Genetically enhancing mitochondrial antioxidant activity improves muscle function in aging

Age-related skeletal muscle dysfunction is a leading cause of morbidity that affects up to half the population aged 80 or greater. Here we tested the effects of increased mitochondrial antioxidant activity on age-dependent skeletal muscle dysfunction using transgenic mice with targeted overexpression of the human catalase gene to mitochondria (MCat mice). Aged MCat mice exhibited improved voluntary exercise, increased skeletal muscle specific force and tetanic Ca²⁺ transients, decreased intracellular Ca²⁺ leak and increased sarcoplasmic reticulum (SR) Ca²⁺ load compared with age-matched wild type (WT) littermates. Furthermore, ryanodine receptor 1 (the sarcoplasmic reticulum Ca²⁺ release channel required for skeletal muscle contraction; RyR1) from aged MCat mice was less oxidized, depleted of the channel stabilizing subunit, calstabin1, and displayed increased single channel open probability (P_o). Overall, these data indicate a direct role for mitochondrial free radicals in promoting the pathological intracellular Ca²⁺ leak that underlies age-dependent loss of skeletal muscle function. This study harbors implications for the development of novel therapeutic strategies, including mitochondria-targeted antioxidants for treatment of mitochondrial myopathies and other healthspan-limiting disorders.Age-dependent muscle weakness is a leading cause of morbidity due to frailty, loss of independence, and physical disability that is associated with increased risk of falls and fractures (1, 2). In geriatric populations age-dependent muscle weakness, characterized both by loss of lean muscle mass (sarcopenia) and reduced skeletal muscle function (3–5), has been estimated to affect 30–50% of 80-y-olds (1, 2, 4).The ‘free radical theory’ of aging, first proposed in 1956 by Harman (6), states that an underlying mechanism of age-dependent pathology is the accumulation of partially reduced forms of oxygen (7, 8), collectively known as reactive oxygen species (ROS). Mitochondria are a major source of cellular ROS (7, 9) and have been proposed to play a key role in age-dependent loss of skeletal muscle function (3, 7, 10), likely through the production of oxidative damage (11, 12). However, the molecular mechanisms underlying this process have not been fully determined.Skeletal muscle contraction is dependent upon release of intracellular Ca²⁺ via the sarcoplasmic reticulum (SR) Ca²⁺ release channel, ryanodine receptor 1 (RyR1). Following membrane depolarization, voltage-sensing Ca²⁺ channels in the transverse tubules (Cav1.1) activate RyR1 and the ensuing rise in cytoplasmic [Ca²⁺] causes muscle contraction via the actin-myosin cross bridge cycle (13). The RyR1 is a homotetrameric protein complex composed of four monomers, kinases, a phosphatase (PP1), phosphodiesterase (PDE4D3), calmodulin, and the RyR1 channel-stabilizing subunit calstabin1 (FK506 binding protein 12, FKBP12) (14). Posttranslational modifications of the channel, including oxidation, cysteine-nitrosylation, and cAMP-dependent protein kinase A-mediated phosphorylation have been linked to impaired Ca²⁺ handling and perturbed contractility in chronic muscle fatigue, heart failure and muscular dystrophy (13–15). Furthermore, we have recently reported that both oxidation of RyR1 and the subsequent intracellular Ca²⁺ leak underlie the age-dependent reduction in skeletal muscle specific force (10). Acute induction of RyR1-mediated SR Ca²⁺ leak with rapamycin, which competes the channel-stabilizing subunit, calstabin1, off from RyR1 (14, 16), resulted in defective mitochondrial function associated with elevated free radical production (10). However, the role of mitochondrial ROS in age-dependent reduction in skeletal muscle function and exercise capacity has not been elucidated.Recently, there have been numerous efforts to study mitochondria-derived free radicals in health and lifespan by experimentally expressing catalase, which catalyzes the decomposition of hydrogen peroxide to water and oxygen, in the mitochondria. This has been done using in vitro models (17), adeno-associate viral vectors (AAV) (18), and most recently by genetically engineering its overexpression in mice (19). These transgenic mice, MCat mice, in which the human catalase is targeted to and overexpressed in mitochondria, display a 10–20% increase in maximum and median lifespan (19), reduced age-related insulin resistance (20), and attenuated energy imbalance.Because mitochondrial targeted overexpression of catalase results in reduced mitochondrial ROS (19, 20), we used the MCat mouse model to investigate the relationship between antioxidant activity and skeletal muscle aging and subsequent functional decline. Aged MCat mice displayed improved voluntary exercise, increased skeletal muscle specific force, increased tetanic Ca²⁺ transients, reduced intracellular Ca²⁺ leak and increased SR Ca²⁺ load compared with age-matched wild-type (WT) littermates. RyR1 channels from aged MCat mice were less oxidized, depleted of calstabin1 and exhibited increased single channel open probability (P_o). Furthermore, pharmacological application of an antioxidant to aged WT RyR1 reduced SR Ca²⁺ leak. We have therefore identified mitochondria as a source of ROS involved in the RyR1 oxidation underlying age-associated skeletal muscle dysfunction.     

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.     

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.     

Arrhythmogenesis in a catecholaminergic polymorphic ventricular tachycardia mutation that depresses ryanodine receptor function

Current mechanisms of arrhythmogenesis in catecholaminergic polymorphic ventricular tachycardia (CPVT) require spontaneous Ca²⁺ release via cardiac ryanodine receptor (RyR2) channels affected by gain-of-function mutations. Hence, hyperactive RyR2 channels eager to release Ca²⁺ on their own appear as essential components of this arrhythmogenic scheme. This mechanism, therefore, appears inadequate to explain lethal arrhythmias in patients harboring RyR2 channels destabilized by loss-of-function mutations. We aimed to elucidate arrhythmia mechanisms in a RyR2-linked CPVT mutation (RyR2-A4860G) that depresses channel activity. Recombinant RyR2-A4860G protein was expressed equally as wild type (WT) RyR2, but channel activity was dramatically inhibited, as inferred by [³H]ryanodine binding and single channel recordings. Mice heterozygous for the RyR2-A4860G mutation (RyR2-A4860G^+/−) exhibited basal bradycardia but no cardiac structural alterations; in contrast, no homozygotes were detected at birth, suggesting a lethal phenotype. Sympathetic stimulation elicited malignant arrhythmias in RyR2-A4860G^+/− hearts, recapitulating the phenotype originally described in a human patient with the same mutation. In isoproterenol-stimulated ventricular myocytes, the RyR2-A4860G mutation decreased the peak of Ca²⁺ release during systole, gradually overloading the sarcoplasmic reticulum with Ca²⁺. The resultant Ca²⁺ overload then randomly caused bursts of prolonged Ca²⁺ release, activating electrogenic Na⁺-Ca²⁺ exchanger activity and triggering early afterdepolarizations. The RyR2-A4860G mutation reveals novel pathways by which RyR2 channels engage sarcolemmal currents to produce life-threatening arrhythmias.In the heart, ryanodine receptor (RyR2) channels release massive amounts of Ca²⁺ from the sarcoplasmic reticulum (SR) in response to membrane depolarization, in turn modulating cardiac excitability and triggering ventricular contractions (1, 2). In their intracellular milieu, RyR2 channels are regulated by a variety of cytosolic and luminal factors so that their output signal (i.e., Ca²⁺) finely grades cardiac contractions (3). However, RyR2 channels operate within a limited margin of safety because conditions that demand higher RyR2 activity (such as sympathetic stimulation) also increase the vulnerability of the heart to life-threatening arrhythmias (4), and this risk is higher in hearts harboring mutant RyR2 channels. Indeed, point mutations in RYR2, the gene encoding for the cardiac RyR channel, are associated with catecholaminergic polymorphic ventricular tachycardia (CPVT) (5), a highly arrhythmogenic syndrome triggered by sympathetic stimulation that may lead to sudden cardiac death, especially in children and young adults (6).To date, delayed afterdepolarizations (DADs) triggered by spontaneous Ca²⁺ release stand as the most accepted cellular mechanism to explain cardiac arrhythmias in CPVT. In this scheme, RyR2 channels destabilized by gain-of-function mutations release Ca²⁺ during diastole, generating a depolarizing transient inward current (I_ti) as the sarcolemmal Na⁺-Ca²⁺ exchanger (NCX) extrudes the released Ca²⁺. This electrogenic inward current then causes DADs, which, if sufficiently large, reach the threshold to initiate untimely action potentials (APs) and generate triggered activity (6–8). Hence, hyperactive RyR2 channels eager to release Ca²⁺ on their own appear as essential components of this arrhythmogenic scheme. In fact, most RyR2-linked CPVT mutations characterized to date produce hyperactive RyR2 channels (9–12). This scheme therefore appears inadequate to explain lethal arrhythmias in patients harboring RyR2 channels destabilized by loss-of-function mutations (13).How do hypoactive RyR2 channels trigger lethal arrhythmias? Here we studied the RyR2-A4860G mutation, which was initially detected in a young girl presenting idiopathic catecholaminergic ventricular fibrillation (VF) (14). When expressed in HEK293 cells, recombinant RyR2-A4860G channels displayed a dramatic depression of activity, manifested mainly as a loss of luminal Ca²⁺ sensitivity (13). However, this in vitro characterization was insufficient to elucidate the mechanisms by which these hypoactive channels generate cellular substrates favorable for cardiac arrhythmias. We thus generated a mouse model of CPVT harboring the RyR2-A4860G mutation. Inbreeding of mice heterozygous for the mutation (RyR2-A4860G^+/−) yields only WT and heterozygous mice, indicating that the mutation is too strong to be harbored in the two RYR2 alleles. Ventricular myocytes from RyR2-A4860G^+/− mice have constitutively lower Ca²⁺ release than WT littermates, and undergo apparently random episodes of prolonged systolic Ca²⁺ release upon β-adrenergic stimulation, giving rise to early afterdepolarizations (EADs). Thus, this unique RYR2 mutation reveals novel pathways whereby RyR2 channels engage sarcolemmal currents to trigger VF. Although exposed in the setting of CPVT, this mechanism may be extended to a variety of settings, including heart failure, atrial fibrillation, and other cardiomyopathies in which RyR2 down-regulation and posttranslational modifications depress RyR2 function.     

From the Cover: Ryanodine receptor fragmentation and sarcoplasmic reticulum Ca2+ leak after one session of high-intensity interval exercise

High-intensity interval training (HIIT) is a time-efficient way of improving physical performance in healthy subjects and in patients with common chronic diseases, but less so in elite endurance athletes. The mechanisms underlying the effectiveness of HIIT are uncertain. Here, recreationally active human subjects performed highly demanding HIIT consisting of 30-s bouts of all-out cycling with 4-min rest in between bouts (≤3 min total exercise time). Skeletal muscle biopsies taken 24 h after the HIIT exercise showed an extensive fragmentation of the sarcoplasmic reticulum (SR) Ca²⁺ release channel, the ryanodine receptor type 1 (RyR1). The HIIT exercise also caused a prolonged force depression and triggered major changes in the expression of genes related to endurance exercise. Subsequent experiments on elite endurance athletes performing the same HIIT exercise showed no RyR1 fragmentation or prolonged changes in the expression of endurance-related genes. Finally, mechanistic experiments performed on isolated mouse muscles exposed to HIIT-mimicking stimulation showed reactive oxygen/nitrogen species (ROS)-dependent RyR1 fragmentation, calpain activation, increased SR Ca²⁺ leak at rest, and depressed force production due to impaired SR Ca²⁺ release upon stimulation. In conclusion, HIIT exercise induces a ROS-dependent RyR1 fragmentation in muscles of recreationally active subjects, and the resulting changes in muscle fiber Ca²⁺-handling trigger muscular adaptations. However, the same HIIT exercise does not cause RyR1 fragmentation in muscles of elite endurance athletes, which may explain why HIIT is less effective in this group.It is increasingly clear that regular physical exercise plays a key role in the general well-being, disease prevention, and longevity of humans. Impaired muscle function manifesting as muscle weakness and premature fatigue development are major health problems associated with the normal aging process as well as with numerous common diseases (1). Physical exercise has a fundamental role in preventing and/or reversing these muscle problems, and training also improves the general health status in numerous diseases (2–4). On the other side of the spectrum, excessive muscle use can induce prolonged force depressions, which may set the limit on training tolerance and performance of top athletes (5, 6).Recent studies imply a key role of the sarcoplasmic reticulum (SR) Ca²⁺ release channel, the ryanodine receptor 1 (RyR1), in the reduced muscle strength observed in numerous physiological conditions, such as after strenuous endurance training (6), in situations with prolonged stress (7), and in normal aging (8, 9). Defective RyR1 function is also implied in several pathological states, including generalized inflammatory disorders (10), heart failure (11), and inherited conditions such as malignant hyperthermia (12) and Duchenne muscular dystrophy (13). In many of the above conditions, there is a link between the impaired RyR1 function and modifications induced by reactive oxygen/nitrogen species (ROS) (6, 8, 10, 12, 13). Conversely, altered RyR1 function may also be beneficial by increasing the cytosolic free [Ca²⁺] ([Ca²⁺]_i) at rest, which can stimulate mitochondrial biogenesis and thereby increase fatigue resistance (14–16). Intriguingly, effective antioxidant treatment hampers beneficial adaptations triggered by endurance training (17–19), and this effect might be due to antioxidants preventing ROS-induced modifications of RyR1 (20).A high-intensity interval training (HIIT) session typically consists of a series of brief bursts of vigorous physical exercise separated by periods of rest or low-intensity exercise. A major asset of HIIT is that beneficial adaptations can be obtained with much shorter exercise duration than with traditional endurance training (21–25). HIIT has been shown to effectively stimulate mitochondrial biogenesis in skeletal muscle and increase endurance in untrained and recreationally active healthy subjects (22, 26), whereas positive effects in elite endurance athletes are less clear (21, 27, 28). Moreover, HIIT improves health and physical performance in various pathological conditions, including cardiovascular disease, obesity, and type 2 diabetes (29, 30). Thus, short bouts of vigorous physical exercise trigger intracellular signaling of large enough magnitude and duration to induce extensive beneficial adaptations in skeletal muscle. The initial signaling that triggers these adaptations is not known.In this study, we tested the hypothesis that a single session of HIIT induces ROS-dependent RyR1 modifications. These modifications might cause prolonged force depression due to impaired SR Ca²⁺ release during contractions. Conversely, they may also initiate beneficial muscular adaptations due to increased SR Ca²⁺ leak at rest.     

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.     

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.     

Arrhythmias,elicited by catecholamines and serotonin,vanish in human chronic atrial fibrillation

Atrial fibrillation (AF) is the most common heart rhythm disorder. Transient postoperative AF can be elicited by high sympathetic nervous system activity. Catecholamines and serotonin cause arrhythmias in atrial trabeculae from patients with sinus rhythm (SR), but whether these arrhythmias occur in patients with chronic AF is unknown. We compared the incidence of arrhythmic contractions caused by norepinephrine, epinephrine, serotonin, and forskolin in atrial trabeculae from patients with SR and patients with AF. In the patients with AF, arrhythmias were markedly reduced for the agonists and abolished for forskolin, whereas maximum inotropic responses were markedly blunted only for serotonin. Serotonin and forskolin produced spontaneous diastolic Ca²⁺ releases in atrial myocytes from the patients with SR that were abolished or reduced in myocytes from the patients with AF. For matching L-type Ca²⁺-current (I_Ca,L) responses, serotonin required and produced ∼100-fold less cAMP/PKA at the Ca²⁺ channel domain compared with the catecholamines and forskolin. Norepinephrine-evoked I_Ca,L responses were decreased by inhibition of Ca²⁺/calmodulin-dependent kinase II (CaMKII) in myocytes from patients with SR, but not in those from patients with AF. Agonist-evoked phosphorylation by CaMKII at phospholamban (Thr-17), but not of ryanodine2 (Ser-2814), was reduced in trabeculae from patients with AF. The decreased CaMKII activity may contribute to the blunting of agonist-evoked arrhythmias in the atrial myocardium of patients with AF.Atrial fibrillation (AF) is the most common cardiac arrhythmia, associated with increased risk of death, congestive heart failure, and stroke (1). AF is characterized by an irregular, often rapid heart rate. Atria contract with reduced force, thereby favoring thrombus formation (2). AF occurs in several cardiac diseases, and its incidence is higher in woman than in men, particularly in those with valvular heart disease (SI Appendix, Table S1). Chronic AF causes structural and electrical remodeling, as well as enlarged atria (SI Appendix, Table S1), which in turn contributes to maintain AF.Atrial contractility is reduced in isolated atrial tissues (3) obtained from patients with chronic AF, attributed to a marked decrease in L-type Ca²⁺ current (I_Ca,L) (3, 4). The inotropic responses to the agonist for β₁-adrenergic receptors (β₁ARs) and β₂-adrenergic receptors (β₂ARs), isoproterenol (ISO), but not the density of βARs and G proteins, are decreased in AF (3); however, whether the function of coexisting β₁ARs and/or β₂ARs is perturbed is unknown. Activation of human atrial β₁ARs, β₂ARs, and serotonin [5-hydroxytryptamine (5-HT)] 5-HT₄ receptors hastens relaxation through phosphorylation of phospholamban (PLB) (5–7) by cAMP-dependent protein kinase (PKA) and Ca²⁺/calmodulin-dependent kinase II (CaMKII) and produces arrhythmias (8, 9) in atrial trabeculae in patients with sinus rhythm (SR). Catecholamines and 5-HT have been proposed to initiate AF (8, 9). The relevance of these in vitro arrhythmias is corroborated by the clinical finding that high sympathetic nervous system activity during and after cardiac surgery causes premature beats and transient postoperative AF in approximately one-third of patients (10).Increased propensity to generate spontaneous impulses is assumed to initiate and/or maintain AF in humans. Arrhythmias may develop through spontaneous impulse generation within individual myocytes and/or reentry around nonexcitable tissue. The traveling electrical impulse of a premature atrial beat can encounter areas of refractoriness, return to its origin in a retrograde way, and through reentry initiate and maintain AF. Spontaneous impulse generation could be related to increased activity of PKA and/or CaMKII, with subsequent uncoordinated release of Ca²⁺ from the sarcoplasmic reticulum. Such a concept is attractive, because Ca²⁺ released from the “leaky” sarcoplasmic reticulum would activate the Na⁺-Ca²⁺ exchanger to extrude Ca²⁺ and to produce a arrhythmogenic depolarizing current, thereby explaining both the contractile dysfunction and the high recurrence rate (11–13).If arrhythmias were initiated and maintained by increased activity of PKA and/or CaMKII, then interventions known to stimulate both kinases would be expected to evoke more arrhythmias in patients with AF. However, in vitro induction of arrhythmias and activation of PKA and CaMKII by catecholamines and 5-HT were not assessed in tissues from patients with AF. Thus, we compared the effects of endogenous agonists on force and arrhythmias in intact trabeculae, as well as the I_Ca,L, Ca²⁺ transients (CaTs), and diastolic Ca²⁺ release, in atrial myocytes obtained from patients with SR and patients with AF. Functional measurements, including relaxation, were supplemented by Western blot analysis of relevant targets of PKA and CaMKII. Chronic AF caused a marked decrease in agonist-evoked arrhythmias, associated with a decrease in some CaMKII-catalyzed functions but unchanged PKA functions.     

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).     

Overexpression of junctophilin-2 does not enhance baseline function but attenuates heart failure development after cardiac stress

Heart failure is accompanied by a loss of the orderly disposition of transverse (T)-tubules and a decrease of their associations with the junctional sarcoplasmic reticulum (jSR). Junctophilin-2 (JP2) is a structural protein responsible for jSR/T-tubule docking. Animal models of cardiac stresses demonstrate that down-regulation of JP2 contributes to T-tubule disorganization, loss of excitation-contraction coupling, and heart failure development. Our objective was to determine whether JP2 overexpression attenuates stress-induced T-tubule disorganization and protects against heart failure progression. We therefore generated transgenic mice with cardiac-specific JP2 overexpression (JP2-OE). Baseline cardiac function and Ca²⁺ handling properties were similar between JP2-OE and control mice. However, JP2-OE mice displayed a significant increase in the junctional coupling area between T-tubules and the SR and an elevated expression of the Na⁺/Ca²⁺ exchanger, although other excitation-contraction coupling protein levels were not significantly changed. Despite similar cardiac function at baseline, overexpression of JP2 provided significantly protective benefits after pressure overload. This was accompanied by a decreased percentage of surviving mice that developed heart failure, as well as preservation of T-tubule network integrity in both the left and right ventricles. Taken together, these data suggest that strategies to maintain JP2 levels can prevent the progression from hypertrophy to heart failure.In working ventricular myocytes, normal excitation-contraction (E-C) coupling requires precise communication between voltage-gated L-type Ca²⁺ channels (Cav1.2) located in clusters within transverse (T)-tubules and, less frequently, on the plasmalemma, and Ca²⁺ release channels/ryanodine receptor channels (RyRs) that are also clustered on the junctional sarcoplasmic reticulum (jSR) membrane (1–4). In normal hearts, flat jSR cisternae containing a continuous row of polymerized calsequestrin (CsQ2) either wrap around a T-tubule segment or abut against the plasmalemma (5, 6) and are coupled to the surface membranes via apposed clusters of RyR2 and Cav1.2 (7). These junctional sites are called dyads. However, although the jSR cisternae constitute a single continuous compartment, the clusters of RyR2 do not occupy the whole jSR surface but are in smaller groups (8, 9). Hence, each dyad is composed of several smaller RyR2/Cav1.2 complexes, also called couplons. Functional interaction between Cav1.2 and RyR2 at these sites ensure synchronous SR Ca²⁺ release and coordinated contraction (1, 10, 11). There is evidence that impaired cardiac E-C coupling/Ca²⁺ handling is a key mediator of heart failure (12, 13). One underlying mechanism for the defective Ca²⁺ release is the progressive loss of T-tubule network organization and of the relationship between RyR2 and Cav1.2 (14–16). Therefore, preventing loss of jSR/T-tubule junctions and of T-tubule organization may represent a new strategy for therapeutic intervention in heart failure.In normal cardiomyocytes, the formation of dyads requires junctophilin 2 (JP2), a structural protein that provides a physical connection between the T-tubule and SR membranes (17). JP2’s eight N-terminal “membrane occupation and recognition nexus” domains bind to the plasmalemma (T-tubules), and its C-terminal transmembrane domain tethers the opposite end to the SR membrane (17). Decreased JP2 levels have been observed in human heart failure patients and in failing hearts from animal models of cardiac disease (16, 18–22). Knockdown of JP2 results in acute heart failure that is associated with the loss of junctional membrane complex, disrupted T-tubule organization, and Ca²⁺ handling dysfunction (23). In addition, embryonic myocytes with JP2 deficiency have defective cardiac dyads, including more SR segments with no T-tubule couplings as well as reduced intracellular Ca²⁺ transients (17). These data collectively suggest that loss of JP2 contributes to the functional defects in heart failure. Therefore, interesting questions are: Is the JP2 deficiency effect linked to the resultant disruption of jSR/T-tubule junctions and of T-tubule network integrity, as suggested by previous findings (16–18, 23)? Conversely, could exogenous overexpression of JP2 in cardiomyocytes improve Ca²⁺ handling and protect against the development of heart failure?To answer this question, we generated transgenic mice with cardiac-specific overexpression of JP2. Moderate overexpression of JP2 led to a significant increase in the junctional coupling area between T-tubule and SR membrane, but surprisingly, it did not enhance cardiac function or increase SR Ca²⁺ release at baseline. However, interestingly, JP2-overexpressing mice were resistant to left ventricular pressure overload-induced heart failure, demonstrating that JP2 overexpression is protective. These data suggest that preventing the loss of JP2 could be a potential therapeutic strategy for heart failure treatment.     

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.     

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.     

Structural insights into the Ca2+ and PI(4,5)P2 binding modes of the C2 domains of rabphilin 3A and synaptotagmin 1

Proteins containing C2 domains are the sensors for Ca²⁺ and PI(4,5)P₂ in a myriad of secretory pathways. Here, the use of a free-mounting system has enabled us to capture an intermediate state of Ca²⁺ binding to the C2A domain of rabphilin 3A that suggests a different mechanism of ion interaction. We have also determined the structure of this domain in complex with PI(4,5)P₂ and IP₃ at resolutions of 1.75 and 1.9 Å, respectively, unveiling that the polybasic cluster formed by strands β3–β4 is involved in the interaction with the phosphoinositides. A comparative study demonstrates that the C2A domain is highly specific for PI(4,5)P₂/PI(3,4,5)P₃, whereas the C2B domain cannot discriminate among any of the diphosphorylated forms. Structural comparisons between C2A domains of rabphilin 3A and synaptotagmin 1 indicated the presence of a key glutamic residue in the polybasic cluster of synaptotagmin 1 that abolishes the interaction with PI(4,5)P₂. Together, these results provide a structural explanation for the ability of different C2 domains to pull plasma and vesicle membranes close together in a Ca²⁺-dependent manner and reveal how this family of proteins can use subtle structural changes to modulate their sensitivity and specificity to various cellular signals.C2 modules are most commonly found in enzymes involved in lipid modifications and signal transduction and in proteins involved in membrane trafficking. They consist of 130 residues and share a common fold composed of two four-stranded β-sheets arranged in a compact β-sandwich connected by surface loops and helices (1–4). Many of these C2 domains have been demonstrated to function in a Ca²⁺-dependent membrane-binding manner and hence act as cellular Ca²⁺ sensors. Calcium ions bind in a cup-shaped invagination formed by three loops at one tip of the β-sandwich where the coordination spheres for the Ca²⁺ ions are incomplete (5–7). This incomplete coordination sphere can be occupied by neutral and anionic (7–9) phospholipids, enabling the C2 domain to dock at the membrane.Previous work in our laboratory has shed light on the 3D structure of the C2 domain of PKCα in complex with both PS and PI(4,5)P₂ simultaneously (10). This revealed an additional lipid-binding site located in the polybasic region formed by β3–β4 strands that preferentially binds to PI(4,5)P₂ (11–15). This site is also conserved in a wide variety of C2 domains of topology I, for example synaptotagmins, rabphilin 3A, DOC2, and PI3KC2α (10, 16–19). Given the importance of PI(4,5)P₂ for bringing the vesicle and plasma membranes together before exocytosis to ensure rapid and efficient fusion upon calcium influx (20–23), it is crucial to understand the molecular mechanisms beneath this event.Many studies have reported different and contradictory results about the membrane binding properties of C2A and C2B domains of synaptotagmin 1 and rabphilin 3A providing an unclear picture about how Ca²⁺ and PI(4,5)P₂ combine to orchestrate the vesicle fusion and repriming processes by acting through the two C2 domains existing in each of these proteins (16, 20, 22, 24–28). A myriad of works have explored the 3D structure of the individual C2 domains of both synaptotagmins and rabphilin 3A (5, 26, 27, 29, 30). However, the impossibility of obtaining crystal structures of these domains in complex with Ca²⁺ and phosphoinositides has hindered the understanding of the molecular mechanism driving the PI(4,5)P₂–C2 domain interaction. Here, we sought to unravel the molecular mechanism of Ca²⁺ and PI(4,5)P₂ binding to the C2A domain of rabphilin 3A by X-ray crystallography. A combination of site-directed mutagenesis together with isothermal titration calorimetry (ITC), fluorescence resonance of energy transfer (FRET), and aggregation experiments has enabled us to propose a molecular mechanism of Ca²⁺/PI(4,5)P₂-dependent membrane interaction through two different motifs that could bend the membrane and accelerate the vesicle fusion process. A comparative analysis revealed the structural basis for the different phosphoinositide affinities of C2A and -B domains. Furthermore, the C2A domain of synaptotagmin 1 lacks one of the key residues responsible for the PI(4,5)P₂ interaction, confirming it is a non-PI(4,5)P₂ responder.     

Synaptotagmin-7 phosphorylation mediates GLP-1–dependent potentiation of insulin secretion from β-cells

Glucose stimulates insulin secretion from β-cells by increasing intracellular Ca²⁺. Ca²⁺ then binds to synaptotagmin-7 as a major Ca²⁺ sensor for exocytosis, triggering secretory granule fusion and insulin secretion. In type-2 diabetes, insulin secretion is impaired; this impairment is ameliorated by glucagon-like peptide-1 (GLP-1) or by GLP-1 receptor agonists, which improve glucose homeostasis. However, the mechanism by which GLP-1 receptor agonists boost insulin secretion remains unclear. Here, we report that GLP-1 stimulates protein kinase A (PKA)-dependent phosphorylation of synaptotagmin-7 at serine-103, which enhances glucose- and Ca²⁺-stimulated insulin secretion and accounts for the improvement of glucose homeostasis by GLP-1. A phospho-mimetic synaptotagmin-7 mutant enhances Ca²⁺-triggered exocytosis, whereas a phospho-inactive synaptotagmin-7 mutant disrupts GLP-1 potentiation of insulin secretion. Our findings thus suggest that synaptotagmin-7 is directly activated by GLP-1 signaling and may serve as a drug target for boosting insulin secretion. Moreover, our data reveal, to our knowledge, the first physiological modulation of Ca²⁺-triggered exocytosis by direct phosphorylation of a synaptotagmin.Glucose-stimulated insulin secretion (GSIS) from pancreatic β-cells follows a biphasic time course consisting of an initial, transient first phase lasting 5–10 min followed by a slowly developing, sustained second phase (1). Type 2 diabetes (T2D) is associated with partial or complete loss of the first insulin secretion phase and a reduction in the second insulin secretion phase (2, 3). Incretins, especially GLP-1, boost GSIS in T2D patients, thereby improving glucose homeostasis (4). GLP-1 exerts its action by activating GLP-1R, a G-protein–coupled receptor expressed on the surface of β-cells, which leads to an increase of adenylate cyclase activity and production of cAMP. Elevated cAMP levels in β-cells enhance GSIS through PKA-dependent and -independent (mediated by Epac2) mechanisms (5, 6). Mouse models with constitutively increased PKA activity have established PKA’s predominant role in the GLP-1–induced potentiation of β-cell GSIS (7, 8), but the downstream effectors remain unidentified.Insulin is secreted in response to glucose by regulated exocytosis of insulin-containing secretory granules. Electrical activity leads to opening of plasmalemmal voltage-gated Ca²⁺ channels (VGCCs) on the β-cell plasma membrane; the resulting increase in [Ca²⁺]_i then triggers Ca²⁺-dependent exocytosis (9). Insulin granule exocytosis is mediated by a multiprotein complex composed of soluble SNAP-receptor (SNARE) proteins (SNAP-25, Syntaxin, and synaptobrevin-2) and Sec1/Munc18-like (SM) proteins (Munc18-1) by a process that shares similarities with synaptic vesicle exocytosis in neurons (10). To date, numerous SNARE isoforms have been implicated in GSIS (11, 12), including Syntaxin-1, Syntaxin-4, SNAP-25 or SNAP-23, and synaptobrevin-2/3 (or VAMP2/3), whereas VAMP8, a nonessential SNARE for GSIS, may be involved in the regulation of GLP-1 potentiation of insulin secretion (13).In addition to SNARE and SM proteins, a Ca²⁺ sensor is required to initiate membrane fusion during exocytosis. Synaptotagmins, expressed mainly in neurons and endocrine cells, share a similar domain structure: a short N-terminal domain, followed by a transmembrane domain, a linker region with variable length, and two tandem Ca²⁺-binding C₂ domains (C₂A and C₂B) at the C terminus (14, 15). Some synaptotagmins bind to phospholipids in a Ca²⁺-dependent manner and have been identified as major Ca²⁺ sensors for regulated exocytosis (14, 16). Synaptotagmin-1, -2, -7, and -9 function as Ca²⁺ sensors for neurotransmitter release, whereas synaptotagmin-1, -7 (Syt7), and -10 regulate hormone secretion and neuropeptide release (9, 17, 18). Specifically, Syt7 regulates insulin granule exocytosis in insulin-secreting cell lines (19, 20). Syt7 is highly expressed in human pancreatic β-cells, and Syt7 KO mice exhibit reduced insulin secretion and consequently impaired glucose tolerance following glucose stimulation (21–23). Collectively, these studies demonstrate that Syt7 is a major Ca²⁺ sensor mediating GSIS in β-cells.Given that GLP-1 potentiates insulin secretion in a glucose-dependent manner, it is highly likely that its insulinotropic action is exerted distally to the initiation of electrical activity, possibly at the level of Ca²⁺ sensing and membrane fusion. Here we report that Syt7 is a stoichiometric substrate for PKA and functions as a downstream target of PKA activated by GLP-1 signaling. Compared with wild-type mice, Syt7 KO mice showed reduced insulin secretion ex vivo and in vivo in response to treatment with the GLP-1 analog exendin-4 in a manner that depended on Syt7 phosphorylation at serine-103. Our data not only provide a mechanism by which GLP-1 stimulates insulin secretion, but also report the physiological regulation of a synaptotagmin by phosphorylation.     

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.     

Three-dimensional architecture of extended synaptotagmin-mediated endoplasmic reticulum–plasma membrane contact sites

The close apposition between the endoplasmic reticulum (ER) and the plasma membrane (PM) plays important roles in Ca²⁺ homeostasis, signaling, and lipid metabolism. The extended synaptotagmins (E-Syts; tricalbins in yeast) are ER-anchored proteins that mediate the tethering of the ER to the PM and are thought to mediate lipid transfer between the two membranes. E-Syt cytoplasmic domains comprise a synaptotagmin-like mitochondrial-lipid–binding protein (SMP) domain followed by five C2 domains in E-Syt1 and three C2 domains in E-Syt2/3. Here, we used cryo-electron tomography to study the 3D architecture of E-Syt–mediated ER–PM contacts at molecular resolution. In vitrified frozen-hydrated mammalian cells overexpressing individual E-Syts, in which E-Syt–dependent contacts were by far the predominant contacts, ER–PM distance (19–22 nm) correlated with the amino acid length of the cytosolic region of E-Syts (i.e., the number of C2 domains). Elevation of cytosolic Ca²⁺ shortened the ER–PM distance at E-Syt1–dependent contacts sites. E-Syt–mediated contacts displayed a characteristic electron-dense layer between the ER and the PM. These features were strikingly different from those observed in cells exposed to conditions that induce contacts mediated by the stromal interaction molecule 1 (STIM1) and the Ca²⁺ channel Orai1 as well as store operated Ca²⁺ entry. In these cells the gap between the ER and the PM was spanned by filamentous structures perpendicular to the membranes. Our results define specific ultrastructural features of E-Syt–dependent ER–PM contacts and reveal their structural plasticity, which may impact on the cross-talk between the ER and the PM and the functions of E-Syts in lipid transport between the two bilayers.The endoplasmic reticulum (ER) consists of a complex network of tubules and cisternae that extends throughout the cell and forms close appositions (“contact sites”) with other membranous organelles and with the plasma membrane (PM) (1, 2). The best characterized function of ER–PM contacts is in Ca²⁺ homeostasis, as ER–PM contacts mediate the excitation–contraction coupling in muscle and store-operated Ca²⁺ entry (SOCE) in all metazoan cells. In SOCE, upon depletion of Ca²⁺ in the ER, the ER protein stromal interaction molecule 1 (STIM1) oligomerizes, binds, and activates Orai1 Ca²⁺ channels at the PM to drive influx of extracellular Ca²⁺, thereby allowing homeostatic regulation of ER Ca²⁺ levels (3, 4). However, growing evidence suggests that ER–PM contacts also play more general roles, including signaling (5, 6) and the regulation of both lipid metabolism and transport between bilayers (1, 7–17).We recently have shown that the three mammalian extended synaptotagmins (E-Syts), homologs of the yeast tricalbins (18, 19), act as ER–PM tethers (20). E-Syts are ER-anchored proteins (via an N-terminal hairpin) containing a synaptotagmin-like mitochondrial-lipid–binding protein (SMP) domain followed by five (E-Syt1) or three (E-Syt2/3) C2 domains. SMP domains are present in proteins that localize at various organelle contact sites, where they have been implicated in lipid transfer between bilayers (19, 21–23). As shown by a recent structural and mass spectrometry study of E-Syt2, the SMP domain dimerizes to form a ∼90-Å-long cylinder traversed by a longitudinally oriented channel lined with hydrophobic residues that harbor glycerophospholipids (23). C2 domains typically are membrane-binding modules whose interaction with the bilayer often is potentiated by elevations in cytosolic Ca²⁺ and/or facilitated by the presence of acidic phospholipids. Accordingly, the property of the E-Syts to tether the ER to the PM is mediated by C2 domain-dependent interactions with phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂] in the PM, and in the case of E-Syt1 is additionally regulated by the elevation of cytosolic Ca²⁺ (11, 20). Because E-Syt1 interacts with both E-Syt2 and E-Syt3, E-Syt1 acts as a Ca²⁺-dependent regulator of E-Syt–mediated ER–PM tethering.The structural features of E-Syt–dependent ER–PM contacts and how the E-Syts and other contact-resident proteins are organized between the bilayers remain unknown. Here we have investigated ER–PM contact architecture in close-to-native conditions using cryo-electron tomography (cryo-ET), a method that enables 3D visualization at molecular resolution of fully hydrated, unstained biological structures in situ, within optimally preserved vitrified cells (24). ER–PM contacts predominantly formed and populated by E-Syt1, E-Syt3, or STIM1 were induced by the transient overexpression of these proteins in COS-7 cells coupled to pharmacological treatments. We found that E-Syt–mediated ER–PM contacts show an electron-dense layer between the two apposed membranes, whereas contacts in which STIM1-mediated tethering to the PM predominates show filaments linking the two membranes. At E-Syt–mediated contacts the ER–PM distance correlates with the length of the cytosolic portion of the specific overexpressed E-Syt isoform and is regulated further by cytosolic Ca²⁺ in the case of E-Syt1.     

Nanoscale patterning of STIM1 and Orai1 during store-operated Ca2+ entry

Stromal interacting molecule (STIM) and Orai proteins constitute the core machinery of store-operated calcium entry. We used transmission and freeze–fracture electron microscopy to visualize STIM1 and Orai1 at endoplasmic reticulum (ER)–plasma membrane (PM) junctions in HEK 293 cells. Compared with control cells, thin sections of STIM1-transfected cells possessed far more ER elements, which took the form of complex stackable cisternae and labyrinthine structures adjoining the PM at junctional couplings (JCs). JC formation required STIM1 expression but not store depletion, induced here by thapsigargin (TG). Extended molecules, indicative of STIM1, decorated the cytoplasmic surface of ER, bridged a 12-nm ER-PM gap, and showed clear rearrangement into small clusters following TG treatment. Freeze–fracture replicas of the PM of Orai1-transfected cells showed extensive domains packed with characteristic “particles”; TG treatment led to aggregation of these particles into sharply delimited “puncta” positioned upon raised membrane subdomains. The size and spacing of Orai1 channels were consistent with the Orai crystal structure, and stoichiometry was unchanged by store depletion, coexpression with STIM1, or an Orai1 mutation (L273D) affecting STIM1 association. Although the arrangement of Orai1 channels in puncta was substantially unstructured, a portion of channels were spaced at ∼15 nm. Monte Carlo analysis supported a nonrandom distribution for a portion of channels spaced at ∼15 nm. These images offer dramatic, direct views of STIM1 aggregation and Orai1 clustering in store-depleted cells and provide evidence for the interaction of a single Orai1 channel with small clusters of STIM1 molecules.Specialized junctions linking the endoplasmic reticulum (ER) to the plasma membrane (PM) were first described by Porter and Palade (1) in skeletal and cardiac muscle. In skeletal muscle, excitation–contraction coupling is mediated by direct physical contact between voltage-gated Ca²⁺ channels (dihydropyridine receptors) in invaginated transverse tubules of the PM and Ca²⁺-release channels (ryanodine receptors) in ER membrane (2). A second type of ER-PM junction mediates inside-out signaling by linking depletion of Ca²⁺ in the ER lumen to Ca²⁺ influx across the PM in a process termed store-operated Ca²⁺ entry (SOCE). In addition to being a mechanism of ionic homeostasis, SOCE supports long-lasting Ca²⁺ signals in many cell types. ER stromal interacting molecule (STIM) and PM Orai proteins were identified by RNAi screening as required for SOCE (3–7). Overexpression of both proteins is required to amplify Ca²⁺ influx through Orai channels (7–10). In Drosophila, STIM and Orai are the sole members of a gene family, which in mammals, includes two STIM and three Orai proteins. STIM1 and Orai1 are predominant in the immune system; human mutations in either gene can cause lethal severe combined immune deficiencies (SCID) (11). ER STIM proteins trigger SOCE by sensing ER Ca²⁺ store depletion, translocating as oligomers to the PM, and binding to PM Orai proteins to promote clustering and channel opening (3, 12–16). These events have been extensively documented by microscopy of cells expressing fluorescently tagged proteins. Numerous studies have defined domains and amino acid residues of STIM1 and Orai1 that are vital for channel function (17, 18).ER-PM junctions underlying SOCE have been visualized by electron microscopy (EM), using either HRP-tagged STIM1 (13, 19) or immunogold labeling of STIM1 (20). However, little is known about the nanometer-scale subcellular organization of STIM and Orai proteins, although they define a basic unit of Ca²⁺ signaling. Here, through a close examination of transmission and freeze–fracture electron micrographs of transfected cells expressing STIM1 and Orai1, we further define the microanatomy of the ER-PM, as well as of ER-ER junctions in store-depleted and untreated cells. These images provide direct candidate signatures for STIM1 molecules bridging the ER-PM and ER-ER gaps and for individual Orai1 channels in puncta. Taken together, our observations provide visual confirmation of STIM1 and Orai1 function, constrain models of STIM1 and Orai1 assembly and interaction, and suggest new aspects of molecular interactions between STIM1 and Orai1.