ATP Regulation of Type-1 Inositol 1,4,5-Trisphosphate Receptor Activity Does Not Require Walker A-type ATP-binding Motifs

ATP is known to increase the activity of the type-1 inositol 1,4,5-trisphosphate receptor (InsP₃R1). This effect is attributed to the binding of ATP to glycine rich Walker A-type motifs present in the regulatory domain of the receptor. Only two such motifs are present in neuronal S2+ splice variant of InsP₃R1 and are designated the ATPA and ATPB sites. The ATPA site is unique to InsP₃R1, and the ATPB site is conserved among all three InsP₃R isoforms. Despite the fact that both the ATPA and ATPB sites are known to bind ATP, the relative contribution of these two sites to the enhancing effects of ATP on InsP₃R1 function is not known. We report here a mutational analysis of the ATPA and ATPB sites and conclude neither of these sites is required for ATP modulation of InsP₃R1. ATP augmented InsP₃-induced Ca²⁺ release from permeabilized cells expressing wild type and ATP-binding site-deficient InsP₃R1. Similarly, ATP increased the single channel open probability of the mutated InsP₃R1 to the same extent as wild type. ATP likely exerts its effects on InsP₃R1 channel function via a novel and as yet unidentified mechanism.Inositol 1,4,5-trisphosphate receptors (InsP₃R)³ are a family of large, tetrameric, InsP₃-gated cation channels. The three members of this family (InsP₃R1, InsP₃R2, and InsP₃R3) are nearly ubiquitously expressed and are localized primarily to the endoplasmic reticulum (ER) membrane (1–3). Numerous hormones, neurotransmitters, and growth factors bind to receptors that stimulate phospholipase C-induced InsP₃ production (4). InsP₃ subsequently binds to the InsP₃R and induces channel opening. This pathway represents a major mechanism for Ca²⁺ liberation from ER stores (5). All three InsP₃R isoforms are dynamically regulated by cytosolic factors in addition to InsP₃ (1). Ca²⁺ is perhaps the most important determinant of InsP₃R activity besides InsP₃ itself and is known to regulate InsP₃R both positively and negatively (6). ATP, in concert with InsP₃ and Ca²⁺, also regulates InsP₃R as do numerous kinases, phosphatases, and protein-binding partners (7–10). This intricate network of regulation allows InsP₃R activity to be finely tuned by the local cytosolic environment (9). As a result, InsP₃-induced Ca²⁺ signals can exhibit a wide variety of spatial and temporal patterns, which likely allows Ca²⁺ to control many diverse cellular processes.Modulation of InsP₃-induced Ca²⁺ release (IICR) by ATP and other nucleotides provides a direct link between intracellular Ca²⁺ signaling and the metabolic state of the cell. Metabolic fluctuations could, therefore, impact Ca²⁺ signaling in many cell types given that InsP₃R are expressed in all cells (11, 12). Consistent with this, ATP has been shown to augment IICR in many diverse cell types including primary neurons (13), smooth muscle cells (14), and exocrine acinar cells (15) as well as in immortalized cell lines (16–18). The effects of ATP on InsP₃R function do not require hydrolysis because non-hydrolyzable ATP analogues are as effective as ATP (7, 14). ATP is thought to bind to distinct regions in the central, coupling domain of the receptors and to facilitate channel opening (2, 19). ATP is not required for channel gating, but instead, increases InsP₃R activity in an allosteric fashion by increasing the open probability of the channel in the presence of activating concentrations of InsP₃ and Ca²⁺ (7, 8, 20).Despite a wealth of knowledge regarding the functional effects of ATP on InsP₃R function, there is relatively little known about the molecular determinants of these actions. ATP is thought to exert effects on channel function by direct binding to glycine-rich regions containing the consensus sequence GXGXXG that are present in the receptors (2). These sequences were first proposed to be ATP-binding domains due to their similarity with Walker A motifs (21). The neuronal S2⁺ splice variant of InsP₃R1 contains two such domains termed ATPA and ATPB. A third site, ATPC, is formed upon removal of the S2 splice site (2, 22). The ATPB site is conserved in InsP₃R2 and InsP₃R3, while the ATPA and ATPC sites are unique to InsP₃R1. Our prior work examining the functional consequences of mutating these ATP-binding sites has yielded unexpected results. For example, mutating the ATPB site in InsP₃R2 completely eliminated the enhancing effects of ATP on this isoform while mutating the analogous site in InsP₃R3 failed to alter the effects of ATP (23). This indicated the presence of an additional locus for ATP modulation of InsP₃R3. In addition, mutation of the ATPC in the S2⁻ splice variant of InsP₃R1 did not alter the ability of ATP to modulate Ca²⁺ release, but instead impaired the ability of protein kinase A to phosphorylate Ser-1755 of this isoform (22).The ATPA and ATPB sites in InsP₃R1 were first identified as putative nucleotide-binding domains after the cloning of the full-length receptor (24). Early binding experiments with 8-azido-[α-³²P]ATP established that ATP cross-linked with receptor purified from rat cerebellum at one site per receptor monomer (19). Later, more detailed, binding experiments on trypsinized recombinant rat InsP₃R1 showed cross-linking of ATP to two distinct regions of the receptor that corresponded with the ATPA and ATPB sites (17). We and others (16, 22, 23) have also reported the binding of ATP analogues to purified GST fusions of small regions of InsP₃R1 surrounding the ATPA and ATPB sites. It is widely accepted, in the context of the sequence similarity to Walker A motifs and biochemical data, that the ATPA and ATPB sites are the loci where ATP exerts its positive functional effects on InsP₃R1 function (1–3, 16). Furthermore, the higher affinity of the ATPA site to ATP is thought to confer the higher sensitivity of InsP₃R1 to ATP versus InsP₃R3, which contains the ATPB site exclusively (25, 26). The purpose of this study, therefore, was to examine the contributions of the ATPA and ATPB sites to ATP modulation of the S2⁺ splice variant of InsP₃R1. We compared the effects of ATP on InsP₃R1 and on ATP-binding site mutated InsP₃R1 using detailed functional analyses in permeabilized cells and in single channel recordings. Here we report that InsP₃R1 is similar to InsP₃R3 in that ATP modulates IICR even at maximal InsP₃ concentrations and that neither the ATPA nor the ATPB site is required for this effect.     

TMEM16 Proteins Produce Volume-regulated Chloride Currents That Are Reduced in Mice Lacking TMEM16A

All vertebrate cells regulate their cell volume by activating chloride channels of unknown molecular identity, thereby activating regulatory volume decrease. We show that the Ca²⁺-activated Cl⁻ channel TMEM16A together with other TMEM16 proteins are activated by cell swelling through an autocrine mechanism that involves ATP release and binding to purinergic P2Y₂ receptors. TMEM16A channels are activated by ATP through an increase in intracellular Ca²⁺ and a Ca²⁺-independent mechanism engaging extracellular-regulated protein kinases (ERK1/2). The ability of epithelial cells to activate a Cl⁻ conductance upon cell swelling, and to decrease their cell volume (regulatory volume decrease) was dependent on TMEM16 proteins. Activation of I_Cl,swell was reduced in the colonic epithelium and in salivary acinar cells from mice lacking expression of TMEM16A. Thus TMEM16 proteins appear to be a crucial component of epithelial volume-regulated Cl⁻ channels and may also have a function during proliferation and apoptotic cell death.Regulation of cell volume is fundamental to all cells, particularly during cell growth and division. External hypotonicity leads to cell swelling and subsequent activation of volume-regulated chloride and potassium channels, to release intracellular ions and to re-shrink the cells, a process termed regulatory volume decrease (RVD)³ (1). Volume-regulated chloride currents (I_Cl,swell) have dual functions during cell proliferation as well as apoptotic volume decrease (AVD), preceding apoptotic cell death (2). Although I_Cl,swell is activated in swollen cells to induce RVD, AVD takes place under normotonic conditions to shrink cells (3, 4). Early work suggested intracellular Ca²⁺ as an important mediator for activation of I_Cl,swell and volume-regulated K⁺ channels (5), whereas subsequent studies only found a permissive role of Ca²⁺ for activation of I_Cl,swell (6), reviewed in Ref. 1. In addition, a plethora of factors and signaling pathways have been implicated in activation of I_Cl,swell, making cell volume regulation an extremely complex process (reviewed in Refs. 1, 3, and 7). These factors include intracellular ATP, the cytoskeleton, phospholipase A2-dependent pathways, and protein kinases such as extracellular-regulated kinase ERK1/2 (reviewed in Refs. 1 and 7). Previous approaches in identifying swelling-activated Cl⁻ channels have been unsuccessful or have produced controversial data. Thus none of the previous candidates such as pI_Cln, the multidrug resistance protein, or ClC-3 are generally accepted to operate as volume-regulated Cl⁻ channels (reviewed in Refs. 8 and 9). Notably, the cystic fibrosis transmembrane conductance regulator (CFTR) had been shown in earlier studies to influence I_Cl,swell and volume regulation (10–12). The variable properties of I_Cl,swell suggest that several gene products may affect I_Cl,swell in different cell types.The TMEM16 transmembrane protein family consists of 10 different proteins with numerous splice variants that contain 8–9 transmembrane domains and have predicted intracellular N- and C-terminal tails (13, 16–18). TMEM16A (also called ANO1) is required for normal development of the murine trachea (14) and is associated with different types of tumors, dysplasia, and nonsyndromic hearing impairment (13, 15). TMEM16A has been identified as a subunit of Ca²⁺-activated Cl⁻ channels that are expressed in epithelial and non-epithelial tissues (16–18). Interestingly, members of the TMEM16 family have been suggested to play a role in osmotolerance in Saccharomyces cerevisiae (19). Here we show that TMEM16 proteins also contribute to I_Cl,swell and regulatory volume decrease.     

Protein-tyrosine Phosphatase-�� and Src Functionally Link Focal Adhesions to the Endoplasmic Reticulum to Mediate Interleukin-1-induced Ca2+ Signaling

Calcium (Ca²⁺) signaling by the pro-inflammatory cytokine interleukin-1 (IL-1) is dependent on focal adhesions, which contain diverse structural and signaling proteins including protein phosphatases. We examined here the role of protein-tyrosine phosphatase (PTP) α in regulating IL-1-induced Ca²⁺ signaling in fibroblasts. IL-1 promoted recruitment of PTPα to focal adhesions and endoplasmic reticulum (ER) fractions, as well as tyrosine phosphorylation of the ER Ca²⁺ release channel IP₃R. In response to IL-1, catalytically active PTPα was required for Ca²⁺ release from the ER, Src-dependent phosphorylation of IP₃R1 and accumulation of IP₃R1 in focal adhesions. In pulldown assays and immunoprecipitations PTPα was required for the association of PTPα with IP₃R1 and c-Src, and this association was increased by IL-1. Collectively, these data indicate that PTPα acts as an adaptor to mediate functional links between focal adhesions and the ER that enable IL-1-induced Ca²⁺ signaling.The interleukin-1 (IL-1)³ family of pro-inflammatory cytokines mediates host responses to infection and injury. Impaired control of IL-1 signaling leads to chronic inflammation and destruction of extracellular matrices (1, 2), as seen in pathological conditions such as pulmonary fibrosis (3), rheumatoid arthritis (4, 5), and periodontitis (6). IL-1 elicits multiple signaling programs, some of which trigger Ca²⁺ release from the endoplasmic reticulum (ER) as well as expression of multiple cytokines and inflammatory factors including c-Fos and c-Jun (7, 8), and matrix metalloproteinases (9, 10), which mediate extracellular matrix degradation via mitogen-activated protein kinase-regulated pathways (11).In anchorage-dependent cells including fibroblasts and chondrocytes, focal adhesions (FAs) are required for IL-1-induced Ca²⁺ release from the ER and activation of ERK (12–14). FAs are actin-enriched adhesive domains composed of numerous (>50) scaffolding and signaling proteins (15–17). Many FA proteins are tyrosine-phosphorylated, including paxillin, focal adhesion kinase, and src family kinases, all of which are crucial for the assembly and disassembly of FAs (18–21). Protein-tyrosine phosphorylation plays a central role in regulating many cellular processes including adhesion (22, 23), motility (24), survival (25), and signal transduction (26–29). Phosphorylation of proteins by kinases is balanced by protein-tyrosine phosphatases (PTP), which can enhance or attenuate downstream signaling by dephosphorylation of tyrosine residues (30–32).PTPs can be divided into two main categories: receptor-like and intracellular PTPs (33). Two receptor-like PTPs have been localized to FA (leukocyte common antigen-related molecule and PTPα). Leukocyte common antigen-related molecule can dephosphorylate and mediate degradation of p130^cas, which ultimately leads to cell death (34, 35). PTPα contains a heavily glycosylated extracellular domain, a transmembrane domain, and two intracellular phosphatase domains (33, 36). The amino-terminal domain predominantly mediates catalytic activity, whereas the carboxyl-terminal domain serves a regulatory function (37, 38). PTPα is enriched in FA (23) and is instrumental in regulating FA dynamics (39) via activation of c-Src/Fyn kinases by dephosphorylating the inhibitory carboxyl tyrosine residue, namely Tyr⁵²⁹ (22, 40–42) and facilitation of integrin-dependent assembly of Src-FAK and Fyn-FAK complexes that regulate cell motility (43). Although PTPα has been implicated in formation and remodeling of FAs (44, 45), the role of PTPα in FA-dependent signaling is not defined.Ca²⁺ release from the ER is a critical step in integrin-dependent IL-1 signal transduction and is required for downstream activation of ERK (13, 46). The release of Ca²⁺ from the ER depends on the inositol 1,4,5-triphosphate receptor (IP₃R), which is an IP₃-gated Ca²⁺ channel (47). All of the IP₃R subtypes (subtypes 1–3) have been localized to the ER, as well as other the plasma membrane and other endomembranes (48–50). Further, IP₃R may associate with FAs, enabling the anchorage of the ER to FAs (51, 52). However, the molecule(s) that provide the structural link for this association has not been defined.FA-restricted, IL-1-triggered signal transduction in anchorage-dependent cells may rely on interacting proteins that are enriched in FAs and the ER (53). Here, we examined the possibility that PTPα associates with c-Src and IP₃R to functionally link FAs to the ER, thereby enabling IL-1 signal transduction.     

CD38/cADPR/Ca2+ Pathway Promotes Cell Proliferation and Delays Nerve Growth Factor-induced Differentiation in PC12 Cells

Intracellular Ca²⁺ mobilization plays an important role in a wide variety of cellular processes, and multiple second messengers are responsible for mediating intracellular Ca²⁺ changes. Here we explored the role of one endogenous Ca²⁺-mobilizing nucleotide, cyclic adenosine diphosphoribose (cADPR), in the proliferation and differentiation of neurosecretory PC12 cells. We found that cADPR induced Ca²⁺ release in PC12 cells and that CD38 is the main ADP-ribosyl cyclase responsible for the acetylcholine (ACh)-induced cADPR production in PC12 cells. In addition, the CD38/cADPR signaling pathway is shown to be required for the ACh-induced Ca²⁺ increase and cell proliferation. Inhibition of the pathway, on the other hand, accelerated nerve growth factor (NGF)-induced neuronal differentiation in PC12 cells. Conversely, overexpression of CD38 increased cell proliferation but delayed NGF-induced differentiation. Our data indicate that cADPR plays a dichotomic role in regulating proliferation and neuronal differentiation of PC12 cells.Mobilization of intracellular Ca²⁺ stores is involved in diverse cell functions, including fertilization, cell proliferation, and differentiation (1–4). At least three endogenous Ca²⁺-mobilizing messengers have been identified, including inositol trisphosphate (IP₃),³ nicotinic adenine acid dinucleotide phosphate (NAADP), and cyclic adenosine diphosphoribose (cADPR). Similar to IP₃, cADPR can mobilize calcium release in a wide variety of cell types and species, from protozoa to animals. The cADPR-mediated Ca²⁺ signaling has been indicated in a variety of cellular processes (5–7), from abscisic acid signaling and regulation of the circadian clock in plants, to mediating long-term synaptic depression in hippocampus.Ample evidence shows that the ryanodine receptors are the main intracellular targets for cADPR (1, 2, 8). Ryanodine receptors (RyRs) are intracellular Ca²⁺ channels widely expressed in various cells and tissues, including muscles and neurons. It is the major cellular mediator of Ca²⁺-induced Ca²⁺ release (CICR) in cells. There are three isoforms of ryanodine receptors: RyR1, RyR2, and RyR3, all of which have been implicated in the cADPR signaling (1, 2, 8). However, evidence regarding cADPR acting directly on the receptors is lacking (9). It has been suggested that accessory proteins, such as calmodulin and FK506-binding protein (FKBP), may be involved instead (10–15).cADPR is formed from nicotinamide adenine dinucleotide (NAD) by ADP-ribosyl cyclases. Six ADP-ribosyl cyclases have been identified so far: Aplysia ADP-ribosyl cyclase, three sea urchin homologues (16, 17), and two mammalian homologues, CD38 and CD157 (18). CD38 is a membrane-bound protein and the main mammalian ADP-ribosyl cyclase. As a novel multifunctional enzyme, CD38 catalyzes the synthesis and hydrolysis of both cADPR and NAADP, two structurally and functionally distinct Ca²⁺ messengers. Virtually all mammalian tissues ever examined have been shown to express CD38. CD38 knock-out mice exhibit multiple physiological defects, ranging from impaired immune responses, metabolic disturbances, to behavioral modifications (1, 6, 18).CD38 was originally identified as a lymphocyte differentiation antigen (18). Indeed, CD38/cADPR has been linked to cell differentiation (5). For example, in human HL-60 cells, CD38 expression and the consequential accumulation of cADPR play a causal role in mediating granulocytic differentiation (19). In addition, expression of CD38 in HeLa and 3T3 cells not only increased intracellular Ca²⁺ concentration but also induced cell proliferation by significantly reducing the S phase duration, leading to shortened cell doubling time (20). The ability of cADPR to increase cell proliferation has also been observed in human T cells (21), human hemopoietic progenitors (22), human peripheral blood mononuclear cells (23), human mesenchymal stem cells (24), and murine mesangial cells (25).The PC12 cell line was derived from rat adrenal medulla and has been used extensively as a neuronal model, since it exhibits many of the functions observed in primary neuronal cultures (26). Most importantly, PC12 cells can be induced by nerve growth factor (NGF) to differentiate into cells with extensive neurite outgrowths, resembling neuronal dendritic trees (26, 27). In contrast to NGF, numerous growth factors and neurotransmitters can induce the proliferation of PC12 cells instead (26). Both IP₃ receptor- and ryanodine receptor-mediated Ca²⁺ stores have been shown to be present in PC12 cells (28–31). The type 2 ryanodine receptor is expressed in PC12 cells and activation of the NO/cGMP pathway in PC12 cells results in calcium mobilization, which is mediated by cADPR and similar to that seen in sea urchin eggs (32). It has been demonstrated that NAADP, another Ca²⁺-mobilizing messenger, is also a potent neuronal differentiation inducer in PC12 cells, while IP₃ exhibits no such role (33, 34). Whether cADPR is involved in the proliferation and differentiation of PC12 cells is unknown.Here we show that activation of the CD38/cADPR/Ca²⁺ signaling is required for the ACh-induced proliferation in PC12 cells, while inhibition of the pathway accelerates NGF-induced neuronal differentiation. Our data indicate that cADPR is important in regulating cell proliferation and neuronal differentiation in PC12 cells.     

Calcium Elevation in Mitochondria Is the Main Ca2+ Requirement for Mitochondrial Permeability Transition Pore (mPTP) Opening

We have investigated in detail the role of intra-organelle Ca²⁺ content during induction of apoptosis by the oxidant menadione while changing and monitoring the Ca²⁺ load of endoplasmic reticulum (ER), mitochondria, and acidic organelles. Menadione causes production of reactive oxygen species, induction of oxidative stress, and subsequently apoptosis. In both pancreatic acinar and pancreatic tumor AR42J cells, menadione was found to induce repetitive cytosolic Ca²⁺ responses because of the release of Ca²⁺ from both ER and acidic stores. Ca²⁺ responses to menadione were accompanied by elevation of Ca²⁺ in mitochondria, mitochondrial depolarization, and mitochondrial permeability transition pore (mPTP) opening. Emptying of both the ER and acidic Ca²⁺ stores did not necessarily prevent menadione-induced apoptosis. High mitochondrial Ca²⁺ at the time of menadione application was the major factor determining cell fate. However, if mitochondria were prevented from loading with Ca²⁺ with 10 μm RU360, then caspase-9 activation did not occur irrespective of the content of other Ca²⁺ stores. These results were confirmed by ratiometric measurements of intramitochondrial Ca²⁺ with pericam. We conclude that elevated Ca²⁺ in mitochondria is the crucial factor in determining whether cells undergo oxidative stress-induced apoptosis.Apoptosis, a mechanism of programmed cell death, usually occurs through intrinsic or extrinsic apoptotic pathways. The caspases involved in apoptosis can be split into two groups, the initiator caspases such as caspase-9 and effector caspases such as caspase-3. Effector caspases are activated by initiator caspases and mediate many of the morphological cellular changes associated with apoptosis (1).Calcium is an important signaling ion involved in the regulation of many physiological as well as pathological cellular responses (2). In the pancreas, we have shown that Ca²⁺ signals elicit enzyme secretion (3), apoptosis (4–6), and pathological intracellular activation of digestive enzymes (7). As such, there must be mechanisms in place by which the cell can differentiate between apoptotic and non-apoptotic Ca²⁺ signals.The spatiotemporal pattern of calcium signaling is crucial for the specificity of cellular responses. For example, repetitive cytosolic calcium spikes confined to the apical region of the pancreatic acinar cell are elicited by physiological stimulation with acetylcholine (ACh) or cholecystokinin (CCK) and result in physiological secretion of zymogen granules (8, 9). However, a sustained global increase in free cytosolic Ca²⁺ induced by supramaximal stimulation with CCK, which resembles prolonged hyperstimulation of pancreatic acinar cells in the pathophysiology of acute pancreatitis, can lead to premature activation of digestive enzymes and vacuole formation within the cell (10–12). Alternatively, global repetitive calcium spikes induced in the pancreatic acinar cell in response to oxidant stress can lead to induction of the mitochondrial permeability transition pore (mPTP)⁴ and apoptosis (4, 5, 13).To understand the role of calcium in apoptosis, several investigators have examined the influence of intracellular stores on the molding of calcium signals that lead to cell death (14–16). It has been well established in a range of cell types that the endoplasmic reticulum (ER) is the major intracellular calcium store required for induction of apoptosis. Pinton et al. (17) have shown that decreasing ER Ca²⁺ concentration with tBuBHQ increased HeLa cell survival in response to oxidant stress induced by ceramide. Scorrano and Korsmeyer (18) also observed that double knock-out Bax and Bak (pro-apoptotic proteins) mouse fibroblasts displayed a reduced resting concentration of ER Ca²⁺ compared with wild type and were resistant to induction of apoptosis by various stimulants, including ceramide. These important studies strongly suggest that the concentration of Ca²⁺ in the ER is a critical determinant of cellular susceptibility to apoptotic stimuli in the cell types studied.A key event in early apoptosis is permeabilization of the mitochondrial membrane. The mPTP is a pore whose molecular composition is still debated (19). Activation of an open pore state can result in swelling of the mitochondrial matrix and release of the apoptogenic proteins from the intermembrane space (20).One important activator of the mPTP is Ca²⁺ (20–22), a function which implicates Ca²⁺ in the initiation of apoptosis (23, 24). Once Ca²⁺ is released from the ER into the cytoplasm, mitochondria take up part of the released Ca²⁺ to prevent propagation of large calcium waves (25–27). This influx is followed by calcium efflux from the mitochondria back into the cytosol (28, 29). An increase in mitochondrial Ca²⁺ concentration in response to physiological stimuli induces increased activity of the mitochondrial respiratory chain and the synthesis of ATP to meet with increasing energy demands on the cell. When mitochondria are exposed to a pathological overload of calcium, opening of the mPTP is triggered, leading to mitochondrial dysfunction and eventually cell death. The mechanism through which calcium can trigger mPTP opening is still unclear and may involve cyclophilin D (30) and voltage-dependent anion channel (31). The mitochondria are endowed with selective and efficient calcium uptake (a calcium-selective uniporter) and release mechanisms (Ca²⁺/Na exchanger, Ca²⁺/H⁺ exchanger, and mPTP) (16, 29, 32, 33).Oxidant stress is a well known inducer of apoptosis in several cell types (34) and is thought to play an important role in the pathogenesis of acute pancreatitis (35). We have used the quinone compound menadione to induce oxidative stress in the pancreatic acinar cell. Menadione is metabolized by flavoprotein reductase to semiquinone and then is oxidized back to quinone, resulting in generation of superoxide anion radicals, hydrogen peroxide, and other reactive oxygen species (ROS) (36). In vivo, menadione causes depolarization and swelling of the mitochondria (37). In pancreatic acinar cells, treatment with menadione not only produces an increase in ROS, but has also been found to evoke cytosolic Ca²⁺ responses, mPTP opening, activation of caspases and apoptotic cell death (4, 5). When cells were pretreated with the calcium chelator BAPTA-AM, menadione was unable to induce apoptosis, indicating that oxidant stress-induced apoptosis in the pancreatic acinar cell is highly calcium-dependent. Here we show that in pancreatic acinar cells, oxidative stress-induced apoptosis is strongly dependent on the Ca²⁺ concentration within mitochondria at the time of ROS production.     

Signaling Processes for Initiating Smooth Muscle Contraction upon Neural Stimulation

Relationships among biochemical signaling processes involved in Ca²⁺/calmodulin (CaM)-dependent phosphorylation of smooth muscle myosin regulatory light chain (RLC) by myosin light chain kinase (MLCK) were determined. A genetically-encoded biosensor MLCK for measuring Ca²⁺-dependent CaM binding and activation was expressed in smooth muscles of transgenic mice. We performed real-time evaluations of the relationships among [Ca²⁺]_i, MLCK activation, and contraction in urinary bladder smooth muscle strips neurally stimulated for 3 s. Latencies for the onset of [Ca²⁺]_i and kinase activation were 55 ± 8 and 65 ± 6 ms, respectively. Both increased with RLC phosphorylation at 100 ms, whereas force latency was 109 ± 3 ms. [Ca²⁺]_i, kinase activation, and RLC phosphorylation responses were maximal by 1.2 s, whereas force increased more slowly to a maximal value at 3 s. A delayed temporal response between RLC phosphorylation and force is probably due to mechanical effects associated with elastic elements in the tissue. MLCK activation partially declined at 3 s of stimulation with no change in [Ca²⁺]_i and also declined more rapidly than [Ca²⁺]_i during relaxation. The apparent desensitization of MLCK to Ca²⁺ activation appears to be due to phosphorylation in its calmodulin binding segment. Phosphorylation of two myosin light chain phosphatase regulatory proteins (MYPT1 and CPI-17) or a protein implicated in strengthening membrane adhesion complexes for force transmission (paxillin) did not change during force development. Thus, neural stimulation leads to rapid increases in [Ca²⁺]_i, MLCK activation, and RLC phosphorylation in phasic smooth muscle, showing a tightly coupled Ca²⁺ signaling complex as an elementary mechanism initiating contraction.Increases in [Ca²⁺]_i3 in smooth muscle cells lead to Ca²⁺/CaM-dependent MLCK activation and RLC phosphorylation. Phosphorylation of RLC increases actin-activated myosin MgATPase activity leading to myosin cross-bridge cycling with force development (1–3).The activation of smooth muscle contraction may be affected by multiple cellular processes. Previous investigations show that free Ca²⁺/CaM is limiting for kinase activation despite the abundance of total CaM (4–6). The extent of RLC phosphorylation is balanced by the actions of MLCK and myosin light chain phosphatase, which is composed of three distinct protein subunits (7). The myosin phosphatase targeting subunit, MYPT1, in smooth muscle binds to myosin filaments, thus targeting the 37-kDa catalytic subunit (type 1 serine/threonine phosphatase, PP1c) to phosphorylated RLC. RLC phosphorylation and muscle force may be regulated by additional signaling pathways involving phosphorylation of RLC by Ca²⁺-independent kinase(s) and inhibition of myosin light chain phosphatase, processes that increase the contraction response at fixed [Ca²⁺]_i (Ca²⁺-sensitization) (8–14). Many studies indicate that agonist-mediated Ca²⁺-sensitization most often reflects decreased myosin light chain phosphatase activity involving two major pathways including MYPT1 phosphorylation by a Rho kinase pathway and phosphorylation of CPI-17 by PKC (8, 14–16). Additionally, phosphorylation of MLCK in its calmodulin-binding sequence by a Ca²⁺/calmodulin-dependent kinase pathway has been implicated in Ca²⁺ desensitization of RLC phosphorylation (17–19). How these signaling pathways intersect the responses of the primary Ca²⁺/CaM pathway during physiological neural stimulation is not known.There is also evidence that smooth muscle contraction requires the polymerization of submembranous cytoskeletal actin filaments to strengthen membrane adhesion complexes involved in transmitting force between actin-myosin filaments and external force-transmitting structures (20–23). In tracheal smooth muscle, paxillin at membrane adhesions undergoes tyrosine phosphorylation in response to contractile stimulation by an agonist, and this phosphorylation increases concurrently with force development in response to agonist. Expression of nonphosphorylatable paxillin mutants in tracheal muscle suppresses acetylcholine-induced tyrosine phosphorylation of paxillin, tension development, and actin polymerization without affecting RLC phosphorylation (24, 25). Thus, paxillin phosphorylation may play an important role in tension development in smooth muscle independently of RLC phosphorylation and cross-bridge cycling.Specific models relating signaling mechanisms in the smooth muscle cell to contraction dynamics are limited when cells in tissues are stimulated slowly and asynchronously by agonist diffusing into the preparation. Field stimulation leading to the rapid release of neurotransmitters from nerves embedded in the tissue avoids these problems associated with agonist diffusion (26, 27). In urinary bladder smooth muscle, phasic contractions are brought about by the parasympathetic nervous system. Upon activation, parasympathetic nerve varicosities release the two neurotransmitters, acetylcholine and ATP, that bind to muscarinic and purinergic receptors, respectively. They cause smooth muscle contraction by inducing Ca²⁺ transients as elementary signals in the process of nerve-smooth muscle communication (28–30). We recently reported the development of a genetically encoded, CaM-sensor for activation of MLCK. The CaM-sensor MLCK contains short smooth muscle MLCK fused to two fluorophores, enhanced cyan fluorescent protein and enhanced yellow fluorescent protein, linked by the MLCK calmodulin binding sequence (6, 14, 31). Upon dimerization, there is significant FRET from the donor enhanced cyan fluorescent protein to the acceptor enhanced yellow fluorescent protein. Ca²⁺/CaM binding dissociates the dimer resulting in a decrease in FRET intensity coincident with activation of kinase activity (31). Thus, CaM-sensor MLCK is capable of directly monitoring Ca²⁺/CaM binding and activation of the kinase in smooth muscle tissues where it is expressed specifically in smooth muscle cells of transgenic mice. We therefore combined neural stimulation with real-time measurements of [Ca²⁺]_i, MLCK activation, and force development in smooth muscle tissue from these mice. Additionally, RLC phosphorylation was measured precisely at specific times following neural stimulation in tissues frozen by a rapid-release electronic freezing device (26, 27). Results from these studies reveal that physiological stimulation of smooth muscle cells by neurotransmitter release leads to rapid increases in [Ca²⁺]_i, MLCK activation, and RLC phosphorylation at similar rates without the apparent activities of Ca²⁺-independent kinases, inhibition of myosin light chain phosphatase, or paxillin phosphorylation. Thus, the elemental processes for phasic smooth muscle contraction are represented by this tightly coupled Ca²⁺ signaling complex.     

Determinants in CaV1 Channels That Regulate the Ca2+ Sensitivity of Bound Calmodulin

Calmodulin binds to IQ motifs in the α₁ subunit of Ca_V1.1 and Ca_V1.2, but the affinities of calmodulin for the motif and for Ca²⁺ are higher when bound to Ca_V1.2 IQ. The Ca_V1.1 IQ and Ca_V1.2 IQ sequences differ by four amino acids. We determined the structure of calmodulin bound to Ca_V1.1 IQ and compared it with that of calmodulin bound to Ca_V1.2 IQ. Four methionines in Ca²⁺-calmodulin form a hydrophobic binding pocket for the peptide, but only one of the four nonconserved amino acids (His-1532 of Ca_V1.1 and Tyr-1675 of Ca_V1.2) contacts this calmodulin pocket. However, Tyr-1675 in Ca_V1.2 contributes only modestly to the higher affinity of this peptide for calmodulin; the other three amino acids in Ca_V1.2 contribute significantly to the difference in the Ca²⁺ affinity of the bound calmodulin despite having no direct contact with calmodulin. Those residues appear to allow an interaction with calmodulin with one lobe Ca²⁺-bound and one lobe Ca²⁺-free. Our data also provide evidence for lobe-lobe interactions in calmodulin bound to Ca_V1.2.The complexity of eukaryotic Ca²⁺ signaling arises from the ability of cells to respond differently to Ca²⁺ signals that vary in amplitude, duration, and location. A variety of mechanisms decode these signals to drive the appropriate physiological responses. The Ca²⁺ sensor for many of these physiological responses is the Ca²⁺-binding protein calmodulin (CaM).² The primary sequence of CaM is tightly conserved in all eukaryotes, yet it binds and regulates a broad set of target proteins in response to Ca²⁺ binding. CaM has two domains that bind Ca²⁺ as follows: an amino-terminal domain (N-lobe) and a carboxyl-terminal domain (C-lobe) joined via a flexible α-helix. Each lobe of CaM binds two Ca²⁺ ions, and binding within each lobe is highly cooperative. The two lobes of CaM, however, have distinct Ca²⁺ binding properties; the C-lobe has higher Ca²⁺ affinity because of a slower rate of dissociation, whereas the N-lobe has weaker Ca²⁺ affinity and faster kinetics (1). CaM can also bind to some target proteins in both the presence and absence of Ca²⁺, and the preassociation of CaM in low Ca²⁺ modulates the apparent Ca²⁺ affinity of both the amino-terminal and carboxyl-terminal lobes. Differences in the Ca²⁺ binding properties of the lobes and in the interaction sites of the amino- and carboxyl-terminal lobes enable CaM to decode local versus global Ca²⁺ signals (2).Even though CaM is highly conserved, CaM target (or recognition) sites are quite heterogeneous. The ability of CaM to bind to very different targets is at least partially due to its flexibility, which allows it to assume different conformations when bound to different targets. CaM also binds to various targets in distinct Ca²⁺ saturation states as follows: Ca²⁺-free (3), Ca²⁺ bound to only one of the two lobes, or fully Ca²⁺-bound (4–7). In addition, CaM may bind with both lobes bound to a target (5, 6) or with only a single lobe engaged (8). If a target site can bind multiple conformers of CaM, CaM may undergo several transitions that depend on Ca²⁺ concentration, thereby tuning the functional response. Identification of stable intermediate states of CaM bound to individual targets will help to elucidate the steps involved in this fine-tuned control.Both Ca_V1.1 and Ca_V1.2 belong to the L-type family of voltage-dependent Ca²⁺ channels, which bind apoCaM and Ca²⁺-CaM at carboxyl-terminal recognition sites in their α₁ subunits (9–14). Ca²⁺ binding to CaM, bound to Ca_V1.2 produces Ca²⁺-dependent facilitation (CDF) (14). Whether Ca_V1.1 undergoes CDF is not known. However, both Ca_V1.2 and Ca_V1.1 undergo Ca²⁺- and CaM-dependent inactivation (CDI) (14, 15). Ca_V1.1 CDI is slower and more sensitive to buffering by 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid than Ca_V1.2 CDI (15). Ca²⁺ buffers are thought to influence CDI and/or CDF in voltage-dependent Ca²⁺ channels by competing with CaM for Ca²⁺ (16).The conformation of the carboxyl terminus of the α₁ subunit is critical for channel function and has been proposed to regulate the gating machinery of the channel (17, 18). Several interactions of this region include intramolecular contacts with the pore inactivation machinery and intermolecular contacts with CaM kinase II and ryanodine receptors (17, 19–22). Ca²⁺ regulation of Ca_V1.2 may involve several motifs within this highly conserved region, including an EF hand motif and three contiguous CaM-binding sequences (10, 12). ApoCaM and Ca²⁺-CaM-binding sites appear to overlap at the site designated as the “IQ motif” (9, 12, 13), which are critical for channel function at the molecular and cellular level (14, 23).Differences in the rate at which 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid affects CDI of Ca_V1.1 and Ca_V1.2 could reflect differences in their interactions with CaM. In this study we describe the differences in CaM interactions with the IQ motifs of the Ca_V1.1 and the Ca_V1.2 channels in terms of crystal structure, CaM affinity, and Ca²⁺ binding to CaM. We find the structures of Ca²⁺-CaM-IQ complexes are similar except for a single amino acid change in the peptide that contributes to its affinity for CaM. We also find that the other three amino acids that differ in Ca_V1.2 and Ca_V1.1 contribute to the ability of Ca_V1.2 to bind a partially Ca²⁺-saturated form of CaM.     

The TRPV4 Cation Channel Mediates Stretch-evoked Ca2+ Influx and ATP Release in Primary Urothelial Cell Cultures

Transient receptor potential channels have recently been implicated in physiological functions in a urogenital system. In this study, we investigated the role of transient receptor potential vanilloid 4 (TRPV4) channels in a stretch sensing mechanism in mouse primary urothelial cell cultures. The selective TRPV4 agonist, 4α-phorbol 12,13-didecanoate (4α-PDD) evoked Ca²⁺ influx in wild-type (WT) urothelial cells, but not in TRPV4-deficient (TRPV4KO) cells. We established a cell-stretch system to investigate stretch-evoked changes in intracellular Ca²⁺ concentration and ATP release. Stretch stimulation evoked intracellular Ca²⁺ increases in a stretch speed- and distance-dependent manner in WT and TRPV4KO cells. In TRPV4KO urothelial cells, however, the intracellular Ca²⁺ increase in response to stretch stimulation was significantly attenuated compared with that in WT cells. Stretch-evoked Ca²⁺ increases in WT urothelium were partially reduced in the presence of ruthenium red, a broad TRP channel blocker, whereas that in TRPV4KO cells did not show such reduction. Potent ATP release occurred following stretch stimulation or 4α-PDD administration in WT urothelial cells, which was dramatically suppressed in TRPV4KO cells. Stretch-dependent ATP release was almost completely eliminated in the presence of ruthenium red or in the absence of extracellular Ca²⁺. These results suggest that TRPV4 senses distension of the bladder urothelium, which is converted to an ATP signal in the micturition reflex pathway during urine storage.Transient receptor potential vanilloid 4 (TRPV4),³ a member of the TRP superfamily of cation channels, is a Ca²⁺-permeable channel activated by a wide variety of physical and chemical stimuli (1, 2). TRPV4 was originally viewed as an osmo- or mechano-sensor, because the channel opens in response to hypotonicity-induced cell swelling (3–5) and shear stress (6). Alternatively, TRPV4 can be activated by diverse chemical stimuli such as synthetic phorbol ester 4α-phorbol 12,13-didecanoate (4α-PDD) (7), a botanical agent (bisandrographolide A), anandamide metabolites such as arachidonic acid and epoxyeicosatrienoic acids, as well as moderate warmth (>27 °C) (8–10). TRPV4 is widely expressed throughout the body, including renal epithelium, auditory hair cells, skin keratinocytes, hippocampus neurons, endothelial cells, and urinary bladder epithelium, thereby contributing to numerous physiological processes such as osmoregulation (11, 12), hearing (13), thermal and mechanical hyperalgesia (14, 15), neural activity in the brain (16), skin barrier recovery (17), and cell volume regulation (18). Therefore, the TRPV4 channel is now considered a multimodal transducer in various tissues and cells.Non-neuronal cells within the urinary bladder wall (notably the transitional epithelial cells (urothelial cells)) function as a barrier against ions, solutes, and infection and also participate in the detection of physical and chemical stimuli (19–21). The urothelium expresses various sensory receptors and channels (bradykinin receptors, adrenergic/cholinergic receptors, nerve growth factor receptors, purinergic receptors, amiloride-sensitive Na⁺ channels, and TRP channels), all of which are substantially implicated in modulating bladder functions (22).Recently, the potential roles of TRP channels have been explored in the bladder. Thus far, expression of TRPV1, TRPV2, TRPV4, TRPA1, and TRPM8 has been reported in different regions of urogenital tracts (21). TRPV1 is reportedly expressed in the epithelial cells lining the urothelium, in interstitial cells, and in sensory nerve terminals. TRPV1-deficient mice displayed a higher frequency of low amplitude non-voiding bladder contractions in comparison with wild-type (WT) mice (22), suggesting that TRPV1 is required for detection of bladder stretch, which involves stretch-evoked release of ATP and nitric oxide. The release of both mediators was reduced in the bladders of TRPV1-deficient mice. In a clinical setting, capsaicin or resiniferatoxin reduces bladder overactivity through desensitization of bladder afferents by acting on TRPV1 (23). Expression of other TRP channels, e.g. TRPM8 and TRPA1, was found in sensory C fibers in the bladder (24–27). The diagnostic ice water test is utilized to determine whether disturbance of bladder function involves neurogenic components, one of which could be related to TRPM8 function, in patients with spinal cord lesion (28). TRPA1 in sensory afferents is activated by several known ligands (allyl isothiocyanate and cinnamaldehyde), thereby inducing bladder overactivity (26). TRPV2 is expressed by several cell types in the rat bladder (29); however, its physiological function has not yet been investigated. TRPV4 is expressed in the urothelium and in smooth muscle cells of the urinary bladder (30, 31). Activation of the channel by specific ligands leads to augmentation of bladder contraction amplitude in cystometry and induction of bladder overactivity in vivo. In a separate cystometry analysis in conjunction with behavioral experiments, the intermicturitional interval was elongated and storage urine volume was increased in TRPV4-deficient mice compared with WT mice (32). Thus, TRPV4 may contribute to bladder function, especially to mediating bladder distention signals to primary afferent nerves during urine storage. However, whether urothelial TRPV4 is required for sensing mechanical stretch, or to what extent urothelial TRPV4 contributes to stretch-evoked ATP release, has not been precisely determined.In the present study, we examined the functional contribution of TRPV4 to stretch-dependent urothelial cell responses and stretch-evoked ATP release in vitro. We first established a primary cell culture for mouse urothelium and retention of TRPV4 expression was confirmed. Because urothelial cells are physically extended during urine storage in vivo, we reproduced this phenomenon in an in vitro experiment using the uni-axial cell stretch system. All the experiments were performed by comparing urothelial cells obtained from WT mice and TRPV4-deficient mice to evaluate the correlation between TRPV4 expression and stretch responses. We demonstrated that urothelial cells sense mechanical stretch stimuli via TRPV4 channels, which induces robust Ca²⁺ influx and contributes to ATP release upon extension.     

Adiponectin Activates AMP-activated Protein Kinase in Muscle Cells via APPL1/LKB1-dependent and Phospholipase C/Ca2+/Ca2+/Calmodulin-dependent Protein Kinase Kinase-dependent Pathways

The binding of the adaptor protein APPL1 to adiponectin receptors is necessary for adiponectin-induced AMP-activated protein kinase (AMPK) activation in muscle, yet the underlying molecular mechanism remains unknown. Here we show that in muscle cells adiponectin and metformin induce AMPK activation by promoting APPL1-dependent LKB1 cytosolic translocation. APPL1 mediates adiponectin signaling by directly interacting with adiponectin receptors and enhances LKB1 cytosolic localization by anchoring this kinase in the cytosol. Adiponectin also activates another AMPK upstream kinase Ca²⁺/calmodulin-dependent protein kinase kinase by activating phospholipase C and subsequently inducing Ca²⁺ release from the endoplasmic reticulum, which plays a minor role in AMPK activation. Our results show that in muscle cells adiponectin is able to activate AMPK via two distinct mechanisms as follows: a major pathway (the APPL1/LKB1-dependent pathway) that promotes the cytosolic localization of LKB1 and a minor pathway (the phospholipase C/Ca²⁺/Ca²⁺/calmodulin-dependent protein kinase kinase-dependent pathway) that stimulates Ca²⁺ release from intracellular stores.Adiponectin, an adipokine abundantly expressed in adipose tissue, exhibits anti-diabetic, anti-inflammatory, and anti-atherogenic properties and hence is a potential therapeutic target for various metabolic diseases (1–3). The beneficial effects of adiponectin are mediated through the direct interaction of adiponectin with its cell surface receptors, AdipoR1 and AdipoR2 (4, 5). Adiponectin increases fatty acid oxidation and glucose uptake in muscle cells by activating AMP-activated protein kinase (AMPK)³ (4, 6), which depends on the interaction of AdipoR1 with the adaptor protein APPL1 (Adaptor protein containing Pleckstrin homology domain, Phosphotyrosine binding domain, and Leucine zipper motif) (5). However, the underlying mechanisms by which APPL1 mediates adiponectin signaling to AMPK activation and other downstream targets remain unclear.AMPK is a serine/threonine protein kinase that acts as a master sensor of cellular energy balance in mammalian cells by regulating glucose and lipid metabolism (7, 8). AMPK is composed of a catalytic α subunit and two noncatalytic regulatory subunits, β and γ. The NH₂-terminal catalytic domain of the AMPKα subunit is highly conserved and contains the activating phosphorylation site (Thr¹⁷²) (9). Two AMPK variants, α1 and α2, exist in mammalian cells that show different localization patterns. AMPKα1 subunit is localized in non-nuclear fractions, whereas the AMPKα2 subunit is found in both nucleus and non-nuclear fractions (10). Biochemical regulation of AMPK activation occurs through various mechanisms. An increase in AMP level stimulates the binding of AMP to the γ subunit, which induces a conformational change in the AMPK heterotrimer and results in AMPK activation (11). Studies have shown that the increase in AMPK activity is not solely via AMP-dependent conformational change, rather via phosphorylation by upstream kinases, LKB1 and CaMKK. Dephosphorylation by protein phosphatases is also important in regulating the activity of AMPK (12).LKB1 has been considered as a constitutively active serine/threonine protein kinase that is ubiquitously expressed in all tissues (13, 14). Under conditions of high cellular energy stress, LKB1 acts as the primary AMPK kinase through an AMP-dependent mechanism (15–17). Under normal physiological conditions, LKB1 is predominantly localized in the nucleus. LKB1 is translocated to the cytosol, either by forming a heterotrimeric complex with Ste20-related adaptor protein (STRADα/β) and mouse protein 25 (MO25α/β) or by associating with an LKB1-interacting protein (LIP1), to exert its biological function (18–22). Although LKB1 has been shown to mediate contraction- and adiponectin-induced activation of AMPK in muscle cells, the underlying molecular mechanisms remain elusive (15, 23).CaMKK is another upstream kinase of AMPK, which shows considerable sequence and structural homology with LKB1 (24–26). The two isoforms of CaMKK, CaMKKα and CaMKKβ, encoded by two distinct genes, share ∼70% homology at the amino acid sequence level and exhibit a wide expression in rodent tissues, including skeletal muscle (27–34). Unlike LKB1, AMPK phosphorylation mediated by CaMKKs is independent of AMP and is dependent only on Ca²⁺/calmodulin (35). Hence, it is possible that an LKB1-independent activation of AMPK by CaMKK exists in muscle cells. However, whether and how adiponectin stimulates this pathway in muscle cells are not known.In this study, we demonstrate that in muscle cells adiponectin induces an APPL1-dependent LKB1 translocation from the nucleus to the cytosol, leading to increased AMPK activation. Adiponectin also activates CaMKK by stimulating intracellular Ca²⁺ release via the PLC-dependent mechanism, which plays a minor role in activation of AMPK. Taken together, our results demonstrate that enhanced cytosolic localization of LKB1 and Ca²⁺-induced activation of CaMKK are the mechanisms underlying adiponectin-stimulated AMPK activation in muscle cells.     

The Antifungal Activity of the Penicillium chrysogenum Protein PAF Disrupts Calcium Homeostasis in Neurospora crassa

The antifungal protein PAF from Penicillium chrysogenum exhibits growth-inhibitory activity against a broad range of filamentous fungi. Evidence from this study suggests that disruption of Ca²⁺ signaling/homeostasis plays an important role in the mechanistic basis of PAF as a growth inhibitor. Supplementation of the growth medium with high Ca²⁺ concentrations counteracted PAF toxicity toward PAF-sensitive molds. By using a transgenic Neurospora crassa strain expressing codon-optimized aequorin, PAF was found to cause a significant increase in the resting level of cytosolic free Ca²⁺ ([Ca²⁺]_c). The Ca²⁺ signatures in response to stimulation by mechanical perturbation or hypo-osmotic shock were significantly changed in the presence of PAF. BAPTA [bis-(aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid], a Ca²⁺ selective chelator, ameliorated the PAF toxicity in growth inhibition assays and counteracted PAF induced perturbation of Ca²⁺ homeostasis. These results indicate that extracellular Ca²⁺ was the major source of these PAF-induced effects. The L-type Ca²⁺ channel blocker diltiazem disrupted Ca²⁺ homeostasis in a similar manner to PAF. Diltiazem in combination with PAF acted additively in enhancing growth inhibition and accentuating the change in Ca²⁺ signatures in response to external stimuli. Notably, both PAF and diltiazem increased the [Ca²⁺]_c resting level. However, experiments with an aequorin-expressing Δcch-1 deletion strain of N. crassa indicated that the L-type Ca²⁺ channel CCH-1 was not responsible for the observed PAF-induced elevation of the [Ca²⁺]_c resting level. This study is the first demonstration of the perturbation of fungal Ca²⁺ homeostasis by an antifungal protein from a filamentous ascomycete and provides important new insights into the mode of action of PAF.The secreted antifungal protein PAF from Penicillium chrysogenum is a small-molecular-mass (6.2 kDa), cationic, and cysteine-rich peptide that inhibits the growth of numerous filamentous fungi (14–16, 21). It belongs to a family of antifungal peptides which show—despite considerable amino acid homology—significant differences in species specificity and modes of action (reviewed in reference 27). Importantly, the primary structures of these antifungals show no similarity to those from higher eukaryotes, e.g., plants, insects, or mammals (see reference 28 for a detailed review on parallels with and differences between PAF and antimicrobial proteins from higher eukaryotes and their mechanisms of action). Apart from the Aspergillus giganteus-derived antifungal protein AFP (19, 45; reviewed in reference 29), PAF is one of the best-studied peptides of this protein family. We have shown that PAF causes rapid hyperpolarization of the plasma membrane at hyphal tips, increased K⁺ efflux, induction of oxidative stress, and apoptotic cell death (21, 25) and that PAF is internalized by hyphae of PAF-sensitive fungi (33). Furthermore, we have shown that PAF interferes with at least two signaling cascades, the protein kinase C/mitogen-activated protein (MAP) kinase and the cyclic AMP (cAMP)/protein kinase A pathways, which play a role in mediating PAF toxicity (5). However, it still has to be elucidated in more detail how these PAF-dependent effects are linked.Recent evidence indicated that the ionic strength of the growth medium interferes with the antifungal activity of PAF (21). It is possible that Ca²⁺ ions may play a major role in influencing protein toxicity in an analogous way to the Ca²⁺-dependent mode of action of antifungal plant defensins. Supplementation of the test medium with low concentrations of CaCl₂ (1 to 5 mM) reversed the antifungal activity of plant defensins (34, 43–44). The defensins RsAFP2 from the seeds of Raphanus sativus and DmAMP1 from the seeds of Dahlia merckii induced K⁺ efflux and Ca²⁺ uptake in Neurospora crassa and caused alkalinization of the growth medium (46). Another seed defensin, MsDef1 from Medicago sativa, was reported to cause Ca²⁺ influx and the inhibition of mammalian L-type Ca²⁺ channels, similar to the Ustilago maydis killer toxin KP4 (13, 40).Ca²⁺ is a universal intracellular second messenger in eukaryotic cells (4, 36). In fungi, there is evidence for Ca²⁺ signaling regulating numerous processes, including spore germination, tip growth, hyphal branching, sporulation, infection structure differentiation, and circadian clocks, as well as responses to osmotic stress, heat shock, mechanical stimuli, oxidative stress, and electrical fields (7, 11, 17, 20, 22, 30–31, 38–39, 41, 49). Ca²⁺ signaling typically involves transient increases in intracellular Ca²⁺ concentrations originating from the extracellular medium and/or mobilization from internal stores (4, 23, 36–37). Little was known about Ca²⁺ dynamics in filamentous fungi until a new method based on the Ca²⁺-sensitive photoprotein aequorin was adapted, allowing routine and easy measurement of intracellular calcium dynamics in these organisms (3, 30).The aim of our study was to investigate the effect of PAF on the level of cytosolic free Ca²⁺ ([Ca²⁺]_c) in the PAF-sensitive target organism N. crassa to characterize its effects on stimulus-specific Ca²⁺ signatures and to define the Ca²⁺ sources responsible for the perturbation of Ca²⁺ homeostasis. By obtaining [Ca²⁺]_c measurements in living cells expressing the bioluminescent Ca²⁺ reporter aequorin, we provide novel insights into the mode of action of this biotechnologically promising antifungal protein.     

Protein Kinase A Increases Type-2 Inositol 1,4,5-Trisphosphate Receptor Activity by Phosphorylation of Serine 937

Protein kinase A (PKA) phosphorylation of inositol 1,4,5-trisphosphate receptors (InsP₃Rs) represents a mechanism for shaping intracellular Ca²⁺ signals following a concomitant elevation in cAMP. Activation of PKA results in enhanced Ca²⁺ release in cells that express predominantly InsP₃R2. PKA is known to phosphorylate InsP₃R2, but the molecular determinants of this effect are not known. We have expressed mouse InsP₃R2 in DT40-3KO cells that are devoid of endogenous InsP₃R and examined the effects of PKA phosphorylation on this isoform in unambiguous isolation. Activation of PKA increased Ca²⁺ signals and augmented the single channel open probability of InsP₃R2. A PKA phosphorylation site unique to the InsP₃R2 was identified at Ser⁹³⁷. The enhancing effects of PKA activation on this isoform required the phosphorylation of Ser⁹³⁷, since replacing this residue with alanine eliminated the positive effects of PKA activation. These results provide a mechanism responsible for the enhanced Ca²⁺ signaling following PKA activation in cells that express predominantly InsP₃R2.Hormones, neurotransmitters, and growth factors stimulate the production of InsP₃³ and Ca²⁺ signals in virtually all cell types (1). The ubiquitous nature of this mode of signaling dictates that this pathway does not exist in isolation; indeed, a multitude of additional signaling pathways can be activated simultaneously. A prime example of this type of “cross-talk” between independently activated signaling systems results from the parallel activation of cAMP and Ca²⁺ signaling pathways (2, 3). Interactions between these two systems occur in numerous distinct cell types with various physiological consequences (3–6). Given the central role of InsP₃R in Ca²⁺ signaling, a major route of modulating the spatial and temporal features of Ca²⁺ signals following cAMP production is potentially through PKA phosphorylation of the InsP₃R isoform(s) expressed in a particular cell type.There are three InsP₃R isoforms (InsP₃R1, InsP₃R2, and InsP₃R3) expressed to varying degrees in mammalian cells (7, 8). InsP₃R1 is the major isoform expressed in the nervous system, but it is less abundant compared with other subtypes in non-neuronal tissues (8). Ca²⁺ release via InsP₃R2 and InsP₃R3 predominate in these tissues. InsP₃R2 is the major InsP₃R isoform in many cell types, including hepatocytes (7, 8), astrocytes (9, 10), cardiac myocytes (11), and exocrine acinar cells (8, 12). Activation of PKA has been demonstrated to enhance InsP₃-induced Ca²⁺ signaling in hepatocytes (13) and parotid acinar cells (4, 14). Although PKA phosphorylation of InsP₃R2 is a likely causal mechanism underlying these effects, the functional effects of phosphorylation have not been determined in cells unambiguously expressing InsP₃R2 in isolation. Furthermore, the molecular determinants of PKA phosphorylation of this isoform are not known.PKA-mediated phosphorylation is an efficient means of transiently and reversibly regulating the activity of the InsP₃R. InsP₃R1 was identified as a major substrate of PKA in the brain prior to its identification as the InsP₃R (15, 16). However, until recently, the functional consequences of phosphorylation were unresolved. Initial conflicting results were reported indicating that phosphoregulation of InsP₃R1 could result in either inhibition or stimulation of receptor activity (16, 17). Mutagenic strategies were employed by our laboratory to clarify this discrepancy. These studies unequivocally assigned phosphorylation-dependent enhanced Ca²⁺ release and InsP₃R1 activity at the single channel level, through phosphorylation at canonical PKA consensus motifs at Ser¹⁵⁸⁹ and Ser¹⁷⁵⁵. The sites responsible were also shown to be specific to the particular InsP₃R1 splice variant (18). These data were also corroborated by replacing the relevant serines with glutamates in a strategy designed to construct “phosphomimetic” InsP₃R1 by mimicking the negative charge added by phosphorylation (19, 20). Of particular note, however, although all three isoforms are substrates for PKA, neither of the sites phosphorylated by PKA in InsP₃R1 are conserved in the other two isoforms (21). Recently, three distinct PKA phosphorylation sites were identified in InsP₃R3 that were in different regions of the protein when compared with InsP₃R1 (22). To date, no PKA phosphorylation sites have been identified in InsP₃R2.Interactions between Ca²⁺ and cAMP signaling pathways are evident in exocrine acinar cells of the parotid salivary gland. In these cells, both signals are important mediators of fluid and protein secretion (23). Multiple components of the [Ca²⁺]_i signaling pathway in these cells are potential substrates for modulation by PKA. Previous work from this laboratory established that activation of PKA potentiates muscarinic acetylcholine receptor-induced [Ca²⁺]_i signaling in mouse and human parotid acinar cells (4, 24, 25). A likely mechanism to explain this effect is that PKA phosphorylation increases the activity of InsP₃R expressed in these cells. Consistent with this idea, activation of PKA enhanced InsP₃-induced Ca²⁺ release in permeabilized mouse parotid acinar cells and also resulted in the phosphorylation of InsP₃R2 (4).Invariably, prior work examining the functional effects of PKA phosphorylation on InsP₃R2 has been performed using cell types expressing multiple InsP₃R isoforms. For example, AR4-2J cells are the preferred cell type for examining InsP₃R2 in relative isolation, because this isoform constitutes more than 85% of the total InsP₃R population (8). InsP₃R1, however, contributes up to ∼12% of the total InsP₃R in AR4-2J cells. An initial report using InsP₃-mediated ⁴⁵Ca²⁺ flux suggested that PKA activation increased InsP₃R activity in AR4-2J cells (21). A similar conclusion was made in a later study, which documented the effects of PKA activation on agonist stimulated Ca²⁺ signals in AR4-2J cells (26). Any effects of phosphorylation observed in these experiments could plausibly have resulted from phosphorylation of the residual InsP₃R1.Although PKA enhances InsP₃-induced calcium release in cells expressing predominantly InsP₃R2, including hepatocytes, parotid acinar cells, and AR4-2J cells (4, 13, 21, 26, 27), InsP₃R2 is not phosphorylated at stoichiometric levels by PKA (21). This observation has called into question the physiological significance of PKA phosphorylation of InsP₃R2 (28). The apparent low levels of InsP₃R2 phosphorylation are clearly at odds with the augmented Ca²⁺ release observed in cells expressing predominantly this isoform. The equivocal nature of these findings probably stems from the fact that, to date, all of the studies demonstrating positive effects of PKA activation on Ca²⁺ release were conducted in cells that also express InsP₃R1. The purpose of the current experiments was to analyze the functional effects of phosphorylation on InsP₃R2 expressed in isolation on a null background. We report that InsP₃R2 activity is increased by PKA phosphorylation under these conditions, and furthermore, we have identified a unique phosphorylation site in InsP₃R2 at Ser⁹³⁷. In total, these results provide a direct mechanism for the cAMP-induced activation of InsP₃R2 via PKA phosphorylation of InsP₃R2.     

A Ca2+ Release-activated Ca2+ (CRAC) Modulatory Domain (CMD) within STIM1 Mediates Fast Ca2+-dependent Inactivation of ORAI1 Channels

STIM1 and ORAI1, the two limiting components in the Ca²⁺ release-activated Ca²⁺ (CRAC) signaling cascade, have been reported to interact upon store depletion, culminating in CRAC current activation. We have recently identified a modulatory domain between amino acids 474 and 485 in the cytosolic part of STIM1 that comprises 7 negatively charged residues. A STIM1 C-terminal fragment lacking this domain exhibits enhanced interaction with ORAI1 and 2–3-fold higher ORAI1/CRAC current densities. Here we focused on the role of this CRAC modulatory domain (CMD) in the fast inactivation of ORAI1/CRAC channels, utilizing the whole-cell patch clamp technique. STIM1 mutants either with C-terminal deletions including CMD or with 7 alanines replacing the negative amino acids within CMD gave rise to ORAI1 currents that displayed significantly reduced or even abolished inactivation when compared with STIM1 mutants with preserved CMD. Consistent results were obtained with cytosolic C-terminal fragments of STIM1, both in ORAI1-expressing HEK 293 cells and in RBL-2H3 mast cells containing endogenous CRAC channels. Inactivation of the latter, however, was much more pronounced than that of ORAI1. The extent of inactivation of ORAI3 channels, which is also considerably more prominent than that of ORAI1, was also substantially reduced by co-expression of STIM1 constructs missing CMD. Regarding the dependence of inactivation on Ca²⁺, a decrease in intracellular Ca²⁺ chelator concentrations promoted ORAI1 current fast inactivation, whereas Ba²⁺ substitution for extracellular Ca²⁺ completely abrogated it. In summary, CMD within the STIM1 cytosolic part provides a negative feedback signal to Ca²⁺ entry by triggering fast Ca²⁺-dependent inactivation of ORAI/CRAC channels.The Ca²⁺ release-activated Ca²⁺ (CRAC)⁵ channel is one of the best characterized store-operated entry pathways (1–7). Substantial efforts have led to identification of two key components of the CRAC channel machinery: the stromal interaction molecule 1 (STIM1), which is located in the endoplasmic reticulum and acts as a Ca²⁺ sensor (8–10), and ORAI1/CRACM1, the pore-forming subunit of the CRAC channel (11–13). Besides ORAI1, two further homologues named ORAI2 and ORAI3 belong to the ORAI channel family (12, 14).STIM1 senses endoplasmic reticulum store depletion primarily by its luminal EF-hand in its N terminus (8, 15), redistributes close to the plasma membrane, where it forms puncta-like structures, and co-clusters with ORAI1, leading to inward Ca²⁺ currents (12, 16–19). The STIM1 C terminus, located in the cytosol, contains two coiled-coil regions overlapping with an ezrin-radixin-moesin (ERM)-like domain followed by a serine/proline- and a lysine-rich region (2, 8, 20–22). Three recent studies have described the essential ORAI-activating region within the ERM domain, termed SOAR (Stim ORAI-activating region) (23), OASF (ORAI-activating small fragment) (24), and CAD (CRAC-activating domain) (25), including the second coiled coil domain and the following ∼55 amino acids. We and others have provided evidence that store depletion leads to a dynamic coupling of STIM1 to ORAI1 (26–28) that is mediated by a direct interaction of the STIM1 C terminus with ORAI1 C terminus probably involving the putative coiled-coil domain in the latter (27).Furthermore, different groups have proven that the C terminus of STIM1 is sufficient to activate CRAC as well as ORAI1 channels independent of store depletion (22–25, 27, 29). We have identified that OASF-(233–474) or shorter fragments exhibit further enhanced coupling to ORAI1 resulting in 3-fold increased constitutive Ca²⁺ currents. A STIM1 fragment containing an additional cluster of anionic amino acids C-terminal to position 474 displays weaker interaction with ORAI1 as well as reduced Ca²⁺ current comparable with that mediated by wild-type STIM1 C terminus. Hence, we have suggested that these 11 amino acids (474–485) act in a modulatory manner onto ORAI1; however, their detailed mechanistic impact within the STIM1/ORAI1 signaling machinery has remained so far unclear.In this study, we focused on the impact of this negative cluster on fast inactivation of STIM1-mediated ORAI Ca²⁺ currents. Lis et al. (30) have shown that all three ORAI homologues display distinct inactivation profiles, where ORAI2 and ORAI3 show a much more pronounced fast inactivation than ORAI1. Moreover, it has been reported (31) that different expression levels of STIM1 to ORAI1 affect the properties of CRAC current inactivation. Yamashita et al. (32) have demonstrated a linkage between the selectivity filter of ORAI1 and its Ca²⁺-dependent fast inactivation. Here we provide evidence that a cluster of acidic residues within the C terminus of STIM1 is involved in the fast inactivation of ORAI1 and further promotes that of ORAI3 and native CRAC currents.     

Characterization of the ATP-dependent Sphingosine 1-Phosphate Transporter in Rat Erythrocytes

Sphingosine 1-phosphate (S1P) is a bioactive lipid signal transmitter present in blood. Blood plasma S1P is supplied from erythrocytes and plays an important role in lymphocyte egress from lymphoid organs. However, the S1P export mechanism from erythrocytes to blood plasma is not well defined. To elucidate the mechanism of S1P export from erythrocytes, we performed the enzymatic characterization of S1P transporter in rat erythrocytes. Rat erythrocytes constitutively released S1P without any stimulus. The S1P release was reduced by an ABCA1 transporter inhibitor, glyburide, but not by a multidrug resistance-associated protein inhibitor, MK571, or a multidrug resistance protein inhibitor, cyclosporine A. Furthermore, we measured S1P transport activity using rat erythrocyte inside-out membrane vesicles (IOVs). Although the effective S1P transport into IOVs was observed in the presence of ATP, this activity was also supported by dATP and adenosine 5′-(β,γ-imido)triphosphate. The rate of S1P transport increased depending on S1P concentration, with an apparent K_m value of 21 μm. Two phosphorylated sphingolipids, dihydrosphingosine 1-phosphate and ceramide 1-phosphate, did not inhibit S1P transport. Similar to the intact erythrocytes, the uptake of S1P into IOVs was inhibited by glyburide and vanadate but not by the other ABC transporter inhibitors. These results suggest that S1P is exported from the erythrocytes by a novel ATP-dependent transporter.Sphingosine 1-phosphate (S1P),² a bioactive lipid molecule present in the blood, plays an important role in diverse cellular responses, such as migration, proliferation, and differentiation (1, 2). These processes are triggered by the binding of S1P to its specific receptors (3), of which five subtypes (S1P₁-S1P₅) have been identified in endothelial and immune cells (4). Studies using S1P₁ receptor-deficient mice showed abnormalities in lymphocyte egress from lymph nodes, spleen, and thymus (5, 6). Whereas blood plasma contains a basal level of S1P from the nanomolar to the micromolar range (7–12), lymphoid tissues maintain a low S1P environment through the activity of S1P lyase (13). It has been proposed that a higher concentration of S1P in the blood plasma than in the lymphoid organs establishes an essential gradient along which lymphocytes expressing the S1P₁ receptor on cell surfaces migrate (2, 5, 6, 13–15).The source of plasma S1P remains unclear despite its importance in the cellular responses of endothelial cells and lymphocytes. Unlike most cells, blood cells, astrocytes, and vascular endothelial cells are reported to release S1P (8, 16–18). These cells contain sphingosine kinase, which synthesizes S1P through the phosphorylation of sphingosine (16, 18, 19). Whereas platelets and mast cells release S1P in a stimulus-dependent manner (17, 20), erythrocytes, neutrophils, and mononuclear cells release S1P in a stimulus-independent manner (16). The roles of S1P derived from erythrocytes, the most abundant of these blood cells, have not been elucidated. However, recent reports suggest that S1P released from erythrocytes is a major source of plasma S1P (7, 9) and promotes lymphocyte egress to blood (9).Previously, we showed that S1P is released from rat platelets upon stimulation by thrombin or Ca²⁺ (21). We proposed that an ATP-dependent transporter plays a key role in S1P release from platelets (21). However, the detailed mechanism of S1P release is unclear because there is no way to assay the transport of S1P across the membrane. In this study we compared the properties of S1P release from erythrocytes with that of platelets and showed that S1P release from erythrocytes does not require any stimuli. We then established an assay to measure the ATP-dependent S1P uptake into inside-out membrane vesicles (IOVs) prepared from rat erythrocytes and characterized S1P transport in erythrocytes.     

Diversity and Developmental Expression of L-type Calcium Channel ��2 Proteins and Their Influence on Calcium Current in Murine Heart

By now, little is known on L-type calcium channel (LTCC) subunits expressed in mouse heart. We show that CaVβ2 proteins are the major CaVβ components of the LTCC in embryonic and adult mouse heart, but that in embryonic heart CaVβ3 proteins are also detectable. At least two CaVβ2 variants of ∼68 and ∼72 kDa are expressed. To identify the underlying CaVβ2 variants, cDNA libraries were constructed from poly(A)⁺ RNA isolated from hearts of 7-day-old and adult mice. Screening identified 60 independent CaVβ2 cDNA clones coding for four types of CaVβ2 proteins only differing in their 5′ sequences. CaVβ2-N1, -N4, and -N5 but not -N3 were identified in isolated cardiomyocytes by RT-PCR and were sufficient to reconstitute the CaVβ2 protein pattern in vitro. Significant L-type Ca²⁺ currents (I_Ca) were recorded in HEK293 cells after co-expression of CaV1.2 and CaVβ2. Current kinetics were determined by the type of CaVβ2 protein, with the ∼72-kDa CaVβ2a-N1 shifting the activation of I_Ca significantly to depolarizing potentials compared with the other CaVβ2 variants. Inactivation of I_Ca was accelerated by CaVβ2a-N1 and -N4, which also lead to slower activation compared with CaVβ2a-N3 and -N5. In summary, this study reveals the molecular LTCC composition in mouse heart and indicates that expression of various CaVβ2 proteins may be used to adapt the properties of LTCCs to changing myocardial requirements during development and that CaVβ2a-N1-induced changes of I_Ca kinetics might be essential in embryonic heart.Cardiac contractions require Ca²⁺ influx in cardiomyocytes from the extracellular fluid, which leads to Ca²⁺ release from the sarcoplasmic reticulum via ryanodine receptors (1).This Ca²⁺-induced Ca²⁺ release (CICR)⁴ causes a marked increase in intracellular Ca²⁺ concentration for short periods of time and underlies cardiac contraction (2, 3). The Ca²⁺ influx into cardiac myocytes is mediated by high voltage-activated L-type Ca²⁺ channels (LTCCs), which are heteromultimeric complexes comprised predominantly of the pore-forming CaVα₁ subunit and the auxiliary CaVβ subunit (4). In heart, the principal CaVα₁ subunit, CaVα₁c (CaV1.2), is encoded by the Cacna1C gene (5). Four genes (Cacnb1-4) encoding CaVβ subunits have been identified that are expressed in the heart of different species including human, rabbit, and rat (6, 7, 8).CaVβ proteins are ∼500 amino acid cytoplasmic proteins that bind to the CaVα₁ I-II intracellular loop (9) and affect channel gating properties (4), trafficking (10, 11), regulation by neurotransmitter receptors through G-protein βγ subunit activation (12), and sensitivity to drugs (13). The CaVβ primary sequence encodes five domains, arranged V1-C1-V2-C2-V3. V1, V2, and V3 are variable domains, whereas C1 and C2 are conserved (14). Structural studies reveal that C1 and C2 form a SH3 domain (Src homology 3 domain) and a NK domain (nucleotide kinase domain), respectively (15). Although C1-V2-C2 makes the CaVβ core, in heart the V1 region appears critical for the kinetics of I_Ca and heart function. Accordingly a mutation in the V1 region of the Cacnb2 gene was recently identified as an underlying cause of Brugada syndrome (16).In mice-targeted deletion of the Cacnb2 gene (17) but not of Cacnb1 (18), Cacnb3 (19, 20), or Cacnb4 (21) leads to a morphologically and functionally compromised heart, which causes severe defective remodeling of intra- and extra-embryonic blood vessels and death at early embryonic stages both when the Cacnb2 gene was targeted globally or in a cardiac myocyte-specific way (17). Although these results point to an essential role of CaVβ2 for I_Ca and cardiac function, the existence of various CaVβ2 splice variants and heterogeneity of the expressed CaVβ2 proteins require further studies on the subunit composition of LTCCs in the mouse heart. In addition and in view of the growing number of preclinical studies using mouse models carrying definite Ca²⁺ channel subunits as transgenes in heart tissue, the identification of the relevant gene products underlying the endogenous mouse cardiac L-type channel is essential. Recent mouse models (e.g. 22, 23, 24) carrying a rat CaVβ2 splice variant (“rat CaVβ2a”) (25) expressed in rat and rabbit brain (26), but not in rabbit heart (26), have only escalated this requirement, because it has never been shown that the mouse orthologue of this variant is endogenously expressed in the mouse heart.So far, five CaVβ2 variants varying only in the V1 domain have been identified from different species (25, 27, 28) and in human heart these variants have been obtained mainly by RT-PCR approaches (29, 30). In contrast, there is little information on the CaVβ proteins present in mouse heart, their respective splice variants, and expression ratios. We therefore started to study CaVβ expression in the murine heart using Western blots and cDNA cloning and to reveal their functional impact on LTCCs formed by the murine CaV1.2 protein.     

Proteomic Analysis of Extracellular ATP-Regulated Proteins Identifies ATP Synthase β-Subunit as a Novel Plant Cell Death Regulator

Extracellular ATP is an important signal molecule required to cue plant growth and developmental programs, interactions with other organisms, and responses to environmental stimuli. The molecular targets mediating the physiological effects of extracellular ATP in plants have not yet been identified. We developed a well characterized experimental system that depletes Arabidopsis cell suspension culture extracellular ATP via treatment with the cell death-inducing mycotoxin fumonisin B1. This provided a platform for protein profile comparison between extracellular ATP-depleted cells and fumonisin B1-treated cells replenished with exogenous ATP, thus enabling the identification of proteins regulated by extracellular ATP signaling. Using two-dimensional difference in-gel electrophoresis and matrix-assisted laser desorption-time of flight MS analysis of microsomal membrane and total soluble protein fractions, we identified 26 distinct proteins whose gene expression is controlled by the level of extracellular ATP. An additional 48 proteins that responded to fumonisin B1 were unaffected by extracellular ATP levels, confirming that this mycotoxin has physiological effects on Arabidopsis that are independent of its ability to trigger extracellular ATP depletion. Molecular chaperones, cellular redox control enzymes, glycolytic enzymes, and components of the cellular protein degradation machinery were among the extracellular ATP-responsive proteins. A major category of proteins highly regulated by extracellular ATP were components of ATP metabolism enzymes. We selected one of these, the mitochondrial ATP synthase β-subunit, for further analysis using reverse genetics. Plants in which the gene for this protein was knocked out by insertion of a transfer-DNA sequence became resistant to fumonisin B1-induced cell death. Therefore, in addition to its function in mitochondrial oxidative phosphorylation, our study defines a new role for ATP synthase β-subunit as a pro-cell death protein. More significantly, this protein is a novel target for extracellular ATP in its function as a key negative regulator of plant cell death.ATP is a ubiquitous, energy-rich molecule of fundamental importance in living organisms. It is a key substrate and vital cofactor in many biochemical reactions and is thus conserved by all cells. However, in addition to its localization and functions inside cells, ATP is actively secreted to the extracellular matrix where it forms a halo around the external cell surface. The existence of this extracellular ATP (eATP)¹ has been reported in several organisms including bacteria (1), primitive eukaryotes (2), animals (3), and plants (4–6). This eATP is not wasted, but harnessed at the cell surface as a potent signaling molecule enabling cells to communicate with their neighbors and regulate crucial growth and developmental processes.In animals, eATP is a crucial signal molecule in several physiological processes such as neurotransmission (7, 8), regulation of blood pressure (9), enhanced production of reactive oxygen species (ROS) (10), protein translocation (11), and apoptosis (12). Extracellular ATP signal perception at the animal cell surface is mediated by P2X and P2Y receptors, which bind ATP extracellularly and recruit intracellular second messengers (13, 14). P2X receptors are ligand-gated ion channels that provide extracellular Ca²⁺ a corridor for cell entry after binding eATP, facilitating a surge in cytosolic [Ca²⁺] that is essential in activating down-stream signaling. P2Y receptors transduce the eATP signal by marshalling heteromeric G-proteins on the cytosolic face of the plasma membrane and activating appropriate downstream effectors.Although eATP exists in plants, homologous P2X/P2Y receptors for eATP signal perception have not yet been identified, even in plant species with fully sequenced genomes. Notwithstanding the obscurity of plant eATP signal sensors, some of the key downstream messengers recruited by eATP-mediated signaling are known. For example, eATP triggers a surge in cytosolic Ca²⁺ concentration (15–17) and a heightened production of nitric oxide (18–20) and reactive oxygen species (17, 21, 22). Altering eATP levels is attended by activation of plant gene expression (16, 21) and changes in protein abundance (5, 23), indicating that eATP-mediated signaling impacts on plant physiology. Indeed eATP has been demonstrated to regulate plant growth (20, 24–26), gravitropic responses (27), xenobiotic resistance (4), plant-symbiont interactions (28), and plant-pathogen interactions (23, 29). However, the mechanism by which eATP regulates these processes remains unclear, largely because the eATP signal sensors and downstream signal regulatory genes and proteins have not been identified.We previously reported that eATP plays a central regulatory role in plant cell death processes (5). Therefore, an understanding of the signaling components galvanized by eATP in cell death regulation might serve a useful purpose in providing mechanistic detail of how eATP signals in plant physiological processes. We found that eATP-mediated signaling negatively regulates cell death as its removal by application of ATP-degrading enzymes to the apoplast activates plant cell death (5). Remarkably, fumonisin B1 (FB1), a pathogen-derived molecule that activates defense gene expression in Arabidopsis (30), commandeers this eATP-regulated signaling to trigger programmed cell death (5). FB1 is a mycotoxin secreted by fungi in the genus Fusarium and initiates programmed cell death in both animal and plant cells (31, 32). In Arabidopsis, FB1 inaugurates cell death by inactivating eATP-mediated signaling via triggering a drastic collapse in the levels of eATP (5). FB1-induced Arabidopsis programmed cell death is dependent on the plant signaling hormone salicylic acid (33), which is a key regulator of eATP levels (29). Because concurrent application of FB1 and exogenous ATP to remedy the FB1-induced eATP deficit blocks death, FB1 and exogenous ATP treatments can therefore be used as probes to identify the key signal regulators downstream of eATP in cell death control. This is vital for achieving the global objective of elucidating the mechanism of eATP signaling in plant physiology.Gel-based proteomic analyses have been previously applied to successfully identify the novel role of eATP in the regulation of plant defense gene expression and disease resistance (23, 29). We have now employed FB1 and ATP treatments together with two-dimensional difference in-gel electrophoresis (DIGE) and matrix-assisted laser desorption-time of flight MS (MALDI-TOF MS) to identify the changes in Arabidopsis protein profiles associated with a shift from normal to cell death-inception metabolism. Additional reverse genetic analyses enabled us to definitively identify a putative ATP synthase β-subunit as a target for eATP-mediated signaling with an unexpected function in the regulation of plant programmed cell death.     

Phosphorylation-dependent Autoinhibition of Myosin Light Chain Phosphatase Accounts for Ca2+ Sensitization Force of Smooth Muscle Contraction

The reversible regulation of myosin light chain phosphatase (MLCP) in response to agonist stimulation and cAMP/cGMP signals plays an important role in the regulation of smooth muscle (SM) tone. Here, we investigated the mechanism underlying the inhibition of MLCP induced by the phosphorylation of myosin phosphatase targeting subunit (MYPT1), a regulatory subunit of MLCP, at Thr-696 and Thr-853 using glutathione S-transferase (GST)-MYPT1 fragments having the inhibitory phosphorylation sites. GST-MYPT1 fragments, including only Thr-696 and only Thr-853, inhibited purified MLCP (IC₅₀ = 1.6 and 60 nm, respectively) when they were phosphorylated with RhoA-dependent kinase (ROCK). The activities of isolated catalytic subunits of type 1 and type 2A phosphatases (PP1 and PP2A) were insensitive to either fragment. Phospho-GST-MYPT1 fragments docked directly at the active site of MLCP, and this was blocked by a PP1/PP2A inhibitor microcystin (MC)-LR or by mutation of the active sites in PP1. GST-MYPT1 fragments induced a contraction of β-escin-permeabilized ileum SM at constant pCa 6.3 (EC₅₀ = 2 μm), which was eliminated by Ala substitution of the fragment at Thr-696 or by ROCK inhibitors or 8Br-cGMP. GST-MYPT1-(697–880) was 5-times less potent than fragments including Thr-696. Relaxation induced by 8Br-cGMP was not affected by Ala substitution at Ser-695, a known phosphorylation site for protein kinase A/G. Thus, GST-MYPT1 fragments are phosphorylated by ROCK in permeabilized SM and mimic agonist-induced inhibition and cGMP-induced activation of MLCP. We propose a model in which MYPT1 phosphorylation at Thr-696 and Thr-853 causes an autoinhibition of MLCP that accounts for Ca²⁺ sensitization of smooth muscle force.The contractile state of smooth muscle (SM)³ is driven by phosphorylation of the regulatory myosin light chain and reflects the balance of the Ca²⁺-calmodulin-dependent myosin light chain kinase and myosin light chain phosphatase (MLCP) activities (1). The stoichiometry between force and [Ca²⁺] varies with different agonists (2), reflecting other signaling pathways that modulate the MLCP or myosin light chain kinase activities (3–5). Agonist activation of G-protein-coupled receptors triggers Ca²⁺ release from the sarcoplasmic reticulum. Simultaneously, G-protein-coupled receptor signals are mediated by Ca²⁺-independent phospholipase A2 (6) and initiate kinase signals, such as PKC, phosphoinositide 3-kinase (7), and ROCK. These lead to inhibition of MLCP activity resulting in an increase in regulatory myosin light chain phosphorylation independent of a change in Ca²⁺ (Ca²⁺ sensitization) (for review, see Ref. 1). K⁺ depolarization can also activate RhoA in a Ca²⁺-dependent manner (8). Conversely, Ca²⁺ desensitization occurs when nitric oxide production and the activation of Gas elevate cGMP and cAMP levels in SM, leading to dis-inhibition and restoration of MLCP activity (9–15). Thus, MLCP plays a pivotal role in controlling phosphorylation of myosin, in response to physiological stimulation.MLCP is a trimeric holoenzyme consisting of a catalytic subunit of protein phosphatase 1 (PP1) δ isoform and a regulatory complex of MYPT1 and an accessory M21 subunit (16). A PP1 binding site, KVKF³⁸, is located at the N terminus of MYPT1 followed by an ankyrin-repeat domain. This N-terminal domain forms a part of the active site together with the catalytic subunit and controls the substrate specificity via allosteric interaction and targeting to loci (17). The C-terminal region of MYPT1 directly binds to substrates such as myosin and ezrin/radixin/moecin proteins as well as, under some conditions, the plasma membrane, tethering the catalytic subunit to multiple targets (18, 19). Furthermore, MYPT1 is involved in the regulation of MLCP activity. Alternative splicing of MYPT1 occurs in SM depending on the tissue and the developmental stage (20). An exon 13 splicing of MYPT1 is involved in Ca²⁺ sensitization that occurs in response to GTP (21), whereas a splice variant of MYPT1, containing the C-terminal Leu-zipper sequence, correlates with cGMP-dependent relaxation of smooth muscle (22). Direct binding of PKG to MYPT1 at the Leu-zipper domain and/or Arg/Lys-rich domain is involved in the activation of MLCP (23–25). In addition, a myosin phosphatase-Rho interacting protein (M-RIP) is directly associated with the MYPT1 C-terminal domain, proposed to recruit RhoA to the MLCP complex (26). The C-terminal region also binds to ZIP kinase, which phosphorylates MYPT1 at Thr-696⁴ (27). Thus, the C-terminal domain of MYPT1 functions as a scaffold for multiple phosphatase regulatory proteins.Phosphorylation of MYPT1 at Thr-696 and Thr-853 and the phosphatase inhibitory protein CPI-17 at Thr-38 play dominant roles in the agonist-induced inhibition of MLCP (18, 28–34), yet the molecular mechanism(s) of MYPT1 inhibitory phosphorylation is poorly understood. Receptor activation induces biphasic contraction of SM, reflecting a sequential activation of PKC and ROCK. Phosphorylation of CPI-17 occurs first in parallel with Ca²⁺ release and the activation of a conventional PKC that causes Ca²⁺-dependent Ca²⁺ sensitization (35). A delayed activation of ROCK increases the phosphorylation of MYPT1 at Thr-853. These phosphorylation events maintain the sustained phase of contraction after the fall in [Ca²⁺]_i (35). Phosphorylation of MYPT1 at Thr-853 is elevated in response to various agonists (35, 36). Unlike the Thr-853 site, phosphorylation of MYPT1 at Thr-696 is often spontaneously phosphorylated under resting conditions and insensitive to stimuli with most agonists (36). Nonetheless, up-regulation of MYPT1 phosphorylation at Thr-696 is reported in some types of hypertensive animals and patients, suggesting an importance of the site under pathological conditions (37–39). Phosphorylation of CPI-17 and MYPT1 at Thr-696 is reversed in response to nitric oxide production and cGMP elevation, which parallels relaxation (14, 15). Upon cGMP elevation, MYPT1 at Ser-695 is phosphorylated, and the Ser phosphorylation blocks the adjacent phosphorylation at Thr-696, causing dis-inhibition of MLCP (27, 40). However, Ser-695 phosphorylation does not cause the dephosphorylation at Thr-696 in intact cerebral artery (41). Thus, phosphorylation of MYPT1 governs Ca²⁺ sensitization and desensitization of SM, although the underlying mechanisms are still controversial. In addition, telokin, a dominant protein in visceral and phasic vascular SM tissues, is phosphorylated by PKG and PKA, activating MLCP by an unknown mechanism and inducing SM relaxation (42).Multiple mechanisms have been suggested for the phosphorylation-dependent inhibition of MLCP. Thiophosphorylation of MYPT1 results in lower V_m and higher K_m values of MLCP activity, suggesting that allosteric modulation of the active site is necessary for the thiophosphorylation-dependent inhibition of MLCP (43). On the other hand, translocation of MYPT1 to the plasma membrane region occurs in parallel with the phosphorylation of MYPT1 at Thr-696 (44, 45), but the amount translocated and the functional meaning remain controversial (41). Phosphorylation of MYPT1 at Thr-853 in vitro reduces its affinity for phospho-myosin, thus suppressing the phosphatase activity (18). It has also been demonstrated that reconstitution of thiophosphorylated MYPT1 at Thr-696 or Thr-853 with isolated PP1δ produces a less-active form of MLCP complex (46). This supports the kinetic analysis (43) that suggests an allosteric effect of MYPT1 phosphorylation on the phosphatase activity. In contrast, a thiophosphopeptide mimicking the phosphorylation site of MBS85, a homolog of MYPT1 and not present in SM, inhibits the activity of MBS85·PP1 complex, suggesting the direct interaction between the MBS85 site and PP1 (47). In the crystal structure model of MYPT1-(1–229). PP1δ complex, the electrostatic potential map at the MLCP active site complements amino acid profiles around the phosphorylation sites (17). Therefore, it is possible that the inhibitory phosphorylation sites directly dock at the active site of MLCP and inhibit the activity. Here, we examine mechanisms underlying the inhibition of MLCP through the phosphorylation of MYPT1 at Thr-696 and Thr-853 using GST fusion versions of various MYPT1 fragments including or excluding either or both of these phosphorylation sites. Phosphorylated MYPT1 fragments including either Thr-696 or Thr-853 potently and specifically inhibit MLCP purified from pig aorta and the enzyme associated with myofilaments in permeabilized ileum SM tissues. We further show that inhibition of MLCP in SM tissues is eliminated by activation of PKA/PKG, suggesting that the GST-MYPT1 fragments mimic agonist-induced autoinhibition and cAMP/cGMP-dependent dis-autoinhibition of MLCP in SM.