Ca2+ release-activated Ca2+ channel blockade as a potential tool in antipancreatitis therapy

Alcohol-related acute pancreatitis can be mediated by a combination of alcohol and fatty acids (fatty acid ethyl esters) and is initiated by a sustained elevation of the Ca²⁺ concentration inside pancreatic acinar cells ([Ca²⁺]_i), due to excessive release of Ca²⁺ stored inside the cells followed by Ca²⁺ entry from the interstitial fluid. The sustained [Ca²⁺]_i elevation activates intracellular digestive proenzymes resulting in necrosis and inflammation. We tested the hypothesis that pharmacological blockade of store-operated or Ca²⁺ release-activated Ca²⁺ channels (CRAC) would prevent sustained elevation of [Ca²⁺]_i and therefore protease activation and necrosis. In isolated mouse pancreatic acinar cells, CRAC channels were activated by blocking Ca²⁺ ATPase pumps in the endoplasmic reticulum with thapsigargin in the absence of external Ca²⁺. Ca²⁺ entry then occurred upon admission of Ca²⁺ to the extracellular solution. The CRAC channel blocker developed by GlaxoSmithKline, GSK-7975A, inhibited store-operated Ca²⁺ entry in a concentration-dependent manner within the range of 1 to 50 μM (IC₅₀ = 3.4 μM), but had little or no effect on the physiological Ca²⁺ spiking evoked by acetylcholine or cholecystokinin. Palmitoleic acid ethyl ester (100 μM), an important mediator of alcohol-related pancreatitis, evoked a sustained elevation of [Ca²⁺]_i, which was markedly reduced by CRAC blockade. Importantly, the palmitoleic acid ethyl ester-induced trypsin and protease activity as well as necrosis were almost abolished by blocking CRAC channels. There is currently no specific treatment of pancreatitis, but our data show that pharmacological CRAC blockade is highly effective against toxic [Ca²⁺]_i elevation, necrosis, and trypsin/protease activity and therefore has potential to effectively treat pancreatitis.     

Action potential prolongation in cardiac myocytes of old rats is an adaptation to sustain youthful intracellular Ca2+ regulation

Advanced age in rats is accompanied by reduced expression of the sarcoplasmic reticulum (SR) Ca2+ pump (SERCA-2). The amplitudes of intracellular Ca2+ (Ca2+(i)) transients and contractions in ventricular myocytes isolated from old (23-24-months) rats (OR), however, are similar to those of young (4-6-months) rat myocytes (YR). OR myocytes also manifest slowed inactivation of L-type Ca2+ current (I(CaL)) and marked prolongation of action potential (AP) duration. To determine whether and how age-associated AP prolongation preserves the Ca2+(i) transient amplitude in OR myocytes, we employed an AP-clamp technique with simultaneous measurements of I(CaL) (with Na+ current, K+ currents and Ca2+ influx via sarcolemmal Na+-Ca2+ exchanger blocked) and Ca2+(i) transients in OR rat ventricular myocytes dialyzed with the fluorescent Ca2+ probe, indo-1. Myocytes were stimulated with AP-shaped voltage clamp waveforms approximating the configuration of prolonged, i.e. the native, AP of OR cells (AP-L), or with short AP waveforms (AP-S), typical of YR myocytes. Changes in SR Ca2+ load were assessed by rapid, complete SR Ca2+ depletions with caffeine. As expected, during stimulation with AP-S vs AP-L, peak I(CaL) increased, by 21+/-4%, while the I(CaL) integral decreased, by 19+/-3% (P<0.01 for each). Compared to AP-L, stimulation of OR myocytes with AP-S reduced the amplitudes of the Ca2+(i) transient by 31+/-6%, its maximal rate of rise (+dCa2+(i)/dt(max); a sensitive index of SR Ca2+ release flux) by 37+/-4%, and decreased the SR Ca2+ load by 29+/-4% (P<0.01 for each). Intriguingly, AP-S also reduced the maximal rate of the Ca2+(i) transient relaxation and prolonged its time to 50% decline, by 35+/-5% and 33+/-7%, respectively (P<0.01 for each). During stimulation with AP-S, the gain of Ca2+-induced Ca2+ release (CICR), indexed by +dCa2+(i)/dt(max)/I(CaL), was reduced by 46+/-4% vs AP-L (P<0.01). We conclude that the effects of an application of a shorter AP to OR myocytes to reduce +dCa2+(i)/dt(max) and the Ca2+ transient amplitude are attributable to a reduction in SR Ca2+ load, presumably due to a reduced I(CaL) integral and likely also to an increased Ca2+ extrusion via sarcolemmal Na+-Ca2+ exchanger. The decrease in the Ca2+(i) transient relaxation rate in OR cells stimulated with shorter APs may reflect a reduction of Ca2+/calmodulin-kinase II-regulated modulation of Ca2+ uptake via SERCA-2, consequent to a reduced local Ca2+ release in the vicinity of SERCA-2, also attributable to reduced SR Ca2+ load. Thus, the reduction of CICR gain during stimulation with AP-S is the net result of both a diminished SR Ca2+ release and an increased peak I(CaL). These results suggest that ventricular myocytes of old rats utilize AP prolongation to preserve an optimal SR Ca2+ loading, CICR gain and relaxation of Ca2+(i) transients.     

Ca2+ regulation in the Na+/Ca2+ exchanger features a dual electrostatic switch mechanism

Regulation of ion-transport in the Na⁺/Ca²⁺ exchanger (NCX) occurs via its cytoplasmic Ca²⁺-binding domains, CBD1 and CBD2. Here, we present a mechanism for NCX activation and inactivation based on data obtained using NMR, isothermal titration calorimetry (ITC) and small-angle X-ray scattering (SAXS). We initially determined the structure of the Ca²⁺-free form of CBD2-AD and the structure of CBD2-BD that represent the two major splice variant classes in NCX1. Although the apo-form of CBD2-AD displays partially disordered Ca²⁺-binding sites, those of CBD2-BD are entirely unstructured even in an excess of Ca²⁺. Striking differences in the electrostatic potential between the Ca²⁺-bound and -free forms strongly suggest that Ca²⁺-binding sites in CBD1 and CBD2 form electrostatic switches analogous to C₂-domains. SAXS analysis of a construct containing CBD1 and CBD2 reveals a conformational change mediated by Ca²⁺-binding to CBD1. We propose that the electrostatic switch in CBD1 and the associated conformational change are necessary for exchanger activation. The response of the CBD1 switch to intracellular Ca²⁺ is influenced by the closely located cassette exons. We further propose that Ca²⁺-binding to CBD2 induces a second electrostatic switch, required to alleviate Na⁺-dependent inactivation of Na⁺/Ca²⁺ exchange. In contrast to CBD1, the electrostatic switch in CBD2 is isoform- and splice variant-specific and allows for tailored exchange activities.     

Voltage-gating and cytosolic Ca2+ activation mechanisms of Arabidopsis two-pore channel AtTPC1

Arabidopsis thaliana two-pore channel AtTPC1 is a voltage-gated, Ca²⁺-modulated, nonselective cation channel that is localized in the vacuolar membrane and responsible for generating slow vacuolar (SV) current. Under depolarizing membrane potential, cytosolic Ca²⁺ activates AtTPC1 by binding at the EF-hand domain, whereas luminal Ca²⁺ inhibits the channel by stabilizing the voltage-sensing domain II (VSDII) in the resting state. Here, we present 2.8 to 3.3 Å cryoelectron microscopy (cryo-EM) structures of AtTPC1 in two conformations, one in closed conformation with unbound EF-hand domain and resting VSDII and the other in a partially open conformation with Ca²⁺-bound EF-hand domain and activated VSDII. Structural comparison between the two different conformations allows us to elucidate the structural mechanisms of voltage gating, cytosolic Ca²⁺ activation, and their coupling in AtTPC1. This study also provides structural insight into the general voltage-gating mechanism among voltage-gated ion channels.

Voltage-gated ion channels (VGICs), such as voltage-gated potassium channel (Kv), sodium channel (Nav), and calcium channel (Cav), are activated by depolarization of membrane potential and play essential roles in electrical signal transduction (1–3). VGICs sense the membrane potential by voltage-sensing domains (VSDs), which consist of four transmembrane helices S1 to S4. In most VGICs, VSDs are stabilized in the resting state by hyperpolarizing (negative) membrane potential, and the channel gate stays closed. At depolarizing (relatively positive) membrane potential, VSDs are activated, and their depolarization-induced conformational changes are coupled to the S5–S6 pore domain, resulting in the opening of the channel gate.While dozens of VGIC structures have been determined over the past two decades, only very few VSDs in these structures were captured in the resting state. That is because VGICs for structural studies in vitro are solubilized in detergent micelle or lipid nanodisc, making it difficult to recapitulate the resting state under hyperpolarizing (negative) membrane potential. The structure of plant two-pore channel (TPC) from Arabidopsis thaliana (AtTPC1) was among the first to capture a resting-state VSD (4, 5). In addition, mutagenesis combined with cross-linking, ion bridge, or toxin binding have been used to trap the structures of VGICs in resting state in several recent studies, including structures of the bacterial sodium channel NavAb (6), the eukaryotic sodium channel chimera Nav1.7-NavPaS (7), and the hyperpolarization-activated cyclic nucleotide-gated ion channel HCN (8). To fully understand the similarities and differences of the voltage-gating mechanism among different VGICs, it will be essential to visualize the structures of various VGICs in both activated and resting state. To this end, we are using AtTPC1 as a model to elucidate the structural mechanism of voltage gating.TPCs belong to the VGIC superfamily and are ubiquitously expressed in animals and plants (9, 10). While animal TPCs (TPC1 and TPC2) are endolysosomal sodium channels, the plant TPC (TPC1) is a nonselective cation channel responsible for generating the slow vacuole (SV) current (11, 12). TPCs function as homodimer with each subunit comprising two homologous Shaker-like 6-transmembrane segment domains (6-TM I and 6-TM II) (9, 10, 13), thereby equivalent to a classical VGIC with four VSDs and one pore domain.Plant TPC is involved in many important physiological processes, such as germination and stomatal opening (12), jasmonate biosynthesis (14, 15), modulation of Ca²⁺ waves induced by salinity stress (16), and plant–pathogen interaction (17). AtTPC1, the most well-studied plant TPC from A. thaliana, is activated by the membrane depolarization and cytosolic Ca²⁺ but inhibited by vacuolar Ca²⁺ (18, 19). Previously, we determined the crystal structure of AtTPC1 in closed state (Protein Data Bank [PDB]: 5E1J, AtTPC1_5E1J) (4). We demonstrated that between the two VSDs within each AtTPC1 subunit, only the second one (VSDII) senses the membrane potential and adopts a resting state in the structure whereas the first one (VSDI) lacks several key features essential for voltage sensing and therefore does not contribute to the voltage-dependent gating. Ca²⁺ activation occurs at the EF-hand domain containing two EF-hand motifs. However, Ca²⁺ binding at EF hand 1 appears to play a structural role and does not contribute to Ca²⁺ activation; Ca²⁺ binding at EF hand 2 is central for Ca²⁺ activation and it adopts an unbound state in the structure (4, 19). We also identified the luminal divalent inhibition site in AtTPC1 where Ca²⁺ or Ba²⁺ binding can stabilize the voltage-sensing VSDII in a resting state. Based on our structural and electrophysiological analysis, we proposed that the conformational changes triggered by the binding of Ca²⁺ to cytosolic EF-hand domain are coupled with the pair pore-lining inner helices from the 6-TM I (IS6), whereas the conformational changes of VSDII activated by membrane potential are coupled with the pair of inner helices from the 6-TM II (IIS6) (4). In order to understand the structural basis underlying multistimuli gating of AtTPC1, here we determined AtTPC1 structures in both closed and partially open conformation under different Ca²⁺ conditions, revealing the structural mechanism of voltage gating and Ca²⁺ modulation of AtTPC1.     

Src family tyrosine kinases inhibit single L-type: Ca2+ channel activity in human atrial myocytes

OBJECTIVE: Tyrosine kinases (TKs) are important regulators of the L-type Ca(2+) channel (LTCC) current in various cell types. However, there are no data addressing the role of TKs in the control of single LTCC activity in human atrial cardiac myocytes, where changes in LTCC gating properties have been described in a number of disease states. METHODS AND RESULTS: Single LTCC activity was recorded in isolated human atrial myocytes. The broad-spectrum TK inhibitor genistein and the Src family-selective TK inhibitor PP1 significantly enhanced single LTCC ensemble average current, availability, and open probability; the latter was due to significant increases of mean open time and mode 2 gating. Conversely, the tyrosine phosphatase inhibitor bisperoxo-phenanthroline-vanadate inhibited single LTCC activity, indicating that LTCC gating properties in human atrial myocytes are controlled by TKs and tyrosine phosphatases in a reciprocal fashion. The effects of genistein on single LTCC activity were not affected by stimulation (8Br-cAMP) or inhibition (Rp-8-CPT-cAMPS) of protein kinase A (PKA) or by inhibition of serine/threonine phosphatases types I and IIa (okadaic acid), indicating that TKs inhibit LTCC gating in human atrial myocytes independent of PKA and phosphatases types I and IIa. However, inhibition of protein kinase C (PKC) by staurosporine or bisindolylmaleimide reversed the stimulatory effects of genistein on single LTCC gating properties, indicating that PKC is required for the inhibitory effect of TKs on single LTCC activity. CONCLUSION: Src family TKs inhibit single LTCC activity in human atrial myocytes via PKC-dependent, but PKA and phosphatase types I and IIa-independent, molecular pathways.     

Ca2+-dependent release of Munc18-1 from presynaptic mGluRs in short-term facilitation

Short-term synaptic facilitation plays an important role in information processing in the central nervous system. Although the crucial requirement of presynaptic Ca²⁺ in the expression of this plasticity has been known for decades, the molecular mechanisms underlying the plasticity remain controversial. Here, we show that presynaptic metabotropic glutamate receptors (mGluRs) bind and release Munc18-1 (also known as rbSec1/nSec1), an essential protein for synaptic transmission, in a Ca²⁺-dependent manner, whose actions decrease and increase synaptic vesicle release, respectively. We found that mGluR4 bound Munc18-1 with an EC₅₀ for Ca²⁺ of 168 nM, close to the resting Ca²⁺ concentration, and that the interaction was disrupted by Ca²⁺-activated calmodulin (CaM) at higher concentrations of Ca²⁺. Consistently, the Munc18-1-interacting domain of mGluR4 suppressed both dense-core vesicle secretion from permeabilized PC12 cells and synaptic transmission in neuronal cells. Furthermore, this domain was sufficient to induce paired-pulse facilitation. Obviously, the role of mGluR4 in these processes was independent of its classical function of activation by glutamate. On the basis of these experimental data, we propose the following model: When neurons are not active, Munc18-1 is sequestered by mGluR4, and therefore the basal synaptic transmission is kept low. After the action potential, the increase in the Ca²⁺ level activates CaM, which in turn liberates Munc18-1 from mGluR4, causing short-term synaptic facilitation. Our findings unite and provide a new insight into receptor signaling and vesicular transport, which are pivotal activities involved in a variety of cellular processes.     

Structural basis for the high Ca2+ affinity of the ubiquitous SERCA2b Ca2+ pump

Sarco(endo)plasmic reticulum Ca²⁺ ATPase (SERCA) Ca²⁺ transporters pump cytosolic Ca²⁺ into the endoplasmic reticulum, maintaining a Ca²⁺ gradient that controls vital cell functions ranging from proliferation to death. To meet the physiological demand of the cell, SERCA activity is regulated by adjusting the affinity for Ca²⁺ ions. Of all SERCA isoforms, the housekeeping SERCA2b isoform displays the highest Ca²⁺ affinity because of a unique C-terminal extension (2b-tail). Here, an extensive structure–function analysis of SERCA2b mutants and SERCA1a2b chimera revealed how the 2b-tail controls Ca²⁺ affinity. Its transmembrane (TM) segment (TM11) and luminal extension functionally cooperate and interact with TM7/TM10 and luminal loops of SERCA2b, respectively. This stabilizes the Ca²⁺-bound E1 conformation and alters Ca²⁺-transport kinetics, which provides the rationale for the higher apparent Ca²⁺ affinity. Based on our NMR structure of TM11 and guided by mutagenesis results, a structural model was developed for SERCA2b that supports the proposed 2b-tail mechanism and is reminiscent of the interaction between the α- and β-subunits of Na⁺,K⁺-ATPase. The 2b-tail interaction site may represent a novel target to increase the Ca²⁺ affinity of malfunctioning SERCA2a in the failing heart to improve contractility.     

Crystal structure of Ca2+/H+ antiporter protein YfkE reveals the mechanisms of Ca2+ efflux and its pH regulation

Ca²⁺ efflux by Ca²⁺ cation antiporter (CaCA) proteins is important for maintenance of Ca²⁺ homeostasis across the cell membrane. Recently, the monomeric structure of the prokaryotic Na⁺/Ca²⁺ exchanger (NCX) antiporter NCX_Mj protein from Methanococcus jannaschii shows an outward-facing conformation suggesting a hypothesis of alternating substrate access for Ca²⁺ efflux. To demonstrate conformational changes essential for the CaCA mechanism, we present the crystal structure of the Ca²⁺/H⁺ antiporter protein YfkE from Bacillus subtilis at 3.1-Å resolution. YfkE forms a homotrimer, confirmed by disulfide crosslinking. The protonated state of YfkE exhibits an inward-facing conformation with a large hydrophilic cavity opening to the cytoplasm in each protomer and ending in the middle of the membrane at the Ca²⁺-binding site. A hydrophobic “seal” closes its periplasmic exit. Four conserved α-repeat helices assemble in an X-like conformation to form a Ca²⁺/H⁺ exchange pathway. In the Ca²⁺-binding site, two essential glutamate residues exhibit different conformations compared with their counterparts in NCX_Mj, whereas several amino acid substitutions occlude the Na⁺-binding sites. The structural differences between the inward-facing YfkE and the outward-facing NCX_Mj suggest that the conformational transition is triggered by the rotation of the kink angles of transmembrane helices 2 and 7 and is mediated by large conformational changes in their adjacent transmembrane helices 1 and 6. Our structural and mutational analyses not only establish structural bases for mechanisms of Ca²⁺/H⁺ exchange and its pH regulation but also shed light on the evolutionary adaptation to different energy modes in the CaCA protein family.     

CaBP1, a neuronal Ca2+ sensor protein,inhibits inositol trisphosphate receptors by clamping intersubunit interactions

Calcium-binding protein 1 (CaBP1) is a neuron-specific member of the calmodulin superfamily that regulates several Ca²⁺ channels, including inositol 1,4,5-trisphosphate receptors (InsP₃Rs). CaBP1 alone does not affect InsP₃R activity, but it inhibits InsP₃-evoked Ca²⁺ release by slowing the rate of InsP₃R opening. The inhibition is enhanced by Ca²⁺ binding to both the InsP₃R and CaBP1. CaBP1 binds via its C lobe to the cytosolic N-terminal region (NT; residues 1–604) of InsP₃R1. NMR paramagnetic relaxation enhancement analysis demonstrates that a cluster of hydrophobic residues (V101, L104, and V162) within the C lobe of CaBP1 that are exposed after Ca²⁺ binding interact with a complementary cluster of hydrophobic residues (L302, I364, and L393) in the β-domain of the InsP₃-binding core. These residues are essential for CaBP1 binding to the NT and for inhibition of InsP₃R activity by CaBP1. Docking analyses and paramagnetic relaxation enhancement structural restraints suggest that CaBP1 forms an extended tetrameric turret attached by the tetrameric NT to the cytosolic vestibule of the InsP₃R pore. InsP₃ activates InsP₃Rs by initiating conformational changes that lead to disruption of an intersubunit interaction between a “hot-spot” loop in the suppressor domain (residues 1–223) and the InsP₃-binding core β-domain. Targeted cross-linking of residues that contribute to this interface show that InsP₃ attenuates cross-linking, whereas CaBP1 promotes it. We conclude that CaBP1 inhibits InsP₃R activity by restricting the intersubunit movements that initiate gating.     

Experimental diabetes enhances Ca2+ mobilization and glutamate exocytosis in cerebral synaptosomes from mice

The present study was conducted to investigate the effects of the diabetic condition on the Ca²⁺ mobilization and glutamate release in cerebral nerve terminals (synaptosomes). Diabetes was induced in male mice by intraperitoneal injection of streptozotocin. Cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) and glutamate release in synaptosomes were determined using fura-2 and enzyme-linked fluorometric assay, respectively. Diabetes significantly enhanced the ability of the depolarizing agents K⁺ and 4-aminopyridine (4-AP) to increase [Ca²⁺]_i. In addition, diabetes significantly enhanced K⁺- and 4-AP-evoked Ca²⁺-dependent glutamate release. The pretreatment of synaptosomes with a combination of ω-agatoxin IVA (a P-type Ca²⁺ channel blocker) and ω-conotoxin GVIA (an N-type Ca²⁺ channel blocker) inhibited K⁺- or 4-AP-induced increases in [Ca²⁺]_i and Ca²⁺-dependent glutamate release in synaptosomes from the control and diabetic mice to a similar extent, respectively. These results indicate that diabetes enhances a K⁺- or 4-AP-evoked Ca²⁺-dependent glutamate release by increasing [Ca²⁺]_i via stimulation of Ca²⁺ entry through both P- and N-type Ca²⁺ channels.     

Hydrogen peroxide-induced vascular relaxation in porcine coronary arteries is mediated by Ca2+-activated K+ channels

Summary Hydrogen peroxide (H₂O₂) elicited concentration-dependent relaxation of endothelium-denuded rings of porcine coronary arteries. The relaxation induced by the H₂O₂ was markedly attenuated by 10μM 1H-[1,2,4]oxadiazolo [4,3,a]quinoxalin-1-one (ODQ), an inhibitor of soluble guanylate cyclase, or by 100nM charybdotoxin, an inhibitor of large-conductance Ca²⁺-activated K⁺ (K_Ca) channels. A combination of the ODQ and charybdotoxin abolished the H₂O₂-induced relaxation. Pretreatment with 25 μM of an Rp stereoisomer of adenosine-3′,5′-cyclic monophosphothioate (Rp-cAMPS), 20μM glibenclamide, or 1mM 4-aminopyridine did not affect the vascular response to H₂O₂. The presence of catalase at 1000U/ml significantly attenuated the H₂O₂-induced relaxation. Exposure of cultured smooth muscle cells to H₂O₂ activated K_Ca channels in a concentration-dependent manner in cell-attached patches. Pretreatment with catalase significantly inhibited the activation of K_Ca channels. Rp-cAMPS did not inhibit the H₂O₂-induced activation of K_Ca channels. The activation of K_Ca channels by H₂O₂ was markedly decreased in the presence of ODQ. However, even in the presence of ODQ, H₂O₂ activated K_Ca channels in a concentration-dependent manner. In inside-out patches, H₂O₂ significantly activated K_Ca channels through a process independent of cyclic guanosine 3′,5′-monophosphate (cGMP). In conclusion, H₂O₂ elicits vascular relaxation due to activation of K_Ca channels, which is mediated partly by a direct action on the channel and partly by activation of soluble guanylate cyclase, resulting in the generation of cGMP.     

L-type Ca channel facilitation mediated by H2O2-induced activation of CaMKII in rat ventricular myocytes

The Ca²⁺-dependent facilitation (CDF) of L-type Ca²⁺ channels, a major mechanism for force-frequency relationship of cardiac contraction, is mediated by Ca²⁺/CaM-dependent kinase II (CaMKII). Recently, CaMKII was shown to be activated by methionine oxidation. We investigated whether oxidation-dependent CaMKII activation is involved in the regulation of L-type Ca²⁺ currents (I_Ca,L) by H₂O₂ and whether Ca²⁺ is required in this process. Using patch clamp, I_Ca,L was measured in rat ventricular myocytes. H₂O₂ induced an increase in I_Ca,L amplitude and slowed inactivation of I_Ca,L. This oxidation-dependent facilitation (ODF) of I_Ca,L was abolished by a CaMKII blocker KN-93, but not by its inactive analog KN-92, indicating that CaMKII is involved in ODF. ODF was not affected by replacement of external Ca²⁺ with Ba²⁺ or presence of EGTA in the internal solutions. However, ODF was abolished by adding BAPTA to the internal solution or by depleting sarcoplasmic reticulum (SR) Ca²⁺ stores using caffeine and thapsigargin. Alkaline phosphatase, β-iminoadenosine 5′-triphosphate (AMP-PNP), an autophosphorylation inhibitor autocamtide-2-related inhibitory peptide (AIP), or a catalytic domain blocker (CaM-KIINtide) did not affect ODF. In conclusion, oxidation-dependent facilitation of L-type Ca²⁺ channels is mediated by oxidation-dependent CaMKII activation, in which local Ca²⁺ increases induced by SR Ca²⁺ release is required.     

Intracellular Ca2+ signaling and store-operated Ca2+ entry are required in Drosophila neurons for flight

Neuronal Ca²⁺ signals can affect excitability and neural circuit formation. Ca²⁺ signals are modified by Ca²⁺ flux from intracellular stores as well as the extracellular milieu. However, the contribution of intracellular Ca²⁺ stores and their release to neuronal processes is poorly understood. Here, we show by neuron-specific siRNA depletion that activity of the recently identified store-operated channel encoded by dOrai and the endoplasmic reticulum Ca²⁺ store sensor encoded by dSTIM are necessary for normal flight and associated patterns of rhythmic firing of the flight motoneurons of Drosophila melanogaster. Also, dOrai overexpression in flightless mutants for the Drosophila inositol 1,4,5-trisphosphate receptor (InsP₃R) can partially compensate for their loss of flight. Ca²⁺ measurements show that Orai gain-of-function contributes to the quanta of Ca²⁺-release through mutant InsP₃Rs and elevates store-operated Ca²⁺ entry in Drosophila neurons. Our data show that replenishment of intracellular store Ca²⁺ in neurons is required for Drosophila flight.     

Synaptotagmin-1 is a bidirectional Ca2+ sensor for neuronal endocytosis

Exocytosis and endocytosis are tightly coupled. In addition to initiating exocytosis, Ca²⁺ plays critical roles in exocytosis–endocytosis coupling in neurons and nonneuronal cells. Both positive and negative roles of Ca²⁺ in endocytosis have been reported; however, Ca²⁺ inhibition in endocytosis remains debatable with unknown mechanisms. Here, we show that synaptotagmin-1 (Syt1), the primary Ca²⁺ sensor initiating exocytosis, plays bidirectional and opposite roles in exocytosis–endocytosis coupling by promoting slow, small-sized clathrin-mediated endocytosis but inhibiting fast, large-sized bulk endocytosis. Ca²⁺-binding ability is required for Syt1 to regulate both types of endocytic pathways, the disruption of which leads to inefficient vesicle recycling under mild stimulation and excessive membrane retrieval following intense stimulation. Ca²⁺-dependent membrane tubulation may explain the opposite endocytic roles of Syt1 and provides a general membrane-remodeling working model for endocytosis determination. Thus, Syt1 is a primary bidirectional Ca²⁺ sensor facilitating clathrin-mediated endocytosis but clamping bulk endocytosis, probably by manipulating membrane curvature to ensure both efficient and precise coupling of endocytosis to exocytosis.

Endocytosis and subsequent vesicle recycling are spatiotemporally coupled to exocytosis, which is critical for neurons and endocrinal cells to maintain the integrity of plasma membrane architecture, intracellular homeostasis, and sustained neurotransmission (1–3). In addition to triggering vesicular exocytosis, neural activity/Ca²⁺ also play an executive role in the coupling of endocytosis to exocytosis (1, 2, 4–6). Following a pioneering study 40 y ago (7), extensive studies have been conducted and showed that Ca²⁺ triggers and facilitates vesicle endocytosis in neurons and nonneuronal secretory cells (1, 8–11). Accumulating evidence also shows that intracellular Ca²⁺ may inhibit endocytosis (12–15), which has been challenged greatly due to the apparently lower occurrences in few preparations and the missing underlining mechanisms, making the endocytic role of Ca²⁺ a four-decades–long dispute (1, 2, 4, 6).Machineries and regulators involved in exocytosis–endocytosis coupling have been extensively studied for over 30 y. The soluble N-ethylmaleimide–sensitive factor attachment protein receptors (SNAREs) and synaptophysin play critical dual roles in exocytosis and endocytosis during neurotransmission (2, 3, 16, 17). Calmodulin and synaptotagmin-1 (Syt1) are currently known primary Ca²⁺ sensors facilitating endocytosis (1, 9, 16, 18, 19). Ca²⁺/calmodulin activate calcineurin, which dephosphorylates endocytic proteins (e.g., dynamin, synaptojanin, and amphiphysin) to facilitate clathrin-mediated endocytosis (CME) and clathrin-independent fast endocytosis (1, 2). Syt1 is a dual Ca²⁺ sensor for both exocytosis and endocytosis (5, 16, 18–20). It promotes CME through binding with the endocytic adaptors adaptor protein-2 (AP-2) and stonin-2 (21–24). In contrast to the well-established Ca²⁺ sensors that promote endocytosis, the mechanism of Ca²⁺-dependent inhibition in endocytosis remains unknown.CME is the classical but slow endocytosis pathway for vesicle retrieval under resting conditions or in response to mild stimulation, while the accumulated Ca²⁺ also triggers calmodulin/calcineurin-dependent bulk endocytosis, which takes up a large area of plasma membrane to fulfill the urgent requirement for high-speed vesicle exocytosis (1–3). They cooperate with kiss-and-run and ultrafast endocytosis to ensure both sufficient and precise membrane retrieval following exocytosis (3, 25–27). These endocytic pathways are all initiated from membrane invagination and are critically controlled by neural activity. However, how the switch between different endocytic modes is precisely determined remains largely unknown.Here, by combining electrophysiological recordings, confocal live imaging, superresolution stimulated emission depletion (STED) imaging, in vitro liposome manipulation, and electron microscope imaging of individual endocytic vesicles, we define Syt1 as a primary and bidirectional Ca²⁺ sensor for endocytosis, which promotes CME but inhibits bulk endocytosis, probably by mediating membrane remodeling. The balance between the facilitatory and inhibitory effects of Syt1 on endocytosis offers a fine-tuning mechanism to ensure both efficient and precise coupling of endocytosis to exocytosis. By including a non-Ca²⁺–binding Syt as the constitutive brake, this work also explains the four-decades–long puzzle about the positive and negative Ca²⁺ effects on endocytosis.     

The store-operated Ca2+ entry complex comprises a small cluster of STIM1 associated with one Orai1 channel

Increases in cytosolic Ca²⁺ concentration regulate diverse cellular activities and are usually evoked by opening of Ca²⁺ channels in intracellular Ca²⁺ stores and the plasma membrane (PM). For the many signals that evoke formation of inositol 1,4,5-trisphosphate (IP₃), IP₃ receptors coordinate the contributions of these two Ca²⁺ sources by mediating Ca²⁺ release from the endoplasmic reticulum (ER). Loss of Ca²⁺ from the ER then activates store-operated Ca²⁺ entry (SOCE) by causing dimers of STIM1 to cluster and unfurl cytosolic domains that interact with the PM Ca²⁺ channel, Orai1, causing its pore to open. The relative concentrations of STIM1 and Orai1 are important, but most analyses of their interactions use overexpressed proteins that perturb the stoichiometry. We tagged endogenous STIM1 with EGFP using CRISPR/Cas9. SOCE evoked by loss of ER Ca²⁺ was unaffected by the tag. Step-photobleaching analysis of cells with empty Ca²⁺ stores revealed an average of 14.5 STIM1 molecules within each sub-PM punctum. The fluorescence intensity distributions of immunostained Orai1 puncta were minimally affected by store depletion, and similar for Orai1 colocalized with STIM1 puncta or remote from them. We conclude that each native SOCE complex is likely to include only a few STIM1 dimers associated with a single Orai1 channel. Our results, demonstrating that STIM1 does not assemble clusters of interacting Orai channels, suggest mechanisms for digital regulation of SOCE by local depletion of the ER.

In generating the cytosolic Ca²⁺ signals that regulate cellular activities, cells call upon two sources of Ca²⁺: the extracellular space, accessed through Ca²⁺ channels in the plasma membrane (PM), and Ca²⁺ sequestered within intracellular stores, primarily within the endoplasmic reticulum (ER). In animal cells, the many receptors that stimulate formation of inositol 1,4,5-trisphosphate (IP₃) provide coordinated access to both Ca²⁺ sources (1). IP₃ stimulates the opening of IP₃ receptors (IP₃R), which are large Ca²⁺-permeable channels expressed mostly within ER membranes. IP₃ thereby triggers Ca²⁺ release from the ER (2, 3). The link to extracellular Ca²⁺ is provided by store-operated Ca²⁺ entry (SOCE), which is activated by loss of Ca²⁺ from the ER. The reduction in ER free-Ca²⁺ concentration causes Ca²⁺ to dissociate from the luminal Ca²⁺-binding sites of stromal interaction molecule 1 (STIM1), a dimeric protein embedded in ER membranes. This loss of Ca²⁺ causes STIM1 to unfurl cytosolic domains that interact with the PM Ca²⁺ channel, Orai1, causing its pore to open and Ca²⁺ to flow into the cell through the SOCE pathway (Fig. 1A) (4, 5). Available evidence suggests that STIM1 must bind to the C-terminal tail of each of the six subunits of an Orai1 channel for optimal activity, with lesser occupancies reducing activity and modifying channel properties (6–10). The interactions between STIM1 and Orai1 occur at membrane contact sites (MCS), where the two membranes are organized to provide a gap of about 10–30 nm, across which the two proteins directly interact (11–13). Orai channels are unusual in having no structural semblance to other ion channels and in having their opening controlled by direct interactions between proteins in different membranes (Fig. 1A). Competing models suggest that dimeric STIM1 binds either to a pair of C-terminal tails within a single channel (6 STIM1 molecules per hexameric Orai1 channel) (Fig. 1 B, a), or that each dimer interacts with only a single C-terminal tail leaving the remaining STIM1 subunit free to cross-link with a different Orai1 channel (12 STIM1 molecules around a single Orai1 channel) (Fig. 1 B, b) (see references in ref. 14). The latter arrangement has been proposed to allow assembly of close-packed Orai1 clusters (Fig. 1 B, c) and to explain the variable stoichiometry of Orai1 to STIM1 at MCS (14).Open in a separate windowFig. 1.SOCE is unaffected by tagging of endogenous STIM1. (A) SOCE is activated when loss of Ca²⁺ from the ER, usually mediated by IP₃Rs, causes Ca²⁺ to dissociate from the EF hands of dimeric STIM1. This causes STIM1 to unfurl its cytosolic domain, unmasking the C-terminal polybasic tail (PBT) and CRAC (Ca²⁺-release-activated channel)-activation domain (CAD) Association of the PBT with PM phosphoinositides causes STIM1 to accumulate at MCS, where the CAD captures the C-terminal tail of Orai1. Binding of STIM1 to each of the six subunits of Orai1 opens the Ca²⁺ channel, allowing SOCE to occur (9). (B) Orai1 is a hexamer, comprising three pairs of dimers (33). Dimeric STIM1 may activate Orai1 by binding as three dimers (B, a), or as six dimers (B, b) with the residual STIM1 subunit free to interact with another Orai1 channel (B, c) (14). (C) Structure of the edited STIM1-EGFP. (D) TIRF images of STIM1-EGFP HeLa cells treated with STIM1 or nonsilencing (NS) shRNA before emptying of Ca²⁺ stores. (Scale bar, 10 µm.) (E) Summary results (individual values, mean ± SD, n = 3 independent experiments, each with ∼30 cells analyzed) show whole-cell fluorescence intensities from TIRF images of STIM1-EGFP HeLa cells treated with the indicated shRNA. Results from WT cells are also shown (n = 4). ****P < 0.0001, ANOVA with Bonferroni test, relative to WT cells. (F) In-gel fluorescence of lysates from WT or STIM1-EGFP HeLa cells (protein loadings in μg). The STIM1-EGFP band (arrow) and molecular mass markers (kDa) are shown. Similar results were obtained in four independent analyses. (G) WB for STIM1 and β-actin for WT and STIM1-EGFP HeLa cells. Protein loadings (μg) and molecular mass markers (kDa) are shown. Arrows show positions of native and EGFP-tagged STIM1. (H) Summary results (individual values, mean ± SD, n = 9) show expression of STIM1-EGFP relative to all STIM1 in STIM1-EGFP HeLa cells (red), and total STIM1 expression in WT and edited cells (black). (I) Effects of histamine in Ca²⁺-free HBS on the peak increase in [Ca²⁺]_c (Δ[Ca²⁺]_c) in populations of WT and STIM1-EGFP HeLa cells. Mean ± SEM from four experiments, each with six determinations. (J) Effects of CPA in Ca²⁺-free HBS on the peak increase in [Ca²⁺]_c (Δ[Ca²⁺]_c) in populations of WT and STIM1-EGFP HeLa cells. Mean ± SEM from four experiments, each with six determinations. (K) Populations of cells were treated (5 min) with CPA in Ca²⁺-free HBS to evoke graded depletion of ER Ca²⁺ stores before addition of extracellular Ca²⁺ (final free [Ca²⁺] ∼10 mM). Results (mean ± SEM, n = 6, each with six determinations) show the amplitude of the SOCE in WT and STIM1-EGFP HeLa cells. See also SI Appendix, Figs. S1 and S2.Opening of most ion channels is regulated by changes in membrane potential or by binding of soluble stimuli, where the relationship between stimulus intensity and response is readily amenable to experimental analysis. The unusual behavior of SOCE, where direct interactions between proteins embedded in different membranes control channel opening (Fig. 1A), makes it more difficult to define stimulus–response relationships and highlights the need to understand the amounts of STIM1 and Orai1 within the MCS where the interactions occur. When STIM1 or Orai1 are overexpressed their behaviors are perturbed, yet most analyses of their interactions have involved overexpression of the proteins. These difficulties motivated the present study, which was designed to determine the number of native STIM1 molecules associated with each SOCE signaling complex.     

Regulation of sarcolemmal Ca2+ pump by endogenous protein phosphatases

Summary The function of several key sarcolemmal proteins is modulated through phosphorylation-dephosphorylation of serine/threonine residues. While the involvement of sarcolemma-associated protein kinases in the phosphorylation of these proteins has been established, the nature of the protein phosphatases controlling these proteins has not been investigated. Rat heart sarcolemma contains two protein phosphatase isozymes, protein phosphatase 1 and 2A, which are distinguished on the basis of their susceptibility of inhibitor 2. Both isozymes elute from a Bio Gel A-0.5 column in association with the highest molecular weight protein fraction. However, some protein phosphatase 1 activity elutes with a smaller molecular weight fraction of about 37000, suggesting that the native enzyme exists as a catalytic subunit in complex with an anchor protein. Inhibition of the protein phosphatases using standard inhibitors leads to a stimulation in both the rate and extent of³²P incorporation into isolated sarcolemma. Also affected by inhibition of protein phosphatase activity is the rate of ATP-dependent calcium uptake, which is stimulated following exposure to either inhibitor 2, a classical protein phosphatase 1 inhibitor, and microcystin, a protein phosphatase 1 and 2A inhibitor. The data suggest that the protein phosphatases regulate the dephosphorylation of sarcolemmal proteins Through this mechanism they serve as important modulators of the sarcolemmal Ca²⁺ pump.     

Loss of luminal Ca2+ activation in the cardiac ryanodine receptor is associated with ventricular fibrillation and sudden death

Different forms of ventricular arrhythmias have been linked to mutations in the cardiac ryanodine receptor (RyR)2, but the molecular basis for this phenotypic heterogeneity is unknown. We have recently demonstrated that an enhanced sensitivity to luminal Ca(2+) and an increased propensity for spontaneous Ca(2+) release or store-overload-induced Ca(2+) release (SOICR) are common defects of RyR2 mutations associated with catecholaminergic polymorphic or bidirectional ventricular tachycardia. Here, we investigated the properties of a unique RyR2 mutation associated with catecholaminergic idiopathic ventricular fibrillation, A4860G. Single-channel analyses revealed that, unlike all other disease-linked RyR2 mutations characterized previously, the A4860G mutation diminished the response of RyR2 to activation by luminal Ca(2+), but had little effect on the sensitivity of the channel to activation by cytosolic Ca(2+). This specific impact of the A4860G mutation indicates that the luminal Ca(2+) activation of RyR2 is distinct from its cytosolic Ca(2+) activation. Stable, inducible HEK293 cells expressing the A4860G mutant showed caffeine-induced Ca(2+) release but exhibited no SOICR. Importantly, HL-1 cardiac cells transfected with the A4860G mutant displayed attenuated SOICR activity compared with cells transfected with RyR2 WT. These observations provide the first evidence that a loss of luminal Ca(2+) activation and SOICR activity can cause ventricular fibrillation and sudden death. These findings also indicate that although suppressing enhanced SOICR is a promising antiarrhythmic strategy, its oversuppression can also lead to arrhythmias.     

CSN5/Jab1 inhibits cardiac L-type Ca2+ channel activity through protein-protein interactions

L-type Ca(2+) channels have a wide tissue distribution and play essential roles in physiological responses. Recent studies have indicated that regulation of L-type Ca(2+) channels involves the assembly of macromolecular signaling complexes such as the beta(2)-adrenergic receptor signaling complex, the small G-protein kir/Gem and the BK channel. Here, we report the previously unidentified role of another protein in binding to the II-III linker of the alpha(1C) subunit of the L-type Ca(2+) channel. This protein is COP9 signalosome subunit 5 (CSN5)/Jun activation domain-binding protein 1 (Jab1). We have demonstrated that CSN5 interacts specifically with the II-III linker of the alpha(1C) subunit in a yeast two-hybrid system. The alpha(1C) subunit and CSN5 were coimmunoprecipitated in rat heart and both proteins were colocalized in sarcolemmal membranes and transverse tubules of cardiac myocytes. Silencing of CSN5 mRNA using siRNA decreased the endogenous protein level of CSN5 and activated L-type Ca(2+) channels expressed in COS7 cells. These data indicate that CSN5 is a protein that plays a newly defined functional role in association with the cardiac L-type Ca(2+) channel.