V1.2 PM expression (15), while recent studies performed in HEK and HL-1 cells reported that endocytic recycling of cardiac Ca_V1.2 channels, regulates their surface abundance (10, 16). Despite this crucial information from other cell-types, there has been a lack of rigorous investigations, at the molecular level, into how Ca_V1.2 channel recycling is regulated in cardiac myocytes.Here, we identify a dynamic, subsarcolemmal pool of Ca_V1.2-cargo–carrying endosomes that are rapidly mobilized to the ventricular myocyte sarcolemma along targeted Rab4a and Rab11a GTPase-regulated recycling pathways in response to βAR-stimulation. Using electrophysiology, cell biology, total internal reflection fluorescence (TIRF), and superresolution microscopy, we report that enhanced t-tubule sarcolemmal Ca_V1.2 abundance via targeted, isoproterenol (ISO)-stimulated recycling of these channels is essential for βAR-regulation of cardiac Ca_V1.2.     

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.     

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.     

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.     

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.     

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.     

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

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

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

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.     

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

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

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

Enhanced Ca2+ signaling,mild primary aldosteronism,and hypertension in a familial hyperaldosteronism mouse model (Cacna1hM1560V/+)

Gain-of-function mutations in the CACNA1H gene (encoding the T-type calcium channel Ca_V3.2) cause autosomal-dominant familial hyperaldosteronism type IV (FH-IV) and early-onset hypertension in humans. We used CRISPR/Cas9 to generate Cacna1h^M1560V/+ knockin mice as a model of the most common FH-IV mutation, along with corresponding knockout mice (Cacna1h^−/−). Adrenal morphology of both Cacna1h^M1560V/+ and Cacna1h^−/− mice was normal. Cacna1h^M1560V/+ mice had elevated aldosterone:renin ratios (a screening parameter for primary aldosteronism). Their adrenal Cyp11b2 (aldosterone synthase) expression was increased and remained elevated on a high-salt diet (relative autonomy, characteristic of primary aldosteronism), but plasma aldosterone was only elevated in male animals. The systolic blood pressure of Cacna1h^M1560V/+ mice was 8 mmHg higher than in wild-type littermates and remained elevated on a high-salt diet. Cacna1h^−/− mice had elevated renal Ren1 (renin-1) expression but normal adrenal Cyp11b2 levels, suggesting that in the absence of Ca_V3.2, stimulation of the renin-angiotensin system activates alternative calcium entry pathways to maintain normal aldosterone production. On a cellular level, Cacna1h^M1560V/+ adrenal slices showed increased baseline and peak intracellular calcium concentrations in the zona glomerulosa compared to controls, but the frequency of calcium spikes did not rise. We conclude that FH-IV, on a molecular level, is caused by elevated intracellular Ca²⁺ concentrations as a signal for aldosterone production in adrenal glomerulosa cells. We demonstrate that a germline Cacna1h gain-of-function mutation is sufficient to cause mild primary aldosteronism, whereas loss of Ca_V3.2 channel function can be compensated for in a chronic setting.

As a key regulator of blood pressure and electrolyte homeostasis, the mineralocorticoid hormone aldosterone is synthesized from its precursor cholesterol in the zona glomerulosa of the adrenal cortex. Cortisol and aldosterone differ by modification at C-17 in cortisol and C-18 in aldosterone. C-18 modification is catalyzed by aldosterone synthase (encoded by the CYP11B2 gene in humans). Altered CYP11B2 expression governs chronic changes in aldosterone production (1). The two main physiological stimuli of aldosterone production are volume depletion and elevated blood potassium (hyperkalemia). Volume depletion activates the renin-angiotensin system (RAS), leading to production of the peptide hormone angiotensin II (Ang II). Ang II binds to its selective G protein-coupled receptor in the zona glomerulosa, triggering calcium release from intracellular stores, depolarizing cells by inhibition of potassium channels, and activating voltage-gated calcium influx from the extracellular space. Hyperkalemia directly depolarizes glomerulosa cells, similarly promoting calcium entry. The resulting elevated intracellular calcium levels are the key signal for aldosterone production (1, 2). In response to aldosterone, the kidney increases NaCl reabsorption and potassium secretion, restoring balance.When increased production of aldosterone is at least partially uncoupled from its main physiological stimuli, primary aldosteronism ensues. This disorder is present in about 6% of hypertensives in a primary care setting and is thus considered the most prevalent cause of secondary hypertension (3). Its diagnosis is based on an elevated aldosterone:renin ratio (ARR) and a positive confirmatory test, such as the failure to suppress aldosterone levels upon administration of NaCl (“saline suppression test”) (4). About one-third of patients with primary aldosteronism have a benign tumor of the adrenal gland (aldosterone-producing adenoma, APA), and the majority of the remainder have bilateral adrenal hyperplasia (4). On a molecular level, about 90% of APAs are explained by heterozygous or hemizygous somatic mutations in one of seven genes (5–7). Mutations in L-type (CACNA1D) (8, 9) and T-type (CACNA1H) (10) calcium channel genes directly cause increased calcium influx (8, 11). Mutations in other ion channels [KCNJ5 (12), CLCN2 (13)] and ion pump genes (ATP1A1, ATP2B3) (14) depolarize the cell membrane and indirectly increase voltage-gated calcium influx. CTNNB1 (15) mutations prevent differentiation of glomerulosa cells into fasciculata cells, leading to increased glomerulosa cell mass (16).In addition, recent studies suggest that somatic mutations in APA genes, in particular CACNA1D and ATP1A1, can cause so-called aldosterone-producing cell clusters, small nodules that protrude into the zona fasciculata and occur in healthy subjects (17) and, more prominently, in subjects with bilateral adrenal hyperplasia (18, 19). Heterozygous mutations in APA genes KCNJ5, CACNA1D, CACNA1H, and CLCN2 can also occur in the germline, often at the identical positions affected by somatic mutations (8, 11, 12, 20–22). Such mutations cause an autosomal-dominant Mendelian disorder called familial hyperaldosteronism (FH). Additional cases are caused by mutations that create a chimeric CYP11B1/CYP11B2 gene (23). Patients with FH typically present with early-onset primary aldosteronism and hypertension. Collectively, these findings characterize primary aldosteronism as a largely genetic disorder, with somatic mutations in APAs and bilateral adrenal hyperplasia and germline mutations in FH.This study focuses on CACNA1H, which we initially described as a disease gene for FH type IV (FH-IV) (11) in five unrelated kindreds. All carried the identical Met1549Val mutation. Whereas the five index cases all presented with hypertension by age 10 y and showed biochemical evidence of primary aldosteronism, two of five mutation-carrier relatives were normotensive, suggesting incomplete penetrance, as occasionally seen in FH (22). In one, both plasma renin activity and aldosterone levels were normal, in the other, plasma renin activity was at the lower limit of normal, and aldosterone was normal. Upon noncurative removal of one adrenal gland in a clinically affected mutation carrier, microscopic glomerulosa hyperplasia was observed. Daniil et al. (24) identified a different de novo mutation at the identical position (Met1549Ile) in a patient with early-onset primary aldosteronism and multiplex developmental disorder, along with other variants of less certain pathogenicity. More recently, Nanba et al. (10) found three adenomas with somatic Ile1430Thr CACNA1H mutations in a cohort of 75 APAs, establishing them as a rare cause of APAs.CACNA1H encodes the T-type calcium channel α-subunit Ca_V3.2. T-type calcium channels activate at membrane potentials positive to −70 mV. A steady-state so-called window current occurs at potentials slightly positive to the glomerulosa resting membrane potential at which channels activate, but do not inactivate completely. It has been suggested that this property, together with oscillations in the zona glomerulosa membrane potential, allows Ca_V3.2 to transduce the calcium current that is necessary for aldosterone production (25). Like other calcium channel α-subunits, Ca_V3.2 contains four repeats, each with six transmembrane segments, and Met1549 mutated in FH-IV is located in the S6 segment of repeat III. Its mutation causes loss of normal channel inactivation and a slight shift of activation to less-depolarizing potentials (11), which in an adrenocortical cancer cell model is associated with increased aldosterone production (26).Thus, strong evidence links CACNA1H mutations to primary aldosteronism. Yet, potential effects of CACNA1H gain-of-function mutations on adrenal cell mass (11) or on calcium signaling in the zona glomerulosa have not been studied.Herein, we generated and characterized a mouse model carrying a heterozygous knockin mutation at a position that is homologous to human Met1549 (Cacna1h^M1560V/+). These mice showed an elevated ARR and elevated systolic and mean arterial blood pressure as signs of mild primary aldosteronism, but glomerulosa histology was normal. Intracellular glomerulosa calcium concentrations were elevated. We also used a corresponding knockout model (Cacna1h^−/−) to further investigate the physiological role of Ca_V3.2 in the adrenal gland.     

Ammonium transporter expression in sperm of the disease vector Aedes aegypti mosquito influences male fertility

The ammonium transporter (AMT)/methylammonium permease (MEP)/Rhesus glycoprotein (Rh) family of ammonia (NH₃/NH₄⁺) transporters has been identified in organisms from all domains of life. In animals, fundamental roles for AMT and Rh proteins in the specific transport of ammonia across biological membranes to mitigate ammonia toxicity and aid in osmoregulation, acid–base balance, and excretion have been well documented. Here, we observed enriched Amt (AeAmt1) mRNA levels within reproductive organs of the arboviral vector mosquito, Aedes aegypti, prompting us to explore the role of AMTs in reproduction. We show that AeAmt1 is localized to sperm flagella during all stages of spermiogenesis and spermatogenesis in male testes. AeAmt1 expression in sperm flagella persists in spermatozoa that navigate the female reproductive tract following insemination and are stored within the spermathecae, as well as throughout sperm migration along the spermathecal ducts during ovulation to fertilize the descending egg. We demonstrate that RNA interference (RNAi)-mediated AeAmt1 protein knockdown leads to significant reductions (∼40%) of spermatozoa stored in seminal vesicles of males, resulting in decreased egg viability when these males inseminate nonmated females. We suggest that AeAmt1 function in spermatozoa is to protect against ammonia toxicity based on our observations of high NH₄⁺ levels in the densely packed spermathecae of mated females. The presence of AMT proteins, in addition to Rh proteins, across insect taxa may indicate a conserved function for AMTs in sperm viability and reproduction in general.

Ammonium transporters (AMTs), methylammonium permeases (MEPs), and Rhesus glycoproteins (Rh proteins) comprise a protein family with three clades, and homologs from each have been identified in virtually all domains of life (1). AMT proteins were first identified in plants (2) with the simultaneous discovery of MEP proteins in fungi (3), followed by Rh proteins in humans (4). Ammonia (NH₃/NH₄⁺) is vital for growth in plants and microorganisms and is retained in some animals for use as an osmolyte (5, 6), for buoyancy (7, 8), and for those lacking sufficient dietary nitrogen (9). In the majority of animals, however, ammonia is the toxic by-product of amino acid and nucleic acid metabolism and, accordingly, requires efficient mechanisms for its regulation, transport, and excretion (10–13). AMT, MEP, and Rh proteins are responsible for the selective movement of ammonia (NH₃) or ammonium (NH₄⁺) across biological membranes, a process that all organisms require. Unlike their vertebrate, bacterial, and fungal counterparts which function as putative NH₃ gas channels (14–18), a myriad of evidence suggests that plant AMT proteins and closely related members in some animals are functionally distinct and facilitate electrogenic ammonium (NH₄⁺) transport (17, 19–22). In contrast to vertebrates which only possess Rh proteins (23), many invertebrates are unique in that they express both AMT and Rh proteins, sometimes in the same cell (24–28). Among insects, the presence of both AMT and Rh proteins has been described in Drosophila melanogaster (29, 30) and mosquitoes that vector disease-causing pathogens, Anopheles gambiae (22, 31) and Aedes aegypti (32, 33). It is unclear whether, in these instances, AMT and Rh proteins can functionally substitute for one another, but in the anal papillae of A. aegypti larvae, knockdown of either Amt or Rh proteins causes decreases in ammonia transport, suggesting that they do not (32–34). To date, studies on ammonia transporter (AMT and Rh) function in insects have focused on ammonia sensing and tasting in sensory structures (22, 30, 31, 35), ammonia detoxification and acid–base balance in muscle, digestive, and excretory organs (15, 36), and ammonia excretion in a variety of organs involved in ion and water homeostasis (9, 24, 32–34).A. aegypti is the primary vector for the transmission of the human arboviral diseases Zika, yellow fever, chikungunya, and dengue virus, which are of global health concern due to rapid increases in the geographical distribution of this species, presently at its highest ever (37, 38). In light of the well-documented evolution of insecticide resistance in mosquitoes (39–42), more recent methods to control disease transmission such as the sterile insect technique (43), transinfection and sterilization of mosquitoes with the bacterium Wolbachia (44), and targeted genome editing rendering adult males sterile (45) have proven effective. These methods take advantage of various aspects of mosquito reproductive biology; however, an understanding of male reproductive biology and the male contributions to female reproductive processes is still in its infancy (46). Here, we describe the expression of an A. aegypti ammonium transporter (AeAmt1) in the sperm during all stages of spermatogenesis, spermiogenesis, and egg fertilization, which is critical for fertility.     

Surface plasmon mediates the visible light–responsive lithium–oxygen battery with Au nanoparticles on defective carbon nitride

Aprotic lithium-oxygen (Li-O₂) batteries have gained extensive interest in the past decade, but are plagued by slow reaction kinetics and induced large-voltage hysteresis. Herein, we use a plasmonic heterojunction of Au nanoparticle (NP)–decorated C₃N₄ with nitrogen vacancies (Au/N_V-C₃N₄) as a bifunctional catalyst to promote oxygen cathode reactions of the visible light–responsive Li-O₂ battery. The nitrogen vacancies on N_V-C₃N₄ can adsorb and activate O₂ molecules, which are subsequently converted to Li₂O₂ as the discharge product by photogenerated hot electrons from plasmonic Au NPs. While charging, the holes on Au NPs drive the reverse decomposition of Li₂O₂ with a reduced applied voltage. The discharge voltage of the Li-O₂ battery with Au/N_V-C₃N₄ is significantly raised to 3.16 V under illumination, exceeding its equilibrium voltage, and the decreased charge voltage of 3.26 V has good rate capability and cycle stability. This is ascribed to the plasmonic hot electrons on Au NPs pumped from the conduction bands of N_V-C₃N₄ and the prolonged carrier life span of Au/N_V-C₃N₄. This work highlights the vital role of plasmonic enhancement and sheds light on the design of semiconductors for visible light–mediated Li-O₂ batteries and beyond.

The aprotic lithium-oxygen (Li-O₂) battery promises ultrahigh theoretical energy density (∼3,600 Wh·kg⁻¹) and is operated with oxygen reduction to generate the product of Li₂O₂ and its reverse oxidation (2Li⁺ + O₂ + 2e⁻ ↔ Li₂O₂, E⁰ = 2.96 V) (1–5). The sluggish oxygen cathode reactions, including the oxygen evolution reaction (OER) and the oxygen reduction reaction (ORR), lead to a high discharge/charge overvoltage (∼1.0 V) during cycles and low round-trip efficiency (6–9). Since the pioneering work on the photoinvolved Li-O₂ battery using TiO₂ (10) or C₃N₄ (11) under ultraviolet (UV)-light irradiation, reduction of the charge/discharge overvoltage via a photomediated strategy has been extensively studied and is anticipated to solve the kinetic issues of the Li-O₂ battery (12–18). However, the light absorption of most semiconductors used is confined in the region of UV light, accounting for only ca. 4% of the solar spectrum (14–16). Expanding the light harvesting from UV to visible light is the long-term goal and challenge of photocatalysis (17–20). Simultaneously, high carrier recombination consumes the majority of photoelectrons and holes before catalyzing the targeted reactions, resulting in a mismatch between the carrier lifetime and kinetics of ORR or OER (19–21). This necessitates a structural design of semiconducting materials for visible-light harvesting to accelerate the cathode reactions in Li-O₂ batteries.Localized surface plasmon resonance (LSPR), which refers to the collective oscillation of conduction band (CB) electrons in metal nanocrystals under resonant excitation, has recently gained much attention (22–25). The decay of excited LSPR can produce hot electrons and holes, which initiate various chemical reactions (22, 23). Intriguingly, when plasmonic metal (e.g., Au, Ag) nanoparticles (NPs) come into contact with a semiconductor such as MoS₂, TiO₂, etc., an interfacial Schottky barrier forms; this barrier functions as a filter to force the energetic electrons or holes to migrate across the interface while inhibiting their reverse movement, thereby leading to effective electron–hole separation and suppressed charge–carrier recombination (26–30). LSPR systems generally are composed of plasmonic metal and semiconductors and exhibit the benefits of a low electron–hole recombination rate, enhanced light harvesting, and tailored response wavelengths from the visible to the near-infrared region (22). Recently, Au/CdSe (31) and Au/Ni(OH)₂ (32) heterojunctions have been attempted for a photocatalytic hydrogen evolution reaction and OER with the aid of hot electrons and holes under visible light. Coupling the plasmonic metal with suitable semiconductors for broadened light harvesting and a plasmon-enhanced effect is highly desirable for both ORR and OER in the Li-O₂ battery.Herein, we report defective C₃N₄ (Au/N_V-C₃N₄) decorated with plasmonic Au NPs as a bifunctional heterojunction catalyst that promotes cathode reactions of the Li-O₂ battery under visible light. The N_V on N_V-C₃N₄ is prone to adsorb and activate O₂, and the plasmon-excited electrons on Au migrate to the CB of N_V-C₃N₄ and relax to the N_V-induced defect band (DB) for O₂ reduction to LiO₂; then it undergoes electron reduction to Li₂O₂. Reversely, the Li₂O₂ is removed by the holes on the Au NPs driven by the applied voltage. The discharge voltage is raised to 3.16 V, and the charge voltage is lowered to 3.26 V at 0.05 mA·cm⁻² with a good rate capability and cycle stability. This investigation integrates a plasmonic heterojunction into the aprotic Li-O₂ battery and illustrates photoenergy conversion and storage under visible light.     

A limited role of NKCC1 in telencephalic glutamatergic neurons for developing hippocampal network dynamics and behavior

NKCC1 is the primary transporter mediating chloride uptake in immature principal neurons, but its role in the development of in vivo network dynamics and cognitive abilities remains unknown. Here, we address the function of NKCC1 in developing mice using electrophysiological, optical, and behavioral approaches. We report that NKCC1 deletion from telencephalic glutamatergic neurons decreases in vitro excitatory actions of γ-aminobutyric acid (GABA) and impairs neuronal synchrony in neonatal hippocampal brain slices. In vivo, it has a minor impact on correlated spontaneous activity in the hippocampus and does not affect network activity in the intact visual cortex. Moreover, long-term effects of the developmental NKCC1 deletion on synaptic maturation, network dynamics, and behavioral performance are subtle. Our data reveal a neural network function of NKCC1 in hippocampal glutamatergic neurons in vivo, but challenge the hypothesis that NKCC1 is essential for major aspects of hippocampal development.

Intracellular chloride concentration ([Cl⁻]_i) is a major determinant of neuronal excitability, as synaptic inhibition is primarily mediated by chloride-permeable receptors (1). In the mature brain, [Cl⁻]_i is maintained at low levels by chloride extrusion, which renders γ-aminobutyric acid (GABA) hyperpolarizing (2) and counteracts activity-dependent chloride loads (3). GABAergic inhibition in the adult is crucial not only for preventing runaway excitation of glutamatergic cells (4) but also for entraining neuronal assemblies into oscillations underlying cognitive processing (5). However, the capacity of chloride extrusion is low during early brain development (6, 7). Additionally, immature neurons are equipped with chloride uptake mechanisms, particularly with the Na⁺/K⁺/2Cl⁻ cotransporter NKCC1 (8–12). NKCC1 contributes to the maintenance of high [Cl⁻]_i in the developing brain (13), favoring depolarization through GABA_A receptor (GABA_AR) activation in vivo (14, 15).When GABA acts as a depolarizing neurotransmitter, neural circuits generate burst-like spontaneous activity (16–20), which is crucial for their developmental refinement (21–24). In vitro evidence indicates that GABAergic interneurons promote neuronal synchrony in an NKCC1-dependent manner (10, 12, 25–28). However, the in vivo developmental functions of NKCC1 are far from understood (29, 30). One fundamental question is to what extent NKCC1 and GABAergic depolarization supports correlated spontaneous activity in the neonatal brain. In the neocortex, GABA imposes spatiotemporal inhibition on network activity already in the neonatal period (14, 25, 31, 32). Whether a similar situation applies to other brain regions is unknown, as two recent chemo- and optogenetic studies in the hippocampus yielded opposing results (25, 33). Manipulations of the chloride driving force are potentially suited to resolve these divergent findings, but pharmacological (34–36) or conventional knockout (10, 11, 37) strategies suffer from unspecific effects that complicate interpretations.Here, we overcome this limitation by selectively deleting Slc12a2 (encoding NKCC1) from telencephalic glutamatergic neurons. We show that chloride uptake via NKCC1 promotes synchronized activity in acute hippocampal slices, but has weak and event type-dependent effects in CA1 in vivo. Long-term loss of NKCC1 leads to subtle changes of network dynamics in the adult, leaving synaptic development unperturbed and behavioral performance intact. Our data suggest that NKCC1-dependent chloride uptake is largely dispensable for several key aspects of hippocampal development in vivo.