Allosteric regulation of SERCA by phosphorylation-mediated conformational shift of phospholamban

The membrane protein complex between the sarcoplasmic reticulum Ca²⁺-ATPase (SERCA) and phospholamban (PLN) controls Ca²⁺ transport in cardiomyocytes, thereby modulating cardiac contractility. β-Adrenergic-stimulated phosphorylation of PLN at Ser-16 enhances SERCA activity via an unknown mechanism. Using solid-state nuclear magnetic resonance spectroscopy, we mapped the physical interactions between SERCA and both unphosphorylated and phosphorylated PLN in membrane bilayers. We found that the allosteric regulation of SERCA depends on the conformational equilibrium of PLN, whose cytoplasmic regulatory domain interconverts between three different states: a ground T state (helical and membrane associated), an excited R state (unfolded and membrane detached), and a B state (extended and enzyme-bound), which is noninhibitory. Phosphorylation at Ser-16 of PLN shifts the populations toward the B state, increasing SERCA activity. We conclude that PLN’s conformational equilibrium is central to maintain SERCA’s apparent Ca²⁺ affinity within a physiological window. This model represents a paradigm shift in our understanding of SERCA regulation by posttranslational phosphorylation and suggests strategies for designing innovative therapeutic approaches to enhance cardiac muscle contractility.The sarcoplasmic reticulum Ca²⁺-ATPase (SERCA)/phospholamban (PLN) complex regulates Ca²⁺ translocation into the sarcoplasmic reticulum (SR) of cardiomyocytes and constitutes the main mechanism of cardiac relaxation (diastole) (1–3). SERCA is a P-type ATPase that translocates two Ca²⁺ ions per ATP molecule hydrolyzed in exchange for three H⁺ (Fig. 1) (4, 5). PLN binds and allosterically inhibits SERCA function, decreasing its apparent affinity for Ca²⁺ ions (3, 6). On β-adrenergic stimulation, cAMP-dependent protein kinase A phosphorylates PLN at Ser-16, reversing the inhibition and augmenting cardiac output (3). Disruptions in this regulatory mechanism degenerate into Ca²⁺ mishandling and heart failure (3).Open in a separate windowFig. 1.Structures of PLN and SERCA. (A) Primary sequence and domains of PLN^AFA. The S16 phosphorylation site is marked with an arrow, and mutation sites (C36A, C41F, C46A) are indicated with asterisks. (B) 3D structures of PLN pentamer [Protein Data Bank (PDB) ID code 2KYV], PLN^AFA monomer in the T (PDB ID code 2KB7) and R states (PDB ID code 2LPF), and SERCA in complex with sarcolipin (PDB ID code 3W5A). SERCA includes 10 TM helices and three cytoplasmic domains: nucleotide binding (N domain), actuator (A domain), and phosphorylation (P domain). (C) Fluorescence energy transfer (FRET) between SERCA^AEDANS (donor) and PLN^Dab-AFA (acceptor), showing the formation of the complex. (D) Ca²⁺-dependent ATPase activity of the SERCA/PLN complex in PC/PE/PA lipid vesicles at lipid:PLN:SERCA molar ratios of 700:1:1 and 700:5:1.Several X-ray structures of SERCA have been determined along its enzymatic coordinates, providing atomic details on the structural transitions in the absence of PLN (4, 5). The first image of the SERCA/PLN complex resulted from cryo-EM studies (7), but the low-resolution data prevent an atomic view of PLN structure and architecture within the complex. In addition, mutagenesis and cross-linking data were used to model the complex, suggesting that the inhibitory transmembrane (TM) region of PLN is positioned into a binding groove far from the putative Ca²⁺ entry, as well as the ATP binding site, and located between TM helices M2, M4, M6, and M9 of SERCA. The location of PLN''s TM domain agrees with a recent crystal structure of the SERCA/PLN complex (8) and is remarkably similar to the one recently identified for a PLN homolog, sarcolipin, in complex with SERCA (9, 10) (Fig. 1).In the SERCA/PLN model, which was further refined using NMR constraints (11), the loop bridging the TM and cytoplasmic domain of PLN adopts an unfolded configuration, stretching toward the N domain of the enzyme (12, 13). Also, PLN’s cytoplasmic domain binds SERCA’s N domain in an α-helical conformation, suggesting that the inhibitory effect may be elicited via an induced fit mechanism.Interestingly, PLN''s cytoplasmic domain in the recent X-ray structure of the complex is completely unresolved (8), leaving many questions regarding its regulatory function unanswered. Moreover, there have been few structural data on the complex between SERCA and phosphorylated PLN. On the basis of cross-linking and sparse spectroscopic data, two different mechanistic models have been proposed: a dissociative model, in which phosphorylation causes PLN detachment from SERCA, reestablishing Ca²⁺ flux (14), and the subunit model, in which phosphorylation induces conformational rearrangements of PLN’s cytoplasmic domain without dissociation (15–17). Nevertheless, the two models fall short in the interpretation of the recent structure–function correlations (15, 18–20).In lipid bilayers, PLN adopts a helix-turn-helix structure arranged in an L-shaped configuration (Fig. 1) (21), with the helical membrane-spanning region (inhibitory) divided into hydrophilic domain Ib and hydrophobic domain II and an amphipathic region (regulatory) domain Ia, which harbors the recognition sequence for cAMP-dependent protein kinase A. A prominent feature of PLN is its conformational dynamics. Although domain II is relatively rigid and membrane-embedded, the cytoplasmic domain Ia, domain Ib, and the intervening loop are more dynamic. Importantly, domain Ia is metamorphic, adopting a helical structure when in contact with membrane (T or ground state) and becoming unfolded when detached (R or high energy, conformationally excited state) (11, 15, 18, 22). On average, the L-shaped T state is the most populated in either monomeric or pentameric PLN (Fig. 1) (21, 23), with the R state accounting for 16–20% of the conformational ensemble (24). Remarkably, PLN’s conformational equilibrium has been detected in both synthetic and natural SR lipid membranes (22) and persists in the presence of SERCA (15, 25). Phosphorylation of PLN at Ser-16 shifts the equilibrium, inducing an order-to-disorder transition and increasing the R state population (15, 18, 26, 27). To date, however, PLN’s conformational equilibrium and its different structural states have never been correlated to SERCA’s regulation.Here, we used magic angle spinning (MAS) solid-state NMR spectroscopy in combination with different isotopic and spin-labeling schemes to elucidate the role of PLN’s conformational equilibrium and phosphorylation in the context of the allosteric regulation of SERCA. We found that in the presence of SERCA, the TM domain of PLN remains anchored to the ATPase, whereas the cytoplasmic domain Ia populates three conformational states: T, R, and SERCA-bound (B). The inhibitory T state is in equilibrium with both the excited R state and a sparsely populated B state, which is noninhibitory and maintains the Ca²⁺ flux within a physiological window. Phosphorylation at Ser-16 shifts the equilibrium toward the B state, reversing PLN inhibitory action. We propose that enhancement of SERCA activity on PLN phosphorylation is achieved through a shift of PLN’s conformational equilibrium toward the B state, with the phosphorylated domain Ia binding a site located between the N and P domains of SERCA.     

Dysfunctional conformational dynamics of protein kinase A induced by a lethal mutant of phospholamban hinder phosphorylation

The dynamic interplay between kinases and substrates is crucial for the formation of catalytically committed complexes that enable phosphoryl transfer. However, a clear understanding on how substrates modulate kinase structural dynamics to control catalytic efficiency is still missing. Here, we used solution NMR spectroscopy to study the conformational dynamics of two complexes of the catalytic subunit of the cAMP-dependent protein kinase A with WT and R14 deletion phospholamban, a lethal human mutant linked to familial dilated cardiomyopathy. Phospholamban is a central regulator of heart muscle contractility, and its phosphorylation by protein kinase A constitutes a primary response to β-adrenergic stimulation. We found that the single deletion of arginine in phospholamban’s recognition sequence for the kinase reduces its binding affinity and dramatically reduces phosphorylation kinetics. Structurally, the mutant prevents the enzyme from adopting conformations and motions committed for catalysis, with concomitant reduction in catalytic efficiency. Overall, these results underscore the importance of a well-tuned structural and dynamic interplay between the kinase and its substrates to achieve physiological phosphorylation levels for proper Ca²⁺ signaling and normal cardiac function.Phosphorylation by cAMP-dependent protein kinase A (PKA) is a central signaling pathway in cardiomyocytes, where it modulates the activity of several Ca²⁺ handling proteins (1, 2). In particular, PKA targets phospholamban (PLN), a small membrane protein embedded in the sarcoplasmic reticulum that binds and regulates the activity of the sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA). The SERCA/PLN complex plays a critical role in cardiac contractility, because it is responsible for Ca²⁺ reuptake in the sarcoplasmic reticulum, thereby controlling muscle relaxation, or diastole (1). PLN regulation of SERCA keeps Ca²⁺ flux within a physiological window (3). Unphosphorylated PLN reduces SERCA’s apparent affinity for Ca²⁺, whereas phosphorylation at Ser16 reverses PLN’s inhibitory effect, increasing Ca²⁺ flux and enhancing cardiac muscle relaxation (4). PKA phosphorylation of PLN is a primary response to β-adrenergic stimulation in the heart, affecting cardiac output directly (5). Altered levels of PLN phosphorylation (i.e., hypo- or hyperphosphorylation) cause SERCA’s function to be outside this physiological window. Specifically, aberrant PLN phosphorylation depresses Ca²⁺ cycling, with an attenuation of Ca²⁺ transients in both amplitude and frequency, resulting in cardiac disease (6, 7). Six naturally occurring mutations of PLN have been linked to early and/or late onset dilated cardiomyopathy (DCM), a leading cause of morbidity and mortality worldwide (8, 9). First reported by Kranias et al. (10, 11), the R14del mutation of PLN (PLN^R14del) was found in several DCM patients (10) and was more recently identified in 14% of a cohort of 354 patients diagnosed with either DCM or arrhythmogenic right ventricular cardiomyopathy (12). It has been proposed that the dysfunctional effects of PLN^R14del are due to chronic suppression of SERCA activity, with a synergistic inhibition by the mutant and the WT PLN (PLN^WT) (10, 13). Recent studies, however, show that PLN phosphorylation by PKA-C is significantly hindered in R14del transgenic mice (∼7% of WT levels), causing Ca²⁺ mishandling and progression of DCM (13). Although the phenotype and genotype of the R14 deletion have been identified, the molecular mechanisms for its aberrant phosphorylation are still unknown.The R14 deletion is located at the recognition sequence for the catalytic subunit of PKA (PKA-C) (Fig. 1) (10). From the X-ray structure of the PKA-C/ATPγN/PLN^WT complex [Protein Data Bank (PDB) ID code 3O7L], the PLN consensus sequence for PKA-C (R-R-X₁-S-X₂, where X denotes a variable amino acid) adopts an extended conformation within the binding groove (14, 15). The side chain of R14 (P-2 site) points toward the large lobe and interacts with Y204, E230, and E203, whereas the side chain of R13 (P-3 site) interacts with S51, the ribose ring, and hydroxyl group of Y330, clamping on the small lobe (Fig. 1 A and B) (14). Based on these structural features, we hypothesized that the R14 deletion disrupts intermolecular interactions between enzyme and substrate, affecting the motions responsible for opening and closing the active cleft and ultimately enzyme turnover. To test this hypothesis, we compared the kinetics of phosphorylation, thermodynamics of binding, and enzyme structural dynamics of the PKA-C/PLN^R14del complex to PKA-C/PLN^WT using steady-state kinetic measurements, isothermal titration calorimetry (ITC), NMR spectroscopy, and molecular dynamic (MD) simulations. We found that, thermodynamically and kinetically, PLN^R14del exhibits characteristics of a poor substrate compared with PLN^WT. More importantly, deletion of R14 prevents the formation of a catalytically committed Michaelis complex, causing dysfunctional conformational dynamics that concur with the reduction of catalytic efficiency. These findings explain the molecular mechanisms for the sluggish phosphorylation kinetics, leading to the Ca²⁺ mishandling associated with DCM. Overall, our study emphasizes the importance of well-tuned dynamic interplay between enzyme and substrate to achieve optimal catalysis.Open in a separate windowFig. 1.Molecular interactions between PKA-C and peptide substrates or inhibitors. (A) Primary sequence of the WT and R14del peptides corresponding to the cytoplasmic region of PLN and the high affinity peptide inhibitor, PKI_5–24. (B) X-ray structure of PKA-C (PDB:1ATP) with the architecture of the peptide binding site, with the electrostatic network of interactions between enzyme and substrate.     

Metformin increases degradation of phospholamban via autophagy in cardiomyocytes

Phospholamban (PLN) is an effective inhibitor of the sarco(endo)plasmic reticulum Ca²⁺ ATPase (SERCA). Here, we examined PLN stability and degradation in primary cultured mouse neonatal cardiomyocytes (CMNCs) and mouse hearts using immunoblotting, molecular imaging, and [³⁵S]methionine pulse-chase experiments, together with lysosome (chloroquine and bafilomycin A1) and autophagic (3-methyladenine and Atg5 siRNA) antagonists. Inhibiting lysosomal and autophagic activities promoted endogenous PLN accumulation, whereas accelerating autophagy with metformin enhanced PLN degradation in CMNCs. This reduction in PLN levels was functionally correlated with an increased rate of SERCA2a activity, accounting for an inotropic effect of metformin. Metabolic labeling reaffirmed that metformin promoted wild-type and R9C PLN degradation. Immunofluorescence showed that PLN and the autophagy marker, microtubule light chain 3, became increasingly colocalized in response to chloroquine and bafilomycin treatments. Mechanistically, pentameric PLN was polyubiquitinylated at the K3 residue and this modification was required for p62-mediated selective autophagy trafficking. Consistently, attenuated autophagic flux in HECT domain and ankyrin repeat-containing E3 ubiquitin protein ligase 1-null mouse hearts was associated with increased PLN levels determined by immunoblots and immunofluorescence. Our study identifies a biological mechanism that traffics PLN to the lysosomes for degradation in mouse hearts.Phospholamban (PLN) is a 52-amino acid peptide located in the sarcoplasmic reticulum (SR) membrane in cardiac, slow-twitch skeletal, and smooth muscle, where it exists as a monomer or pentamer. Whereas monomeric PLN physically interacts with sarco(endo)plasmic reticulum Ca²⁺ ATPase type 2a (SERCA2a) to antagonize its function, pentameric PLN complexes are thought to be a reservoir of inactive PLN (1–3). The physical interaction between SERCA2a and PLN reduces the apparent affinity of SERCA2a for Ca²⁺, thereby making SERCA2a less active in transporting Ca²⁺ from the cytoplasm to the lumen of the SR at the same concentration of cytoplasmic Ca²⁺. The physical interaction between the two proteins is regulated by phosphorylation of PLN at Ser16 by protein kinase A or at Thr17 by Ca²⁺/calmodulin-dependent protein kinase II (2). Phosphorylation of PLN reduces its affinity for SERCA2a, thereby increasing SERCA2a activity (2). Evidence from transgenic mice also supports the inhibitory function of PLN. Although targeted PLN deletion enhances baseline cardiac performance, cardiac-specific overexpression of superinhibitory forms of PLN leads to decreases in the affinity of SERCA2a for Ca²⁺ (2). These observations underscore the primary role of PLN as a regulator of SERCA2a activity and, therefore, as a crucial regulator of cardiac contractility. PLN inhibition of SERCA2a can be reversed by either external (i.e., activation of β-adrenergic receptors) or internal (i.e., increased intracellular Ca²⁺ concentration) stimuli.Previous studies identified three PLN mutations in families of patients with hereditary dilated cardiomyopathy. These mutations, the substitution of Cys for Arg9 (R9C) (4), Arg14 deletion (RΔ14) (5), and the substitution of TGA for TAA in the Leu39 codon, creating a stop codon (L39stop) (6), also lead to dilated cardiomyopathy in transgenic mice. At the cellular level, ectopically expressed RΔ14 and L39stop PLN mutants localize at the plasma membrane in HEK-293T cells, cultured mouse neonatal cardiomyocytes, and cardiac fibroblasts, whereas wild-type and the R9C mutant reside within the endoplasmic reticulum (ER)/SR (6, 7). These data, together with a recent study by Sharma et al. (8), suggest a highly ordered trafficking of PLN, ultimately ensuring correct localization, and thus function, within the SR. However, PLN trafficking and degradation mechanisms in mammalian cardiomyocytes have not been clearly established.Protein degradation and clearance of damaged organelles are critical for cellular physiology, and failure in proper clearance has been shown to have pathological repercussions (9). Autophagy is a major mechanism that mediates protein and organelle degradation in response to external and internal signals. External stimulation through pharmacological agonists, such as metformin and rapamycin, promotes autophagy via AMP-activated protein kinase (AMPK) and mammalian target of rapamycin signal pathways, whereas amino acid starvation and an increased intracellular AMP/ATP ratio serve as internal signals to promote autophagy via the Ca²⁺/Calmodulin-dependent kinase kinase-β (10). Steps in the autophagy pathway involve nucleation of targeted macromolecules on the ER membrane, trafficking of autophagosomes to lysosomes and, finally, fusion of the autophagosome-lysosome, resulting in targeted protein degradation (11). In the heart, autophagy plays a crucial role in response to insults, in part by relieving ER stress (12) and removing damaged mitochondria (13). Loss of autophagy could result in irreversible apoptosis and reduced cardiac functioning (14).To characterize PLN degradation, we conducted a series of assays in cultured mouse neonatal cardiomyocytes (CMNCs) and the hearts of HECT domain and ankyrin repeat-containing E3 ubiquitin protein ligase 1 (Hace1)-null mice. Our results show that PLN degradation required both polyubiquitinylation and p62-mediated selective autophagy in CMNCs. Loss of HACE1 was associated with increased PLN levels, supporting the notion that selective autophagy modulates PLN degradation in vivo. Metformin promoted wild-type and R9C PLN degradation through autophagic pathways, resulting in metformin-induced inotropic enhancement.     

STIM1 enhances SR Ca2+ content through binding phospholamban in rat ventricular myocytes

In ventricular myocytes, the physiological function of stromal interaction molecule 1 (STIM1), an endo/sarcoplasmic reticulum (ER/SR) Ca²⁺ sensor, is unclear with respect to its cellular localization, its Ca²⁺-dependent mobilization, and its action on Ca²⁺ signaling. Confocal microscopy was used to measure Ca²⁺ signaling and to track the cellular movement of STIM1 with mCherry and immunofluorescence in freshly isolated adult rat ventricular myocytes and those in short-term primary culture. We found that endogenous STIM1 was expressed at low but measureable levels along the Z-disk, in a pattern of puncta and linear segments consistent with the STIM1 localizing to the junctional SR (jSR). Depleting SR Ca²⁺ using thapsigargin (2–10 µM) changed neither the STIM1 distribution pattern nor its mobilization rate, evaluated by diffusion coefficient measurements using fluorescence recovery after photobleaching. Two-dimensional blue native polyacrylamide gel electrophoresis and coimmunoprecipitation showed that STIM1 in the heart exists mainly as a large protein complex, possibly a multimer, which is not altered by SR Ca²⁺ depletion. Additionally, we found no store-operated Ca²⁺ entry in control or STIM1 overexpressing ventricular myocytes. Nevertheless, STIM1 overexpressing cells show increased SR Ca²⁺ content and increased SR Ca²⁺ leak. These changes in Ca²⁺ signaling in the SR appear to be due to STIM1 binding to phospholamban and thereby indirectly activating SERCA2a (Sarco/endoplasmic reticulum Ca²⁺ ATPase). We conclude that STIM1 binding to phospholamban contributes to the regulation of SERCA2a activity in the steady state and rate of SR Ca²⁺ leak and that these actions are independent of store-operated Ca²⁺ entry, a process that is absent in normal heart cells.Store-operated Ca²⁺ entry (SOCE) is a cellular mechanism to ensure that sufficient levels of Ca²⁺ are present in the intracellular Ca²⁺ stores to enable robust signaling (1). SOCE depends on the presence and interaction of two proteins, STIM1 (stromal interaction molecule 1) and Orai1 (a low conductance plasma/sarcolemmal Ca²⁺ channel), or their equivalents (2–5). STIM1 is an endo/sarcoplasmic reticulum (ER/SR) Ca²⁺-sensitive protein that interacts with Orai1 to activate the channel function of Orai1, a Ca²⁺ selective channel, and thus permit Ca²⁺ entry. SOCE is clearly present in nonexcitable cells such as T lymphocytes and some excitable cells including skeletal muscle cells (4, 6–13). STIM1 is a membrane-spanning ER/SR protein with a single transmembrane domain and a luminal Ca²⁺ ([Ca²⁺]_ER/SR)-sensing domain. When luminal Ca²⁺ is low (i.e., [Ca²⁺]_ER/SR drops to less than 300 µM), then STIM1 self-aggregates and associates with Orai1 to activate it, producing a SOCE current (I_SOCE) (2, 14–16) and Ca²⁺ entry (with a reversal potential E_SOCE ∼ +50 mV or more) (17, 18). Then, as [Ca²⁺]_ER/SR increases in response to the Ca²⁺ influx, the process reverses.In adult skeletal muscle cells, Ca²⁺ influx is normally low, and it has been suggested that SOCE is needed for maintaining an appropriate level of [Ca²⁺]_ER/SR and correct Ca²⁺ signaling (6, 7, 9, 19). In skeletal muscle, it has been hypothesized that STIM1 is prelocalized in the SR terminal cisternae (6, 20) and hence can more rapidly respond to changes in [Ca²⁺]_ER/SR. The putative importance of SOCE in skeletal muscle was further supported by the observation that the skeletal muscle dysfunction is significant in STIM1-null mice where 91% (30/33) of the animals died in the perinatal period from a skeletal myopathy (6). Furthermore, in humans, STIM1 mutations were identified as a genetic cause of tubular aggregate myopathy (21).Despite the clarity of the SOCE paradigm, the canonical SOCE activation process described above does not apply to all conditions in which STIM1 and Orai1 interact. For example, in T lymphocytes, STIM1 clustering is necessary and sufficient to activate SOCE, regardless of whether [Ca²⁺]_ER/SR is low (4). When present, the STIM1 EF hand mutation causes STIM1 oligomerization and constitutive Ca²⁺ influx across the plasma membrane into cells with full Ca²⁺ stores (4). Although this is consistent with the use of STIM1 clusters and puncta to measure the activation of Orai1 (15, 16, 22, 23), it does not necessarily reflect the state of [Ca²⁺]_ER/SR. Furthermore, several small-molecule bioactive reagents, such as 2-APB and FCCP, neither of which causes [Ca²⁺]_ER/SR depletion, induce STIM1 clustering (24). Thus, STIM1 may have actions that are more complicated than simple [Ca²⁺]_ER/SR sensing and Orai1 signaling.Cardiomyocytes have been reported to have SOCE (8, 13, 25, 26) but are very different from many of the cells noted above that exhibit significant [Ca²⁺]_ER/SR depletion-sensitive Ca²⁺ entry through the Ca²⁺-selective Orai1. Cardiac ventricular myocytes are different from the other cells in that they have large, regular, and dynamic changes in [Ca²⁺]_i and robust influx and extrusion pathways across the sarcolemmal membrane. For example, it is not unusual for investigators to measure a 10–20 nA calcium current (I_Ca,L) in single cardiac ventricular myocytes that is readily extruded by the sarcolemmal Na⁺/Ca²⁺ exchanger. Because of these large fluxes, adult ventricular myocytes have no “need” for SOCE and the same logic applies to neonatal cardiomyocytes. Nevertheless, reports of SOCE in neonatal cardiac myocytes are clear (10, 12, 13). Against this background, we have attempted to determine if STIM1 is present in adult cardiomyocytes and, if so, where the protein is located, how it is mobilized, and how it may interact with other Ca²⁺ signal proteins. In the work presented here, we show that STIM1 is present but that its function in heart is distinct from the canonical SOCE behavior and does not contribute to Ca²⁺ influx through I_SOCE. Instead we show that STIM1 binds phospholamban (PLN), an endogenous SERCA2a inhibitor in the heart (27), and by doing so reduces the PLN-dependent inhibition of SERCA2a and thereby indirectly activates SERCA2a.     

Cyclic nucleotide-gated channel 18 is an essential Ca2+ channel in pollen tube tips for pollen tube guidance to ovules in Arabidopsis

In flowering plants, pollen tubes are guided into ovules by multiple attractants from female gametophytes to release paired sperm cells for double fertilization. It has been well-established that Ca²⁺ gradients in the pollen tube tips are essential for pollen tube guidance and that plasma membrane Ca²⁺ channels in pollen tube tips are core components that regulate Ca²⁺ gradients by mediating and regulating external Ca²⁺ influx. Therefore, Ca²⁺ channels are the core components for pollen tube guidance. However, there is still no genetic evidence for the identification of the putative Ca²⁺ channels essential for pollen tube guidance. Here, we report that the point mutations R491Q or R578K in cyclic nucleotide-gated channel 18 (CNGC18) resulted in abnormal Ca²⁺ gradients and strong pollen tube guidance defects by impairing the activation of CNGC18 in Arabidopsis. The pollen tube guidance defects of cngc18-17 (R491Q) and of the transfer DNA (T-DNA) insertion mutant cngc18-1 (+/−) were completely rescued by CNGC18. Furthermore, domain-swapping experiments showed that CNGC18’s transmembrane domains are indispensable for pollen tube guidance. Additionally, we found that, among eight Ca²⁺ channels (including six CNGCs and two glutamate receptor-like channels), CNGC18 was the only one essential for pollen tube guidance. Thus, CNGC18 is the long-sought essential Ca²⁺ channel for pollen tube guidance in Arabidopsis.Pollen tubes deliver paired sperm cells into ovules for double fertilization, and signaling communication between pollen tubes and female reproductive tissues is required to ensure the delivery of sperm cells into the ovules (1). Pollen tube guidance is governed by both female sporophytic and gametophytic tissues (2, 3) and can be separated into two categories: preovular guidance and ovular guidance (1). For preovular guidance, diverse signaling molecules from female sporophytic tissues have been identified, including the transmitting tissue-specific (TTS) glycoprotein in tobacco (4), γ-amino butyric acid (GABA) in Arabidopsis (5), and chemocyanin and the lipid transfer protein SCA in Lilium longiflorum (6, 7). For ovular pollen tube guidance, female gametophytes secrete small peptides as attractants, including LUREs in Torenia fournieri (8) and Arabidopsis (9) and ZmEA1 in maize (10, 11). Synergid cells, central cells, egg cells, and egg apparatus are all involved in pollen tube guidance, probably by secreting different attractants (9–15). Additionally, nitric oxide (NO) and phytosulfokine peptides have also been implicated in both preovular and ovular pollen tube guidance (16–18). Thus, pollen tubes could be guided by diverse attractants in a single plant species.Ca²⁺ gradients at pollen tube tips are essential for both tip growth and pollen tube guidance (19–27). Spatial modification of the Ca²⁺ gradients leads to the reorientation of pollen tube growth in vitro (28, 29). The Ca²⁺ gradients were significantly increased in pollen tubes attracted to the micropyles by synergid cells in vivo, compared with those not attracted by ovules (30). Therefore, the Ca²⁺ gradients in pollen tube tips are essential for pollen tube guidance. The Ca²⁺ gradients result from external Ca²⁺ influx, which is mainly mediated by plasma membrane Ca²⁺ channels in pollen tube tips. Thus, the Ca²⁺ channels are the key components for regulating the Ca²⁺ gradients and are consequently essential for pollen tube guidance. Using electrophysiological techniques, inward Ca²⁺ currents were observed in both pollen grain and pollen tube protoplasts (31–36), supporting the presence of plasma membrane Ca²⁺ channels in pollen tube tips. Recently, a number of candidate Ca²⁺ channels were identified in pollen tubes, including six cyclic nucleotide-gated channels (CNGCs) and two glutamate receptor-like channels (GLRs) in Arabidopsis (37–40). Three of these eight channels, namely CNGC18, GLR1.2, and GLR3.7, were characterized as Ca²⁺-permeable channels (40, 41) whereas the ion selectivity of the other five CNGCs has not been characterized. We hypothesized that the Ca²⁺ channel essential for pollen tube guidance could be among these eight channels.In this research, we first characterized the remaining five CNGCs as Ca²⁺ channels. We further found that CNGC18, out of the eight Ca²⁺ channels, was the only one essential for pollen tube guidance in Arabidopsis and that its transmembrane domains were indispensable for pollen tube guidance.     

Cyst formation following disruption of intracellular calcium signaling

Mutations in polycystin 1 and 2 (PC1 and PC2) cause the common genetic kidney disorder autosomal dominant polycystic kidney disease (ADPKD). It is unknown how these mutations result in renal cysts, but dysregulation of calcium (Ca²⁺) signaling is a known consequence of PC2 mutations. PC2 functions as a Ca²⁺-activated Ca²⁺ channel of the endoplasmic reticulum. We hypothesize that Ca²⁺ signaling through PC2, or other intracellular Ca²⁺ channels such as the inositol 1,4,5-trisphosphate receptor (InsP3R), is necessary to maintain renal epithelial cell function and that disruption of the Ca²⁺ signaling leads to renal cyst development. The cell line LLC-PK1 has traditionally been used for studying PKD-causing mutations and Ca²⁺ signaling in 2D culture systems. We demonstrate that this cell line can be used in long-term (8 wk) 3D tissue culture systems. In 2D systems, knockdown of InsP3R results in decreased Ca²⁺ transient signals that are rescued by overexpression of PC2. In 3D systems, knockdown of either PC2 or InsP3R leads to cyst formation, but knockdown of InsP3R type 1 (InsP3R1) generated the largest cysts. InsP3R1 and InsP3R3 are differentially localized in both mouse and human kidney, suggesting that regional disruption of Ca²⁺ signaling contributes to cystogenesis. All cysts had intact cilia 2 wk after starting 3D culture, but the cells with InsP3R1 knockdown lost cilia as the cysts grew. Studies combining 2D and 3D cell culture systems will assist in understanding how mutations in PC2 that confer altered Ca²⁺ signaling lead to ADPKD cysts.The commonly occurring genetic kidney disorder, autosomal dominant polycystic kidney disease (ADPKD), is the result of mutations in polycystin 1 or 2 (PC1 or PC2). The progressive cyst formation within all segments of the nephron that defines the disorder leads to renal failure requiring treatment by dialysis and/or organ transplantation (1–3). Altered Ca²⁺ signaling is one of several pathways that have been implicated in the disease (4, 5). A major limitation toward elucidating the role of Ca²⁺ signaling in cyst formation has been the lack of easily manipulated, physiologically relevant experimental methodologies.In the past, ADPKD research has relied largely upon data from mouse models and cells maintained in 2D cell culture. Mouse models have played a significant role in understanding the biology of cyst formation but are unable to fully recapitulate the physiology of disease progression in humans due to the inherent differences between the species including life span, genetics, and environment. Two-dimensional cell culture has the ability to provide information on signaling pathways and response to therapies in a fast, high-throughput manner, but is incapable of replicating the inherent 3D nature of cyst formation. Advances in 3D tissue culture over the past 2 decades have improved the ability to model cyst development in vitro. However, previously published 3D tissue models of ADPKD have relied upon short-term culture of Madin-Darby canine kidney (MDCK) cells (6–12) or cells from patients (13–18) or PC1-null mice (19, 20; for review, see ref. 21). Recently, 3D tissues have been developed that incorporate mouse cells containing a shRNA-mediated knockdown of PC1 (9, 19). The benefits of this system include the use of a cell line, thus eliminating the need to isolate primary cells, and the use of cells with a stable genetic background.Ca²⁺ signaling underpins many cellular processes ranging from cell proliferation to cell death. Intracellular Ca²⁺ levels can be modified by opening of the inositol 1, 4, 5-trisphosphate receptor (InsP3R) or other intracellular Ca²⁺ release channels, including PC2. Over 99% of PC2 resides on the endoplasmic reticulum (22), where it is known to act as a modulator of the InsP3R and the ryanodine receptor (RyR) (23), with the remainder on the primary cilia. PC2 itself can function as a Ca²⁺-activated Ca²⁺ release channel (22, 24).Although it was demonstrated in 3D cultures that the knockdown of PC1 leads to cyst development (25), the effect of knocking down PC2 or other Ca²⁺-signaling proteins has not been shown. It has been hypothesized that the disruption of PC2, or the proteins that it interacts with, will result in cyst growth, as Ca²⁺ is a major signaling molecule (26, 27). Cells with decreased PC2 have been linked with decreased Ca²⁺ signaling (28), and overexpression of PC2 has been shown to act as an inhibitor of cell proliferation (29). Changing PC2 expression levels alters the uptake of Ca²⁺ into the endoplasmic reticulum, leading to liver cyst formation (30), but no direct link involving the release of Ca²⁺ from the endoplasmic reticulum has been implicated in renal cyst development. Similarly, changes in the expression of the InsP3R have been correlated with various disease conditions; for example, the InsP3R is upregulated in colorectal cancer (31), but downregulated in bile duct obstruction and cholestasis (32, 33).Here, we demonstrate that cyst formation can be followed for several weeks using a 3D culture system and that the disruption of intracellular Ca²⁺ signaling, through the knockdown of either InsP3R or PC2, leads to cyst development.     

Convergent Ca2+ and Zn2+ signaling regulates apoptotic Kv2.1 K+ currents

A simultaneous increase in cytosolic Zn²⁺ and Ca²⁺ accompanies the initiation of neuronal cell death signaling cascades. However, the molecular convergence points of cellular processes activated by these cations are poorly understood. Here, we show that Ca²⁺-dependent activation of Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) is required for a cell death-enabling process previously shown to also depend on Zn²⁺. We have reported that oxidant-induced intraneuronal Zn²⁺ liberation triggers a syntaxin-dependent incorporation of Kv2.1 voltage-gated potassium channels into the plasma membrane. This channel insertion can be detected as a marked enhancement of delayed rectifier K⁺ currents in voltage clamp measurements observed at least 3 h following a short exposure to an apoptogenic stimulus. This current increase is the process responsible for the cytoplasmic loss of K⁺ that enables protease and nuclease activation during apoptosis. In the present study, we demonstrate that an oxidative stimulus also promotes intracellular Ca²⁺ release and activation of CaMKII, which, in turn, modulates the ability of syntaxin to interact with Kv2.1. Pharmacological or molecular inhibition of CaMKII prevents the K⁺ current enhancement observed following oxidative injury and, importantly, significantly increases neuronal viability. These findings reveal a previously unrecognized cooperative convergence of Ca²⁺- and Zn²⁺-mediated injurious signaling pathways, providing a potentially unique target for therapeutic intervention in neurodegenerative conditions associated with oxidative stress.Calcium has long been recognized as a critical component of neuronal cell death pathways triggered by oxidative, ischemic, and other forms of injury (1). Indeed, Ca²⁺ deregulation has been associated with a variety of detrimental processes in neurons, including mitochondrial dysfunction (2), generation of reactive oxygen species (3), and activation of apoptotic signaling cascades (4). More recently, zinc, a metal crucial for proper cellular functioning (5), has been found to be closely linked to many of the injurious conditions in which Ca²⁺ had been thought to play a prominent role (6–10). In fact, it has been suggested that a number of deleterious properties initially attributed to Ca²⁺ may have significant Zn²⁺-mediated components (11, 12). Although it is virtually impossible to chelate, or remove, Ca²⁺ without disrupting Zn²⁺ levels (13), the introduction of techniques to monitor Ca²⁺ and Zn²⁺ simultaneously in cells (14) has made it increasingly apparent that both cations have important yet possibly distinct roles in neuronal cell death (12, 15–18). However, the relationship between the cell death signaling pathways activated by the cations is unclear, and possible molecular points of convergence between these signaling cascades have yet to be identified.Injurious oxidative and nitrosative stimuli lead to the liberation of intracellular Zn²⁺ from metal binding proteins (19). The released Zn²⁺, in turn, triggers p38 MAPK- and Src-dependent Kv2.1 channel insertion into the plasma membrane, resulting in a prominent increase in delayed rectifier K⁺ currents in dying neurons, with no change in activation voltage, ∼3 h following a brief exposure to the stimulus (20–26). The increase in Kv2.1 channels present in the membrane mediates a pronounced loss of intracellular K⁺, likely accompanied by Cl⁻ (27, 28), that facilitates apoptosome assembly and caspase activation (20, 29–34). Indeed, K⁺ efflux appears to be a requisite event for the completion of many apoptotic programs, including oxidant-induced, Zn²⁺-mediated neuronal death (21).Ca²⁺ has been suggested to regulate the p38 MAPK signaling cascade via Ca²⁺/calmodulin-dependent protein kinase II (CaMKII)-mediated activation of the MAP3K apoptosis signaling kinase-1 (ASK-1) (35). Because ASK-1 is also required for p38-dependent manifestation of the Zn²⁺-triggered, Kv2.1-mediated enhancement of K⁺ currents (36), we hypothesized that the p38 activation cascade may provide a point of convergence between Ca²⁺ and Zn²⁺ signals following oxidative injury. Here, we report that Ca²⁺ and Zn²⁺ signals do, in fact, converge on a cellular event critical for the K⁺ current enhancement, and that CaMKII is required for this process. However, CaMKII does not act upstream of p38 activation as originally hypothesized, but instead interacts with the N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) protein syntaxin, which we showed to be necessary for the insertion of Kv2.1-encoded K⁺ channels following an apoptotic stimulus (23).     

Fine-tuning synaptic plasticity by modulation of CaV2.1 channels with Ca2+ sensor proteins

Modulation of P/Q-type Ca²⁺ currents through presynaptic voltage-gated calcium channels (Ca_V2.1) by binding of Ca²⁺/calmodulin contributes to short-term synaptic plasticity. Ca²⁺-binding protein-1 (CaBP1) and Visinin-like protein-2 (VILIP-2) are neurospecific calmodulin-like Ca²⁺ sensor proteins that differentially modulate Ca_V2.1 channels, but how they contribute to short-term synaptic plasticity is unknown. Here, we show that activity-dependent modulation of presynaptic Ca_V2.1 channels by CaBP1 and VILIP-2 has opposing effects on short-term synaptic plasticity in superior cervical ganglion neurons. Expression of CaBP1, which blocks Ca²⁺-dependent facilitation of P/Q-type Ca²⁺ current, markedly reduced facilitation of synaptic transmission. VILIP-2, which blocks Ca²⁺-dependent inactivation of P/Q-type Ca²⁺ current, reduced synaptic depression and increased facilitation under conditions of high release probability. These results demonstrate that activity-dependent regulation of presynaptic Ca_V2.1 channels by differentially expressed Ca²⁺ sensor proteins can fine-tune synaptic responses to trains of action potentials and thereby contribute to the diversity of short-term synaptic plasticity.Neurons fire repetitively in different frequencies and patterns, and activity-dependent alterations in synaptic strength result in diverse forms of short-term synaptic plasticity that are crucial for information processing in the nervous system (1–3). Short-term synaptic plasticity on the time scale of milliseconds to seconds leads to facilitation or depression of synaptic transmission through changes in neurotransmitter release. This form of plasticity is thought to result from residual Ca²⁺ that builds up in synapses during repetitive action potentials and binds to a Ca²⁺ sensor distinct from the one that evokes neurotransmitter release (1, 2, 4, 5). However, it remains unclear how changes in residual Ca²⁺ cause short-term synaptic plasticity and how neurotransmitter release is regulated to generate distinct patterns of short-term plasticity.In central neurons, voltage-gated calcium (Ca_V2.1) channels are localized in high density in presynaptic active zones where their P/Q-type Ca²⁺ current triggers neurotransmitter release (6–11). Because synaptic transmission is proportional to the third or fourth power of Ca²⁺ entry through presynaptic Ca_V2.1 channels, small changes in Ca²⁺ current have profound effects on synaptic transmission (2, 12). Studies at the calyx of Held synapse have provided important insights into the contribution of presynaptic Ca²⁺ current to short-term synaptic plasticity (13–17). Ca_V2.1 channels are required for synaptic facilitation, and Ca²⁺-dependent facilitation and inactivation of the P/Q-type Ca²⁺ currents are correlated temporally with synaptic facilitation and rapid synaptic depression (13–17).Molecular interactions between Ca²⁺/calmodulin (CaM) and Ca_V2.1 channels induce sequential Ca²⁺-dependent facilitation and inactivation of P/Q-type Ca²⁺ currents in nonneuronal cells (18–21). Facilitation and inactivation of P/Q-type currents are dependent on Ca²⁺/CaM binding to the IQ-like motif (IM) and CaM-binding domain (CBD) of the Ca_V2.1 channel, respectively (20, 21). This bidirectional regulation serves to enhance channel activity in response to short bursts of depolarizations and then to decrease activity in response to long bursts. In synapses of superior cervical ganglion (SCG) neurons expressing exogenous Ca_V2.1 channels, synaptic facilitation is induced by repetitive action potentials, and mutation of the IM and CBD motifs prevents synaptic facilitation and inhibits the rapid phase of synaptic depression (22). Thus, in this model synapse, regulation of presynaptic Ca_V2.1 channels by binding of Ca²⁺/CaM can contribute substantially to the induction of short-term synaptic plasticity by residual Ca²⁺.CaM is expressed ubiquitously, but short-term plasticity has great diversity among synapses, and the potential sources of this diversity are unknown. How could activity-dependent regulation of presynaptic Ca_V2.1 channels contribute to the diversity of short-term synaptic plasticity? CaM is the founding member of a large family of Ca²⁺ sensor (CaS) proteins that are differentially expressed in central neurons (23–25). Two CaS proteins, Ca²⁺-binding protein-1 (CaBP1) and Visinin-like protein-2(VILIP-2), modulate facilitation and inactivation of Ca_V2.1 channels in opposite directions through interaction with the bipartite regulatory site in the C-terminal domain (26, 27), and they have varied expression in different types of central neurons (23, 25, 28). CaBP1 strongly enhances inactivation and prevents facilitation of Ca_V2.1 channel currents, whereas VILIP-2 slows inactivation and enhances facilitation of Ca_V2.1 currents during trains of stimuli (26, 27). Molecular analyses show that the N-terminal myristoylation site and the properties of individual EF-hand motifs in CaBP1 and VILIP-2 determine their differential regulation of Ca_V2.1 channels (27, 29–31). However, the role of CaBP1 and VILIP-2 in the diversity of short-term synaptic plasticity is unknown, and the high density of Ca²⁺ channels and unique Ca²⁺ dynamics at the presynaptic active zone make extrapolation of results from studies in nonneuronal cells uncertain. We addressed this important question directly by expressing CaBP1 and VILIP-2 in presynaptic SCG neurons and analyzing their effects on synaptic plasticity. Our results show that CaM-related CaS proteins can serve as sensitive bidirectional switches that fine-tune the input–output relationships of synapses depending on their profile of activity and thereby maintain the balance of facilitation versus depression by the regulation of presynaptic Ca_V2.1 channels.     

In vivo genetic dissection of O2-evoked cGMP dynamics in a Caenorhabditis elegans gas sensor

cGMP signaling is widespread in the nervous system. However, it has proved difficult to visualize and genetically probe endogenously evoked cGMP dynamics in neurons in vivo. Here, we combine cGMP and Ca²⁺ biosensors to image and dissect a cGMP signaling network in a Caenorhabditis elegans oxygen-sensing neuron. We show that a rise in O₂ can evoke a tonic increase in cGMP that requires an atypical O₂-binding soluble guanylate cyclase and that is sustained until oxygen levels fall. Increased cGMP leads to a sustained Ca²⁺ response in the neuron that depends on cGMP-gated ion channels. Elevated levels of cGMP and Ca²⁺ stimulate competing negative feedback loops that shape cGMP dynamics. Ca²⁺-dependent negative feedback loops, including activation of phosphodiesterase-1 (PDE-1), dampen the rise of cGMP. A different negative feedback loop, mediated by phosphodiesterase-2 (PDE-2) and stimulated by cGMP-dependent kinase (PKG), unexpectedly promotes cGMP accumulation following a rise in O₂, apparently by keeping in check gating of cGMP channels and limiting activation of Ca²⁺-dependent negative feedback loops. Simultaneous imaging of Ca²⁺ and cGMP suggests that cGMP levels can rise close to cGMP channels while falling elsewhere. O₂-evoked cGMP and Ca²⁺ responses are highly reproducible when the same neuron in an individual animal is stimulated repeatedly, suggesting that cGMP transduction has high intrinsic reliability. However, responses vary substantially across individuals, despite animals being genetically identical and similarly reared. This variability may reflect stochastic differences in expression of cGMP signaling components. Our work provides in vivo insights into the architecture of neuronal cGMP signaling.The second messenger cyclic guanosine monophosphate (cGMP) regulates a range of physiological processes. In nervous systems, it can transduce sensory inputs (1) and modulate neuronal excitability and learning (2) and is implicated in control of mood and cognition (3). Precise regulation of cGMP levels ([cGMP]) is thought critical for these functions. This importance has prompted development of genetically encoded cGMP indicators, with the goal of visualizing cGMP dynamics with high temporal and spatial resolution (4, 5). Although these sensors have been used to image pharmacologically evoked changes in cGMP in cultured cells or tissue slices (6–10), endogenous cGMP dynamics have not been visualized and functionally dissected in vivo in any nervous system (4, 5).Local [cGMP] reflects the net activity of guanylate cyclases (GCs) that synthesize cGMP (11) and phosphodiesterases (PDEs) that degrade it (12, 13). Mammals have several families of GCs (14, 15) and eight families of cGMP PDEs (16), each with distinct regulatory properties. Different PDE types are often coexpressed, but little is known about how they work together. cGMP signaling alters cell physiology by controlling cGMP-dependent protein kinases (PKG) (17, 18), cGMP-gated channels (CNGC) (19), and cGMP-regulated PDEs (12). These cGMP effectors can also feed back to control cGMP dynamics.cGMP is a major second messenger in Caenorhabditis elegans, implicated in the function of a third of its sensory neurons, including thermosensory, olfactory, gustatory, and O₂-sensing neurons (20). Genetic and behavioral studies suggest that cGMP mediates sensory transduction in many of these neurons (21–25). Despite this pervasiveness, cGMP has not been visualized in any C. elegans cell: genetic inferences about its roles in signal transduction are untested, and we have no mechanistic insights into cGMP signaling dynamics and feedback control. Consistent with the prominence of cGMP signaling in the nematode, the C. elegans genome encodes 34 GCs (26), six PDE genes, at least one PKG (24, 27, 28), and six CNGC subunits (19, 21, 22, 29–31).Here, we use cGMP and Ca²⁺ sensors to visualize and dissect cGMP signaling dynamics in a C. elegans O₂ sensor. We image single and double mutants defective in a soluble guanylate cyclase (sGC), CNGC subunits, PDE-1, PDE-2, and PKG. Our results reveal a signaling network of interwoven checks and balances. Counterintuitively, cGMP activation of PDE-2 promotes cGMP accumulation by controlling gating of CNGC and limiting Ca²⁺-mediated negative feedback, including activation of PDE-1. We show that cGMP signal transduction is highly reliable when the same individual is stimulated repeatedly but, surprisingly, is highly variable across genetically identical, similarly reared animals. Finally, simultaneous imaging of O₂-evoked cGMP and Ca²⁺ responses suggests that cGMP dynamics can differ in distinct subcellular compartments of a C. elegans neuron, consistent with the existence of cGMP nanodomains.     

Rapid stimulus-evoked astrocyte Ca2+ elevations and hemodynamic responses in mouse somatosensory cortex in vivo

Increased neuron and astrocyte activity triggers increased brain blood flow, but controversy exists over whether stimulation-induced changes in astrocyte activity are rapid and widespread enough to contribute to brain blood flow control. Here, we provide evidence for stimulus-evoked Ca²⁺ elevations with rapid onset and short duration in a large proportion of cortical astrocytes in the adult mouse somatosensory cortex. Our improved detection of the fast Ca²⁺ signals is due to a signal-enhancing analysis of the Ca²⁺ activity. The rapid stimulation-evoked Ca²⁺ increases identified in astrocyte somas, processes, and end-feet preceded local vasodilatation. Fast Ca²⁺ responses in both neurons and astrocytes correlated with synaptic activity, but only the astrocytic responses correlated with the hemodynamic shifts. These data establish that a large proportion of cortical astrocytes have brief Ca²⁺ responses with a rapid onset in vivo, fast enough to initiate hemodynamic responses or influence synaptic activity.Brain function emerges from signaling in and between neurons and associated astrocytes, which causes fluctuations in cerebral blood flow (CBF) (1–5). Astrocytes are ideally situated for controlling activity-dependent increases in CBF because they closely associate with synapses and contact blood vessels with their end-feet (1, 6). Whether or not astrocytic Ca²⁺ responses develop often or rapidly enough to account for vascular signals in vivo is still controversial (7–10). Ca²⁺ responses are of interest because intracellular Ca²⁺ is a key messenger in astrocytic communication and because enzymes that synthesize the vasoactive substances responsible for neurovascular coupling are Ca²⁺-dependent (1, 4). Neuronal activity releases glutamate at synapses and activates metabotropic glutamate receptors on astrocytes, and this activation can be monitored by imaging cytosolic Ca²⁺ changes (11). Astrocytic Ca²⁺ responses are often reported to evolve on a slow (seconds) time scale, which is too slow to account for activity-dependent increases in CBF (8, 10, 12, 13). Furthermore, uncaging of Ca²⁺ in astrocytes triggers vascular responses in brain slices through specific Ca²⁺-dependent pathways with a protracted time course (14, 15). More recently, stimulation of single presynaptic neurons in hippocampal slices was shown to evoke fast, brief, local Ca²⁺ elevations in astrocytic processes that were essential for local synaptic functioning in the adult brain (16, 17). This work prompted us to reexamine the characteristics of fast, brief astrocytic Ca²⁺ signals in vivo with special regard to neurovascular coupling, i.e., the association between local increases in neural activity and the concomitant rise in local blood flow, which constitutes the physiological basis for functional neuroimaging.Here, we describe how a previously undescribed method of analysis enabled us to provide evidence for fast Ca²⁺ responses in a main fraction of astrocytes in mouse whisker barrel cortical layers II/III in response to somatosensory stimulation. The astrocytic Ca²⁺ responses were brief enough to be a direct consequence of synaptic excitation and correlated with stimulation-induced hemodynamic responses. Fast Ca²⁺ responses in astrocyte end-feet preceded the onset of dilatation in adjacent vessels by hundreds of milliseconds. This finding might suggest that communication at the gliovascular interface contributes considerably to neurovascular coupling.     

From the Cover: Mitochondrial calcium overload is a key determinant in heart failure

Calcium (Ca²⁺) released from the sarcoplasmic reticulum (SR) is crucial for excitation–contraction (E–C) coupling. Mitochondria, the major source of energy, in the form of ATP, required for cardiac contractility, are closely interconnected with the SR, and Ca²⁺ is essential for optimal function of these organelles. However, Ca²⁺ accumulation can impair mitochondrial function, leading to reduced ATP production and increased release of reactive oxygen species (ROS). Oxidative stress contributes to heart failure (HF), but whether mitochondrial Ca²⁺ plays a mechanistic role in HF remains unresolved. Here, we show for the first time, to our knowledge, that diastolic SR Ca²⁺ leak causes mitochondrial Ca²⁺ overload and dysfunction in a murine model of postmyocardial infarction HF. There are two forms of Ca²⁺ release channels on cardiac SR: type 2 ryanodine receptors (RyR2s) and type 2 inositol 1,4,5-trisphosphate receptors (IP3R2s). Using murine models harboring RyR2 mutations that either cause or inhibit SR Ca²⁺ leak, we found that leaky RyR2 channels result in mitochondrial Ca²⁺ overload, dysmorphology, and malfunction. In contrast, cardiac-specific deletion of IP3R2 had no major effect on mitochondrial fitness in HF. Moreover, genetic enhancement of mitochondrial antioxidant activity improved mitochondrial function and reduced posttranslational modifications of RyR2 macromolecular complex. Our data demonstrate that leaky RyR2, but not IP3R2, channels cause mitochondrial Ca²⁺ overload and dysfunction in HF.Type 2 ryanodine receptor/Ca²⁺ release channel (RyR2) and type 2 inositol 1,4,5-trisphosphate receptor (IP3R2) are the major intracellular Ca²⁺ release channels in the heart (1–3). RyR2 is essential for cardiac excitation–contraction (E–C) coupling (2), whereas the role of IP3R2 in cardiomyocytes is less well understood (3). E–C coupling requires energy in the form of ATP produced primarily by oxidative phosphorylation in mitochondria (4–8).Both increased and reduced mitochondrial Ca²⁺ levels have been implicated in mitochondrial dysfunction and increased reactive oxygen species (ROS) production in heart failure (HF) (6, 7, 9–17). Albeit Ca²⁺ is required for activation of key enzymes (i.e., pyruvate dehydrogenase phosphatase, isocitrate dehydrogenase, and α-ketoglutarate dehydrogenase) in the tricarboxylic acid (also known as Krebs) cycle (18, 19), excessive mitochondrial Ca²⁺ uptake has been associated with cellular dysfunction (14, 20). Furthermore, the exact source of mitochondrial Ca²⁺ has not been clearly established. Given the intimate anatomical and functional association between the sarcoplasmic reticulum (SR) and mitochondria (6, 21, 22), we hypothesized that SR Ca²⁺ release via RyR2 and/or IP3R2 channels in cardiomyocytes could lead to mitochondrial Ca²⁺ accumulation and dysfunction contributing to oxidative overload and energy depletion.     

Conformational signaling required for synaptic plasticity by the NMDA receptor complex

The NMDA receptor (NMDAR) is known to transmit important information by conducting calcium ions. However, some recent studies suggest that activation of NMDARs can trigger synaptic plasticity in the absence of ion flow. Does ligand binding transmit information to signaling molecules that mediate synaptic plasticity? Using Förster resonance energy transfer (FRET) imaging of fluorescently tagged proteins expressed in neurons, conformational signaling is identified within the NMDAR complex that is essential for downstream actions. Ligand binding transiently reduces FRET between the NMDAR cytoplasmic domain (cd) and the associated protein phosphatase 1 (PP1), requiring NMDARcd movement, and persistently reduces FRET between the NMDARcd and calcium/calmodulin-dependent protein kinase II (CaMKII), a process requiring PP1 activity. These studies directly monitor agonist-driven conformational signaling at the NMDAR complex required for synaptic plasticity.Agonist binding to the NMDAR is required for two major forms of synaptic plasticity: long-term potentiation (LTP) and long-term depression (LTD) (1). Surprisingly, activation of NMDARs can produce plasticity in opposite directions, with LTP enhancing transmission and LTD reducing transmission. A model was developed (2, 3) to explain how activation of NMDAR could produce these opposing phenomena: strong stimuli during LTP induction drive a large flux of Ca²⁺ through NMDARs, leading to a large increase in intracellular calcium ion concentration ([Ca²⁺]_i) that activates one series of biochemical steps leading to synaptic potentiation; a weaker stimulus during LTD induction drives a more reduced flux of Ca²⁺ through NMDARs, producing a modest increase in [Ca²⁺]_i that activates a different series of biochemical steps, leading to synaptic depression. However, this model is not consistent with recent studies suggesting that no change in [Ca²⁺]_i is required for LTD, and instead invokes metabotropic signaling by the NMDAR (4). Studies supporting an ion-flow-independent role for NMDARs in LTD (4–7) and other processes (7–13) stand in contrast to studies proposing that flow of Ca²⁺ through NMDAR is required for LTD (14) (see ref. 15 for additional references). An important test of an ion-flow-independent model would be to measure directly signaling actions by NMDARs in the absence of ion flow.     

VEGF-induced neoangiogenesis is mediated by NAADP and two-pore channel-2–dependent Ca2+ signaling

Vascular endothelial growth factor (VEGF) and its receptors VEGFR1/VEGFR2 play major roles in controlling angiogenesis, including vascularization of solid tumors. Here we describe a specific Ca²⁺ signaling pathway linked to the VEGFR2 receptor subtype, controlling the critical angiogenic responses of endothelial cells (ECs) to VEGF. Key steps of this pathway are the involvement of the potent Ca²⁺ mobilizing messenger, nicotinic acid adenine-dinucleotide phosphate (NAADP), and the specific engagement of the two-pore channel TPC2 subtype on acidic intracellular Ca²⁺ stores, resulting in Ca²⁺ release and angiogenic responses. Targeting this intracellular pathway pharmacologically using the NAADP antagonist Ned-19 or genetically using Tpcn2^−/− mice was found to inhibit angiogenic responses to VEGF in vitro and in vivo. In human umbilical vein endothelial cells (HUVECs) Ned-19 abolished VEGF-induced Ca²⁺ release, impairing phosphorylation of ERK1/2, Akt, eNOS, JNK, cell proliferation, cell migration, and capillary-like tube formation. Interestingly, Tpcn2 shRNA treatment abolished VEGF-induced Ca²⁺ release and capillary-like tube formation. Importantly, in vivo VEGF-induced vessel formation in matrigel plugs in mice was abolished by Ned-19 and, most notably, failed to occur in Tpcn2^−/− mice, but was unaffected in Tpcn1^−/− animals. These results demonstrate that a VEGFR2/NAADP/TPC2/Ca²⁺ signaling pathway is critical for VEGF-induced angiogenesis in vitro and in vivo. Given that VEGF can elicit both pro- and antiangiogenic responses depending upon the balance of signal transduction pathways activated, targeting specific VEGFR2 downstream signaling pathways could modify this balance, potentially leading to more finely tailored therapeutic strategies.In the adult the formation of new capillaries is an uncommon occurrence mostly restricted to pathological rather than physiological conditions, the majority of blood vessels remaining quiescent once organ growth is accomplished (1). Physiological neoangiogenesis is generally restricted to body sites undergoing regeneration or restructuring (e.g., tissue lesion repair and corpus luteum formation), whereas pathological neoangiogenesis takes place in different diseases ranging from macular degeneration to atherosclerosis, and is vital for the highly noxious development of solid tumors, thus representing a promising target for therapeutic strategies (2). Vascular endothelial growth factors (VEGF), and in particular the family member VEGF-A, are major regulators of angiogenesis and regulate ECs, mainly through the stimulation of VEGF receptor-2 (VEGFR2), a receptor tyrosine kinase, to induce cell proliferation, migration, and sprouting in the early stages of angiogenesis (3, 4). Antiangiogenic agents that target VEGF signaling have become an important component of therapies in multiple cancers, but their use is limited by acquisition of resistance to their therapeutic effects (5, 6). When overall VEGF receptor (VEGFR) signaling is experimentally impaired by the use of blocking antibodies or of specific tyrosine kinase inhibitors, alternative cellular and tissue strategies nullify the success of such interventions (5, 7, 8). Resistance to anti-VEGF therapies may occur through a variety of mechanisms, including evocation of alternative compensatory factors, selection of hypoxia-resistant tumor cells, action of proangiogenic circulating cells, and increased circulating nontumor proangiogenic factors. Moreover, cross-interactions (both cellular and humoral) between ECs and other environmental cues have to be taken into account for the ultimate aim of tailoring therapeutic interventions according to the specific pattern of the angiogenic microenvironment and EC conditions (5–7). The search for novel key downstream effectors is therefore of potential significance in the perspective of angiogenesis control in cancer progression.Autophosphorylation of VEGFR2 upon binding VEGF results in the activation of downstream signaling cascades through complex and manifold molecular interactions that transmit signals leading to angiogenic responses. Stimulation of different EC types via VEGFR2 results in increases in intracellular free calcium concentrations [Ca²⁺]_i (9, 10) and the crucial role of this signaling element in the regulation of EC functions and angiogenesis is recognized (11, 12), and thought to be largely mediated by the phospholipase Cγ (PLCγ)/inositol 1,4,5 trisphosphate (IP₃) signaling pathway (10). It has been reported that IP₃ releases Ca²⁺ from intracellular stores in ECs, increasing [Ca²⁺]_i, and is augmented by store-operated Ca²⁺ influx (13). This signaling primes the endothelium for angiogenesis through the activation of downstream effectors such as endothelial nitric oxide synthase (eNOS), protein kinases C (PKC), and mitogen-activated protein kinases (MAPKs). Indeed, it has been reported that the interplay between IP₃-dependent Ca²⁺ mobilization and store-operated Ca²⁺ entry produces Ca²⁺ signals whose inhibition impairs the angiogenic effect of VEGF (14, 15). Given the complexity of both VEGF and Ca²⁺ signaling, and the crucial finding that VEGF evokes pro- and antiangiogenic responses, it is clear that the specificity of VEGF-evoked Ca²⁺ signatures deserves further investigation.Differences in Ca²⁺ signatures, which are key to determining specific Ca²⁺-dependent cellular responses, rely upon often complex spatiotemporal variations in [Ca²⁺]_i (16). A major determinant of these are based on functionally distinct intracellular Ca²⁺-mobilizing messengers, namely IP₃ and cyclic adenosine diphosphoribose (cADPR), which mobilize Ca²⁺ from the endoplasmic reticulum (ER) stores, and nicotinic acid adenine dinucleotide phosphate (NAADP), which triggers Ca²⁺ release from acidic organelles, such as lysosomes and endosomes (17, 18). NAADP likely targets a channel distinct from IP₃ and ryanodine receptors (RyRs), known as two-pore channels (TPCs) (19–25), and the resulting localized NAADP-evoked Ca²⁺ signals may in some cases be globalized via IP₃ and RyRs through Ca²⁺-induced Ca²⁺ release (26, 27). However, in a few cell types, direct activation of RyRs and Ca²⁺ influx channels by NAADP have also been proposed as alternative mechanisms (28, 29). It has been demonstrated that NAADP-sensitive Ca²⁺ stores are present in the endothelium, and that NAADP is capable of regulating vascular smooth muscle contractility and blood pressure by EC-dependent mechanisms (30). In addition, we have previously demonstrated that NAADP is a specific and essential intracellular mediator of ECs histamine H₁ receptors, evoking [Ca²⁺]_i release and secretion of von Willebrand factor, which requires the functional expression of TPCs (31).In the present work, we identify a novel pathway for VEGFR2 signal transduction whereby receptor activation leads to NAADP and TPC2-dependent Ca²⁺ release from acidic Ca²⁺ stores, which in turn controls angiogenic response in vitro and in vivo. These findings demonstrate, to our knowledge for the first time, the direct relationship between NAADP-mediated Ca²⁺ release and the signaling mechanisms underlying ECs angiogenesis mediated by VEGF.     

Rab3-interacting molecules 2α and 2β promote the abundance of voltage-gated CaV1.3 Ca2+ channels at hair cell active zones

Ca²⁺ influx triggers the fusion of synaptic vesicles at the presynaptic active zone (AZ). Here we demonstrate a role of Ras-related in brain 3 (Rab3)–interacting molecules 2α and β (RIM2α and RIM2β) in clustering voltage-gated Ca_V1.3 Ca²⁺ channels at the AZs of sensory inner hair cells (IHCs). We show that IHCs of hearing mice express mainly RIM2α, but also RIM2β and RIM3γ, which all localize to the AZs, as shown by immunofluorescence microscopy. Immunohistochemistry, patch-clamp, fluctuation analysis, and confocal Ca²⁺ imaging demonstrate that AZs of RIM2α-deficient IHCs cluster fewer synaptic Ca_V1.3 Ca²⁺ channels, resulting in reduced synaptic Ca²⁺ influx. Using superresolution microscopy, we found that Ca²⁺ channels remained clustered in stripes underneath anchored ribbons. Electron tomography of high-pressure frozen synapses revealed a reduced fraction of membrane-tethered vesicles, whereas the total number of membrane-proximal vesicles was unaltered. Membrane capacitance measurements revealed a reduction of exocytosis largely in proportion with the Ca²⁺ current, whereas the apparent Ca²⁺ dependence of exocytosis was unchanged. Hair cell-specific deletion of all RIM2 isoforms caused a stronger reduction of Ca²⁺ influx and exocytosis and significantly impaired the encoding of sound onset in the postsynaptic spiral ganglion neurons. Auditory brainstem responses indicated a mild hearing impairment on hair cell-specific deletion of all RIM2 isoforms or global inactivation of RIM2α. We conclude that RIM2α and RIM2β promote a large complement of synaptic Ca²⁺ channels at IHC AZs and are required for normal hearing.Tens of Ca_V1.3 Ca²⁺ channels are thought to cluster within the active zone (AZ) membrane underneath the presynaptic density of inner hair cells (IHCs) (1–4). They make up the key signaling element, coupling the sound-driven receptor potential to vesicular glutamate release (5–7). The mechanisms governing the number of Ca²⁺ channels at the AZ as well as their spatial organization relative to membrane-tethered vesicles are not well understood. Disrupting the presynaptic scaffold protein Bassoon diminishes the numbers of Ca²⁺ channels and membrane-tethered vesicles at the AZ (2, 8). However, the loss of Bassoon is accompanied by the loss of the entire synaptic ribbon, which makes it challenging to distinguish the direct effects of gene disruption from secondary effects (9).Among the constituents of the cytomatrix of the AZ, RIM1 and RIM2 proteins are prime candidates for the regulation of Ca²⁺ channel clustering and function (10, 11). The family of RIM proteins has seven identified members (RIM1α, RIM1β, RIM2α, RIM2β, RIM2γ, RIM3γ, and RIM4γ) encoded by four genes (RIM1–RIM4). All isoforms contain a C-terminal C₂ domain but differ in the presence of additional domains. RIM1 and RIM2 interact with Ca²⁺ channels, most other proteins of the cytomatrix of the AZ, and synaptic vesicle proteins. They interact directly with the auxiliary β (Ca_Vβ) subunits (12, 13) and pore-forming Ca_Vα subunits (14, 15). In addition, RIMs are indirectly linked to Ca²⁺ channels via RIM-binding protein (14, 16, 17). A regulation of biophysical channel properties has been demonstrated in heterologous expression systems for RIM1 (12) and RIM2 (13).A role of RIM1 and RIM2 in clustering Ca²⁺ channels at the AZ was demonstrated by analysis of RIM1/2-deficient presynaptic terminals of cultured hippocampal neurons (14), auditory neurons in slices (18), and Drosophila neuromuscular junction (19). Because α-RIMs also bind the vesicle-associated protein Ras-related in brain 3 (Rab3) via the N-terminal zinc finger domain (20), they are also good candidates for molecular coupling of Ca²⁺ channels and vesicles (18, 21, 22). Finally, a role of RIMs in priming of vesicles for fusion is the subject of intense research (18, 21–27). RIMs likely contribute to priming via disinhibiting Munc13 (26) and regulating vesicle tethering (27). Here, we studied the expression and function of RIM in IHCs. We combined molecular, morphologic, and physiologic approaches for the analysis of RIM2α knockout mice [RIM2α SKO (28); see Methods] and of hair cell-specific RIM1/2 knockout mice (RIM1/2 cDKO). We demonstrate that RIM2α and RIM2β are present at IHC AZs of hearing mice, positively regulate the number of synaptic Ca_V1.3 Ca²⁺ channels, and are required for normal hearing.     

Dysferlin stabilizes stress-induced Ca2+ signaling in the transverse tubule membrane

Dysferlinopathies, most commonly limb girdle muscular dystrophy 2B and Miyoshi myopathy, are degenerative myopathies caused by mutations in the DYSF gene encoding the protein dysferlin. Studies of dysferlin have focused on its role in the repair of the sarcolemma of skeletal muscle, but dysferlin’s association with calcium (Ca²⁺) signaling proteins in the transverse (t-) tubules suggests additional roles. Here, we reveal that dysferlin is enriched in the t-tubule membrane of mature skeletal muscle fibers. Following experimental membrane stress in vitro, dysferlin-deficient muscle fibers undergo extensive functional and structural disruption of the t-tubules that is ameliorated by reducing external [Ca²⁺] or blocking L-type Ca²⁺ channels with diltiazem. Furthermore, we demonstrate that diltiazem treatment of dysferlin-deficient mice significantly reduces eccentric contraction-induced t-tubule damage, inflammation, and necrosis, which resulted in a concomitant increase in postinjury functional recovery. Our discovery of dysferlin as a t-tubule protein that stabilizes stress-induced Ca²⁺ signaling offers a therapeutic avenue for limb girdle muscular dystrophy 2B and Miyoshi myopathy patients.Dysferlinopathies are degenerative myopathies secondary to mutations in the gene encoding the protein dysferlin. These myopathies, most commonly limb girdle muscular dystrophy type 2B (LGMD2B) and Miyoshi myopathy (MM), are independent of motor neuron activation (1), indicating that they are myogenic in origin. Dysferlin is a 230-kDa protein composed of seven C2 domains with homology to synaptotagmin (2, 3) and a single transmembrane domain near its C terminus (4, 5). The complexity of dysferlin’s potential role in muscle is highlighted by the number of its purported functions, including membrane repair (2, 3), vesicle fusion (4), microtubule regulation (5, 6), cell adhesion (7, 8), and intercellular signaling (9). Understanding the contributions of dysferlin to the maintenance of normal skeletal muscle function is critical for the development of appropriate therapies for patients diagnosed with LGMD2B and MM.Recently, we demonstrated the localization of dysferlin at the A-I junction in mature muscle fibers (10). These results agree with earlier reports associating dysferlin with the dihydropyridine receptor (DHPR, L-type Ca²⁺ channel), Ahnak, caveolin 3, and several other proteins involved in Ca²⁺-based signaling and the function of transverse (t-) tubules (11–14). Consistent with this localization and the potential for a functional role in this specialized compartment, dysferlin-deficient murine muscle demonstrates altered transverse tubule (t-tubule) structure (15) as well as increased oxidative stress (16, 17), inflammation, and necrosis (18–20) after injury.Here we demonstrate that dysferlin is enriched in the t-tubule membrane, where it contributes to the maintenance of the t-tubule and Ca²⁺ homeostasis. We show that, although the structure and function of dysferlin-deficient t-tubules are normal at rest, they are more readily disrupted following experimental injury and are protected by reducing extracellular [Ca²⁺] or blocking L-type Ca²⁺ channels with diltiazem. We also demonstrate that treatment of dysferlin-deficient mice with diltiazem significantly improves their recovery from injuries induced by eccentric contractions. These findings support a role for dysferlin in stabilizing the t-tubules of skeletal muscle subjected to stress and suggest that diltiazem treatment may represent a viable therapeutic option for LGMD2B and MM patients.     

Preassociated apocalmodulin mediates Ca2+-dependent sensitization of activation and inactivation of TMEM16A/16B Ca2+-gated Cl? channels

Ca²⁺-activated chloride currents carried via transmembrane proteins TMEM16A and TMEM16B regulate diverse processes including mucus secretion, neuronal excitability, smooth muscle contraction, olfactory signal transduction, and cell proliferation. Understanding how TMEM16A/16B are regulated by Ca²⁺ is critical for defining their (patho)/physiological roles and for rationally targeting them therapeutically. Here, using a bioengineering approach—channel inactivation induced by membrane-tethering of an associated protein (ChIMP)—we discovered that Ca²⁺-free calmodulin (apoCaM) is preassociated with TMEM16A/16B channel complexes. The resident apoCaM mediates two distinct Ca²⁺-dependent effects on TMEM16A, as revealed by expression of dominant-negative CaM₁₂₃₄. These effects are Ca²⁺-dependent sensitization of activation (CDSA) and Ca²⁺-dependent inactivation (CDI). CDI and CDSA are independently mediated by the N and C lobes of CaM, respectively. TMEM16A alternative splicing provides a mechanism for tuning apoCaM effects. Channels lacking splice segment b selectively lost CDI, and segment a is necessary for apoCaM preassociation with TMEM16A. The results reveal multidimensional regulation of TMEM16A/16B by preassociated apoCaM and introduce ChIMP as a versatile tool to probe the macromolecular complex and function of Ca²⁺-activated chloride channels.Calcium (Ca²⁺)-activated chloride (Cl^–) channels (CaCCs) broadly expressed in mammalian cells regulate diverse physiological functions including: epithelial mucus secretion (1, 2), neuronal excitability (3–5), smooth muscle contraction (6), olfactory transduction (7, 8), and cell proliferation (9, 10). Drugs targeting CaCCs are being pursued as therapies for hypertension, cystic fibrosis, asthma, and cancer (1, 9, 11).Three laboratories independently identified the transmembrane protein TMEM16A as the molecular component of a CaCC (12–14). TMEM16A belongs to a protein family with 10 members encoded by distinct genes (15–18). There is universal agreement that TMEM16A, and the closely related TMEM16B, are bona fide CaCCs (2, 12–14, 19). Consistent with this, TMEM16A knockout mice displayed defective CaCC activity in a variety of epithelia (20–22), and the olfactory CaCC current was completely abolished in TMEM16B knockout mice (23). Hydropathy analyses suggest TMEM16 proteins have a similar topology with cytosolic N and C termini and eight predicted transmembrane helices (2, 19). Human TMEM16A has four alternatively spliced segments (a–d), differential inclusion of which modify voltage and Ca²⁺ sensitivity of resultant channel splice variants (24).CaCCs are highly sensitive to intracellular [Ca²⁺], displaying graded increases in Cl⁻ current (I_Cl) amplitude as [Ca²⁺]_i is raised from resting levels (∼100 nM) to the 1- to 2-μM range. In some cases, high [Ca²⁺]_i (>10 μM) leads to decreased I_Cl amplitude (inactivation) (25–27). The Ca²⁺ sensor(s) for Ca²⁺-dependent activation and inactivation (CDA and CDI) of TMEM16A/16B is unknown. There are two possible nonexclusive mechanisms: (i) direct Ca²⁺ binding to the channel or (ii) Ca²⁺ binding through a separate Ca²⁺-sensing protein. The TMEM16A sequence does not reveal any canonical Ca²⁺-binding EF hand motifs (14, 16, 17). A sequence in the first intracellular loop of TMEM16A resembling the “Ca²⁺ bowl” in large conductance Ca²⁺-activated K⁺ (BK) channels was disqualified by mutagenesis as the Ca²⁺ sensor responsible for CDA of TMEM16A (28). A revised TMEM16A topological model suggests the originally predicted extracellular loop 4 is located intracellularly (29), and mutating E702 and E705 within this loop markedly alter Ca²⁺ sensitivity of TMEM16A (29, 30).Some reports have suggested involvement of calmodulin (CaM) in distinct aspects of Ca²⁺-dependent regulation of CaCCs. Tian et al. reported that inhibiting CaM with trifluoperazine or J-8 markedly suppressed CDA of TMEM16A(abc) in HEK293 cells, and mapped the CaM binding site to splice segment b (31). They concluded that CaM is essential for TMEM16A activation. However, this suggestion is contradicted by the robust CDA of TMEM16A(ac), a splice variant lacking the putative CaM binding site on splice segment b (2, 24). Recently, Ca²⁺–CaM was found to bind TMEM16A(ac) in a Ca²⁺-dependent manner and result in an increased permeability of the channel to HCO₃⁻ (32). Deleting the Ca²⁺–CaM binding site did not affect CDA of TMEM16A(ac). Ca²⁺–CaM regulation of TMEM16A HCO₃⁻ permeability conforms to a traditional signaling mode where Ca²⁺ binds to freely diffusing CaM to form a Ca²⁺–CaM complex that then interacts with a target protein.There are several examples of an alternative mode of CaM signaling in which Ca²⁺-free CaM (apoCaM) is preassociated with target proteins under resting [Ca²⁺]_i conditions and acts as a resident Ca²⁺ sensor to regulate function of the host protein in response to increased [Ca²⁺]_i (33). This mode of CaM signaling is used as the activating mechanism for small conductance K⁺ channels (34) and Ca²⁺-dependent regulation of high voltage-gated Ca²⁺ (Ca_V1 and Ca_V2) channels (35, 36). A distinguishing feature of this mode of CaM signaling is that it is impervious to pharmacological inhibitors of Ca²⁺–CaM, but can be eliminated in dominant negative fashion by a CaM mutant, CaM₁₂₃₄, in which all four EF hands have been mutated so they no longer bind Ca²⁺ (34, 36). Whether apocalmodulin preassociates with TMEM16A/TMEM16B channel complexes and participates in Ca²⁺-dependent regulation of these channels is controversial (30, 37, 38).Using a recently developed bioengineering approach—channel inactivation induced by membrane-tethering of an associated protein (ChIMP) (39)—we discovered that apoCaM is functionally preassociated with TMEM16A/16B channel complexes. Whereas the resident apoCaM is not necessary for CDA, it does mediate two distinct Ca²⁺-dependent processes in TMEM16A. First, it causes a leftward shift in the Ca²⁺-dependent activation curve at [Ca²⁺]_i ≤ 1 μM, an effect we term Ca²⁺-dependent “sensitization” of activation (CDSA). Second, it is the Ca²⁺ sensor for CDI observed at [Ca²⁺]_i > 10 μM. The two opposite effects are independently mediated by the two lobes of preassociated apoCaM— Ca²⁺ occupancy of the N lobe leads to CDI, whereas the C lobe mediates CDSA. Alternative splicing of TMEM16A provides a mechanism for regulating apoCaM binding and signaling. TMEM16A splice variants lacking segment a lost apoCaM binding altogether, eliminating both CDSA and CDI, whereas variants specifically lacking segment b were selectively deficient in CDI. Finally, TMEM16A variants defective in apoCaM binding displayed dramatically decreased trafficking to the cell surface.     

Biphasic regulation of InsP3 receptor gating by dual Ca2+ release channel BH3-like domains mediates Bcl-xL control of cell viability

Antiapoptotic Bcl-2 family members interact with inositol trisphosphate receptor (InsP₃R) Ca²⁺ release channels in the endoplasmic reticulum to modulate Ca²⁺ signals that affect cell viability. However, the molecular details and consequences of their interactions are unclear. Here, we found that Bcl-x_L activates single InsP₃R channels with a biphasic concentration dependence. The Bcl-x_L Bcl-2 homology 3 (BH3) domain-binding pocket mediates both high-affinity channel activation and low-affinity inhibition. Bcl-x_L activates channel gating by binding to two BH3 domain-like helices in the channel carboxyl terminus, whereas inhibition requires binding to one of them and to a previously identified Bcl-2 interaction site in the channel-coupling domain. Disruption of these interactions diminishes cell viability and sensitizes cells to apoptotic stimuli. Our results identify BH3-like domains in an ion channel and they provide a unifying model of the effects of antiapoptotic Bcl-2 proteins on the InsP₃R that play critical roles in Ca²⁺ signaling and cell viability.The inositol trisphosphate receptors (InsP₃R) are a family of intracellular cation channels that release Ca²⁺ from the endoplasmic reticulum (ER) in response to a variety of extracellular stimuli (1). Three InsP₃R isoforms are ubiquitously expressed and regulate diverse cell processes, including cell viability (1). Activation of the channels by InsP₃ elicits changes in cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) that provide versatile signals to regulate molecular processes with high spatial and temporal fidelity (1). Regions of close proximity to mitochondria enable localized Ca²⁺ release events to be transduced to mitochondria (2, 3). Ca²⁺ released from the ER during cell stimulation modulates activities of effector molecules and is taken up by mitochondria to stimulate oxidative phosphorylation and enhance ATP production (4–6) to match energetic supply with enhanced demand. In addition, cells in vivo are constantly exposed to low levels of circulating hormones, transmitters, and growth factors that bind to plasma membrane receptors to provide a background level of cytoplasmic InsP₃ (7) that generates low-level stochastic InsP₃R-mediated localized or propagating [Ca²⁺]_i signals (8–10). Such signals also play an important role in maintenance of cellular bioenergetics (8). Nevertheless, under conditions of cell stress the close proximity of mitochondria to Ca²⁺ release sites may result in mitochondrial Ca²⁺ overload and initiate Ca²⁺-dependent forms of cell death, including necrosis and apoptosis (11–13). It has been suggested that high levels of ER Ca²⁺ (14–16) and enhanced activity of the InsP₃R (17–19) promote cell death by providing a higher quantity of released Ca²⁺ to mitochondria (3, 20, 21).Protein interactions modulate the magnitude and quality of InsP₃R-mediated [Ca²⁺]_i signals that regulate apoptosis and cell viability. Notable in this regard is the Bcl-2 protein family. Proapoptotic Bcl-2–related proteins Bax and Bak initiate cytochrome C release from mitochondria in response to diverse apoptotic stimuli, whereas antiapoptotic Bcl-2–related proteins, including Bcl-2 and Bcl-x_L, antagonize Bax/Bak by forming heterodimers that prevent their oligomerization and apoptosis initiation (22, 23). Heterodimerization is mediated by interactions of proapoptotic Bcl-2 homology 3 (BH3) domains with a hydrophobic groove on the surface of antiapoptotic Bcl-2 proteins (23) that is a therapeutic target in diseases, including cancer (22). Whereas a central feature of molecular models of apoptosis is the control of outer mitochondrial membrane permeability by Bcl-2–related proteins, a substantial body of evidence has demonstrated that these proteins localize to the ER (24, 25), bind to InsP₃Rs (26–32) and, by modulating InsP₃R-mediated Ca²⁺ release, regulate ER-mediated cell death and survival (15, 27, 32–34). Nevertheless, a unified understanding of the detailed molecular mechanisms by which Bcl-2 family proteins interact with and regulate InsP₃R channel activity is lacking. The Bcl-2 family member homolog NrZ interacts with the amino-terminal InsP₃-binding region via its helix 1 BH4 domain and inhibits Ca²⁺ release (28). Bcl-2 also interacts with the InsP₃R (26) via its BH4 domain (35), but in contrast it associates with a region in the central coupling domain (35). Whereas this interaction also inhibits Ca²⁺ release (26), Bok interacts with the channel 500 residues C-terminal to the Bcl-2 binding sequence via its BH4 domain but does not affect Ca²⁺ release (29). Conversely, the Bcl-x_L BH4 domain may lack this interaction (36). Inhibition of the Bcl-2 BH4 domain interaction with the channel enhanced InsP₃R-mediated Ca²⁺ signals and apoptosis sensitivity in white blood cells (18, 35, 37). However, it is unclear if Bcl-2 inhibits Ca²⁺ signaling directly by binding to the channel or if it acts indirectly, as a hub in a protein complex that influences channel phosphorylation (38). Conversely, we demonstrated that Bcl-x_L, Bcl-2, and Mcl-1 bind to the carboxyl (C)-terminus of all three InsP₃R isoforms, and showed that these interactions activated single InsP₃R channels and promoted InsP₃R-mediated Ca²⁺ release and apoptosis resistance (27, 31, 32). Furthermore, Bcl-x_L mediates an interaction of oncogenic K-RAS with the InsP₃R C terminus that regulates its biochemical and functional interaction and cell survival (39). However, the molecular details of the interactions of antiapoptotic protein with the InsP₃R C terminus are unknown. Furthermore, the relationship between Bcl-2 family protein binding in the coupling domain and C terminus is unclear. Thus, the mechanisms whereby Bcl-2 and Bcl-x_L affect InsP₃R activity and the effects of this modulation on cell viability remain to be determined.Here, we used single-channel electrophysiology of native ER membranes to explore the detailed mechanisms of the effects of Bcl-x_L on the InsP₃R, and the role of this interaction on cell viability. Surprisingly, our results reveal that whereas Bcl-x_L activates the channel at low concentrations, it inhibits it at higher concentrations, resulting in a biphasic response of channel activation on [Bcl-x_L]. Remarkably, the Bcl-x_L BH3 domain-binding pocket is required for both effects. Low [Bcl-x_L] activates the channel by simultaneous binding to two BH3 domain-like helices in the channel C terminus, whereas channel inhibition at high [Bcl-x_L] requires binding to only one of them and to a site previously identified as the Bcl-2 binding site in the channel-coupling domain. Disruption of these interactions diminishes cell viability. Our results provide a unifying model of the effects of antiapoptotic Bcl-2 proteins on the InsP₃R that play critical roles in Ca²⁺ signaling and cell viability.     

Activating mutations in STIM1 and ORAI1 cause overlapping syndromes of tubular myopathy and congenital miosis

Signaling through the store-operated Ca²⁺ release-activated Ca²⁺ (CRAC) channel regulates critical cellular functions, including gene expression, cell growth and differentiation, and Ca²⁺ homeostasis. Loss-of-function mutations in the CRAC channel pore-forming protein ORAI1 or the Ca²⁺ sensing protein stromal interaction molecule 1 (STIM1) result in severe immune dysfunction and nonprogressive myopathy. Here, we identify gain-of-function mutations in the cytoplasmic domain of STIM1 (p.R304W) associated with thrombocytopenia, bleeding diathesis, miosis, and tubular myopathy in patients with Stormorken syndrome, and in ORAI1 (p.P245L), associated with a Stormorken-like syndrome of congenital miosis and tubular aggregate myopathy but without hematological abnormalities. Heterologous expression of STIM1 p.R304W results in constitutive activation of the CRAC channel in vitro, and spontaneous bleeding accompanied by reduced numbers of thrombocytes in zebrafish embryos, recapitulating key aspects of Stormorken syndrome. p.P245L in ORAI1 does not make a constitutively active CRAC channel, but suppresses the slow Ca²⁺-dependent inactivation of the CRAC channel, thus also functioning as a gain-of-function mutation. These data expand our understanding of the phenotypic spectrum of dysregulated CRAC channel signaling, advance our knowledge of the molecular function of the CRAC channel, and suggest new therapies aiming at attenuating store-operated Ca²⁺ entry in the treatment of patients with Stormorken syndrome and related pathologic conditions.Ca²⁺ influx in response to the depletion of intracellular Ca²⁺ stores, or store-operated Ca²⁺ entry, constitutes one of the major routes of Ca²⁺ entry in all animal cells (1). Under physiological conditions, Ca²⁺ influx is activated in response to numerous G protein-coupled receptors and receptor tyrosine kinases signaling via inositol-1,4,5-trisphosphate as a second messenger (2). Store-operated Ca²⁺ entry is mediated primarily by the Ca²⁺ release-activated Ca²⁺ (CRAC) channel (3), which consists of the pore-forming subunits ORAI1–3 (or CRAC modulators 1–3) and Ca²⁺ sensors, STIM1 and STIM2 (4–7). STIM proteins reside in the membrane of endoplasmic reticulum (ER), whereas ORAI proteins reside in the plasma membrane. STIM1 is a single transmembrane-spanning protein (8–12) that, in resting cells, exists as a dimer that binds Ca²⁺ through two EF hand-containing domains located in the ER lumen (13). Depletion of Ca²⁺ in the ER induces a series of molecular events in the conformation and localization of STIM1, initiated by the formation of higher-order oligomers, protein unfolding, and accumulation at discrete sites in the cell where the ER membrane is in close proximity to the plasma membrane (11, 13–16). In these sites, STIM1 binds to the cytosolic C and N termini of ORAI1 (17, 18), resulting in channel activation and generation of a highly Ca²⁺-selective CRAC current, or I_CRAC (3, 19, 20). I_CRAC is responsible not only for restoring cytosolic and ER Ca²⁺ concentration, thus maintaining the cell in a Ca²⁺ signaling-competent stage (1), but also for many cellular functions such as regulation of gene expression, exocytosis, proliferation, and apoptosis (1).Consistent with a fundamental role of the CRAC channel in cell signaling, loss-of-function mutations in STIM1 or ORAI1 lead to immune deficiency and nonprogressive myopathy (21–23). However, evidence that gain-of-function mutations in STIM1 and ORAI1 can affect human health is only recently starting to emerge. It was shown that mutations in the domain of STIM1 that binds Ca²⁺ (EF hand domain) in resting conditions are associated with nonsyndromic myopathy with tubular aggregates (24). Functional studies demonstrated that these mutations cause hyperactivation of the CRAC channel (24). However, it remains unknown whether myopathy with tubular aggregates is caused by the increased activity of the CRAC channel, increased activity of another Ca²⁺ channel using STIM1 as a sensor (25), or a function of STIM1 that is unrelated to Ca²⁺ signaling, as STIM1 can function independently of ORAI1 (26-28).Stormorken syndrome [Mendelian Inheritance in Man (MIM) 185070] is a rare autosomal-dominant condition with a constellation of symptoms, including congenital miosis, bleeding diathesis, thrombocytopenia, functional (or anatomical) asplenia, and proximal muscle weakness (29). Other manifestations include ichthyosis, headaches, and dyslexia (30). Patients typically display increased creatine kinase (CK) levels and histologic evidence of myopathy with tubular aggregates (30, 31). Here, we show that Stormorken syndrome is caused by an activating mutation in STIM1. We also identify a mutation in the STIM1-interacting molecule, ORAI1, in a Stormorken-like syndrome that presented with miosis and tubular myopathy. Functional analyses reveal that both mutations enhance the activity of the CRAC channel, but by different molecular mechanisms. These data expand the phenotypic spectrum of activating mutations in the CRAC channels from myopathy with tubular aggregates to miosis, bleeding diathesis, thrombocytopenia, asplenia, ichthyosis, headaches, and dyslexia.     

Calcineurin determines toxic versus beneficial responses to α-synuclein

Calcineurin (CN) is a highly conserved Ca²⁺–calmodulin (CaM)-dependent phosphatase that senses Ca²⁺ concentrations and transduces that information into cellular responses. Ca²⁺ homeostasis is disrupted by α-synuclein (α-syn), a small lipid binding protein whose misfolding and accumulation is a pathological hallmark of several neurodegenerative diseases. We report that α-syn, from yeast to neurons, leads to sustained highly elevated levels of cytoplasmic Ca²⁺, thereby activating a CaM-CN cascade that engages substrates that result in toxicity. Surprisingly, complete inhibition of CN also results in toxicity. Limiting the availability of CaM shifts CN''s spectrum of substrates toward protective pathways. Modulating CN or CN''s substrates with highly selective genetic and pharmacological tools (FK506) does the same. FK506 crosses the blood brain barrier, is well tolerated in humans, and is active in neurons and glia. Thus, a tunable response to CN, which has been conserved for a billion years, can be targeted to rebalance the phosphatase’s activities from toxic toward beneficial substrates. These findings have immediate therapeutic implications for synucleinopathies.Cells must tightly regulate Ca²⁺ homeostasis to avoid pathological perturbations and cell death (1). For example, a profound disruption of Ca²⁺ homeostasis is seen in Parkinson disease (PD), the second most common neurodegenerative disorder. Mutations or aberrant expression of α-synuclein (α-syn), a major protein involved in the pathogenesis of PD, can induce Ca²⁺ overload and cell death (2–5). Additional clinical and experimental observations highlight the importance of Ca²⁺ homeostasis in the pathogenesis of PD. Midbrain dopaminergic (DA) neurons that overexpress Ca²⁺-binding proteins, which buffer intracellular Ca²⁺, are characteristically spared from degeneration (6). Patients with hypertension who are treated with the L-type Ca²⁺ channel blocker, isradipine, have a lower incidence of PD (7). Moreover, isradipine protects DA neurons incubated with α-syn fibrils and is protective in animal models of toxin-induced PD (8–10).From yeast to mammals, calcineurin is largely responsible for transducing the signals generated by changes in Ca²⁺ levels (11). Calcineurin (CN) is a calmodulin (CaM)-dependent serine/threonine phosphatase composed of a catalytic subunit (calcineurin A, CNA) and an activating regulatory subunit (calcineurin B, CNB). As intracellular Ca²⁺ levels rise, Ca²⁺ binds to CNB and CaM, another key calcium signaling protein. Together, Ca²⁺-bound CNB and CaM bind CNA, inducing a conformational change that fully activates the phosphatase (11). Signaling through CN plays critical roles in processes ranging from stress response survival in yeast (12) to mammalian development (13).Despite the compelling link between Ca²⁺ homeostasis and PD, we know little about the signaling pathways driven by sustained Ca²⁺ elevations and how they might lead to cell death (4, 5). Yeast provide a powerful model system for such investigations, given their genetic tractability and the remarkable conservation of Ca²⁺-signaling pathways from yeast to humans (14, 15). Moreover, the expression of human α-syn in yeast leads to cellular pathologies directly relevant to neurons and PD, including nitrosative stress (16, 17), defects in vesicle trafficking (18–20), and faulty mitochondrial function (21, 22).     

Calcium sensor regulation of the CaV2.1 Ca2+ channel contributes to short-term synaptic plasticity in hippocampal neurons

Short-term synaptic plasticity is induced by calcium (Ca²⁺) accumulating in presynaptic nerve terminals during repetitive action potentials. Regulation of voltage-gated Ca_V2.1 Ca²⁺ channels by Ca²⁺ sensor proteins induces facilitation of Ca²⁺ currents and synaptic facilitation in cultured neurons expressing exogenous Ca_V2.1 channels. However, it is unknown whether this mechanism contributes to facilitation in native synapses. We introduced the IM-AA mutation into the IQ-like motif (IM) of the Ca²⁺ sensor binding site. This mutation does not alter voltage dependence or kinetics of Ca_V2.1 currents, or frequency or amplitude of spontaneous miniature excitatory postsynaptic currents (mEPSCs); however, synaptic facilitation is completely blocked in excitatory glutamatergic synapses in hippocampal autaptic cultures. In acutely prepared hippocampal slices, frequency and amplitude of mEPSCs and amplitudes of evoked EPSCs are unaltered. In contrast, short-term synaptic facilitation in response to paired stimuli is reduced by ∼50%. In the presence of EGTA-AM to prevent global increases in free Ca²⁺, the IM-AA mutation completely blocks short-term synaptic facilitation, indicating that synaptic facilitation by brief, local increases in Ca²⁺ is dependent upon regulation of Ca_V2.1 channels by Ca²⁺ sensor proteins. In response to trains of action potentials, synaptic facilitation is reduced in IM-AA synapses in initial stimuli, consistent with results of paired-pulse experiments; however, synaptic depression is also delayed, resulting in sustained increases in amplitudes of later EPSCs during trains of 10 stimuli at 10–20 Hz. Evidently, regulation of Ca_V2.1 channels by CaS proteins is required for normal short-term plasticity and normal encoding of information in native hippocampal synapses.Modification of synaptic strength in central synapses is highly dependent upon presynaptic activity. The frequency and pattern of presynaptic action potentials regulates the postsynaptic response through diverse forms of short- and long-term plasticity that are specific to individual synapses and depend upon accumulation of intracellular Ca²⁺ (1–4). Presynaptic plasticity regulates neurotransmission by varying the amount of neurotransmitter released by each presynaptic action potential (1–5). P/Q-type Ca²⁺ currents conducted by voltage-gated Ca_V2.1 Ca²⁺ channels initiate neurotransmitter release at fast excitatory glutamatergic synapses in the brain (6–9) and regulate short-term presynaptic plasticity (3, 10). These channels exhibit Ca²⁺-dependent facilitation and inactivation that is mediated by the Ca²⁺ sensor (CaS) protein calmodulin (CaM) bound to a bipartite site in their C-terminal domain composed of an IQ-like motif (IM) and a CaM binding domain (CBD) (11–14). Ca²⁺-dependent facilitation and inactivation of P/Q-type Ca²⁺ currents correlate with facilitation and rapid depression of synaptic transmission at the Calyx of Held (15–18). Elimination of Ca_V2.1 channels by gene deletion prevents facilitation of synaptic transmission at the Calyx of Held (19, 20). Cultured sympathetic ganglion neurons with presynaptic expression of exogenous Ca_V2.1 channels harboring mutations in their CaS regulatory site have reduced facilitation and slowed depression of postsynaptic responses because of reduced Ca²⁺-dependent facilitation and Ca²⁺-dependent inactivation of Ca_V2.1 currents (21). The CaS proteins Ca²⁺-binding protein 1 (CaBP-1), visinin-like protein-2 (VILIP-2), and neuronal Ca²⁺ sensor-1 (NCS-1) induce different degrees of Ca²⁺-dependent facilitation and inactivation of channel activity (22–26). Expression of these different CaS proteins with Ca_V2.1 channels in cultured sympathetic ganglion neurons results in corresponding bidirectional changes in facilitation and depression of the postsynaptic response (25, 26). Therefore, binding of CaS proteins to Ca_V2.1 channels at specific synapses can change the balance of CaS-dependent facilitation and inactivation of Ca_V2.1 channels, and determine the outcome of synaptic plasticity (27). Currently, it is not known whether such molecular regulation of Ca_V2.1 by CaS proteins induces or modulates synaptic plasticity in native hippocampal synapses.To understand the functional role of regulation of Ca_V2.1 channels by CaS proteins in synaptic plasticity in vivo, we generated knock-in mice with paired alanine substitutions for the isoleucine and methionine residues in the IM motif (IM-AA) in their C-terminal domain. Here we investigated the effects of mutating this CaS regulatory site on hippocampal neurotransmission and synaptic plasticity. This mutation had no effect on basal Ca²⁺ channel function or on basal synaptic transmission. However, we found reduced short-term facilitation in response to paired stimuli in autaptic synapses in hippocampal cultures and in Schaffer collateral (SC)-CA1 synapses in acutely prepared hippocampal slices. Moreover, synaptic facilitation in mutant SC-CA1 synapses developed and decayed more slowly during trains of stimuli. These results identify a critical role for modulation of Ca_V2.1 channels by CaS proteins in short-term synaptic plasticity, which is likely to have important consequences for encoding and transmitting information in the hippocampus.