Roles of Interaction between Actuator and Nucleotide Binding Domains of Sarco(endo)plasmic Reticulum Ca2+-ATPase as Revealed by Single and Swap Mutational Analyses of Serine 186 and Glutamate 439

Roles of hydrogen bonding interaction between Ser¹⁸⁶ of the actuator (A) domain and Glu⁴³⁹ of nucleotide binding (N) domain seen in the structures of ADP-insensitive phosphorylated intermediate (E2P) of sarco(endo)plasmic reticulum Ca²⁺-ATPase were explored by their double alanine substitution S186A/E439A, swap substitution S186E/E439S, and each of these single substitutions. All the mutants except the swap mutant S186E/E439S showed markedly reduced Ca²⁺-ATPase activity, and S186E/E439S restored completely the wild-type activity. In all the mutants except S186E/E439S, the isomerization of ADP-sensitive phosphorylated intermediate (E1P) to E2P was markedly retarded, and the E2P hydrolysis was largely accelerated, whereas S186E/E439S restored almost the wild-type rates. Results showed that the Ser¹⁸⁶-Glu⁴³⁹ hydrogen bond stabilizes the E2P ground state structure. The modulatory ATP binding at sub-mm∼mm range largely accelerated the EP isomerization in all the alanine mutants and E439S. In S186E, this acceleration as well as the acceleration of the ATPase activity was almost completely abolished, whereas the swap mutation S186E/E439S restored the modulatory ATP acceleration with a much higher ATP affinity than the wild type. Results indicated that Ser¹⁸⁶ and Glu⁴³⁹ are closely located to the modulatory ATP binding site for the EP isomerization, and that their hydrogen bond fixes their side chain configurations thereby adjusts properly the modulatory ATP affinity to respond to the cellular ATP level.Sarcoplasmic reticulum Ca²⁺-ATPase (SERCA1a)² is a representative member of P-type ion-transporting ATPases and catalyzes Ca²⁺ transport coupled with ATP hydrolysis (Fig. 1) (1–9). In the catalytic cycle, the enzyme is activated by binding of two Ca²⁺ ions at the transport sites (E2 to E1Ca₂, steps 1–2) and then autophosphorylated at Asp³⁵¹ with MgATP to form ADP-sensitive phosphoenzyme (E1P, step 3), which can react with ADP to regenerate ATP. Upon formation of this EP, the bound Ca²⁺ ions are occluded in the transport sites (E1PCa₂). The subsequent isomeric transition to ADP-insensitive form (E2P) results in a change in the orientation of the Ca²⁺ binding sites and reduction of their affinity, and thus Ca²⁺ release into lumen (steps 4 and 5). Finally, the hydrolysis takes place and returns the enzyme into an unphosphorylated and Ca²⁺-unbound form (E2, step 6). E2P can also be formed from P_i in the presence of Mg²⁺ and the absence of Ca²⁺ by reversal of its hydrolysis.Open in a separate windowFIGURE 1.Reaction cycle of sarco(endo)plasmic reticulum Ca²⁺-ATPase.The cytoplasmic three domains N, A, and P largely move and change their organization states during the Ca²⁺ transport cycle (10–22). These changes are linked with the rearrangements in the transmembrane helices. In the EP isomerization (loss of ADP sensitivity) and Ca²⁺ release, the A domain largely rotates (by ∼110° parallel to membrane plane), intrudes into the space between the N and P domains, and the P domain largely inclines toward the A domain. Thus in E2P, these domains produce the most compactly organized state (see Fig. 2 for the change E1Ca₂·AlF₄⁻·ADP →E2·MgF₄²⁻ as the model for the overall process E1PCa₂·ADP^‡ → E2·P_i).Open in a separate windowFIGURE 2.Structure of SERCA1a and formation of Ser¹⁸⁶-Glu⁴³⁹ hydrogen bond between the A and N domains. The coordinates for the structures E1Ca₂·AlF₄⁻·ADP, (the analog for the transition state of the phosphoryl transfer E1PCa₂·ADP^‡, left panel) and E2·MgF₄²⁻ (E2·P_i analog (21), right panel) of Ca²⁺-ATPase were obtained from the Protein Data Bank (PDB accession code 1T5T and 1WPG, respectively (12, 14)). The arrows indicate approximate movements of the A and P domains in the change from E1Ca₂·AlF₄⁻ ·ADP to E2·MgF₄²⁻. Ser¹⁸⁶ and Glu⁴³⁹ are depicted as van der Waals spheres. These two residues form a hydrogen bond in E2·MgF₄²⁻ (see inset). The phosphorylation site Asp³⁵¹, two Ca²⁺ at the transport sites and ADP with AlF₄⁻ at the catalytic site in E1Ca₂·AlF₄⁻·ADP, MgF₄²⁻ bound at the catalytic site in E2·MgF₄²⁻ are depicted. The TGES¹⁸⁴ loop and Val²⁰⁰ loop of the A domain and Tyr¹²² on the top part of M2 are shown. These elements produce three interaction networks between A and P domains and M2 (Tyr¹²²) in E2·MgF₄²⁻ (23–26). M1′ and M1-M10 are also indicated.We have found that the interactions between the A and P domains at the Val²⁰⁰-loop (Asp¹⁹⁶-Asp²⁰³) with the residues of the P domain (Arg⁶⁷⁸/Glu⁶⁸⁰/Arg⁶⁵⁶/Asp⁶⁶⁰) (23) and at the Tyr¹²² hydrophobic cluster (24–26) (see Fig. 2) play critical roles for Ca²⁺ deocclusion/release in E2PCa₂ → E2P + 2Ca²⁺ after the loss of ADP sensitivity (E1PCa₂ to E2PCa₂ isomerization). The proper length of the A/M1′ linker is critical for inducing the inclining motion of the A and P domains for the Ca²⁺ deocclusion and release from E2PCa₂ (27, 28). The importance of the interdomain interaction between Arg⁶⁷⁸ (P) and Asp²⁰³ (A) in stabilizing the E2P and E2 intermediates and its influence on modulatory ATP activation were pointed out by the mutation R678A (29). Regarding the N domain, the importance of Glu⁴³⁹ in the EP isomerization and E2P hydrolysis was previously noted by its alanine substitution, and possible importance of its interaction with Ser¹⁸⁶ on the A domain has been suggested since Glu⁴³⁹ forms a hydrogen bond with Ser¹⁸⁶ in the E2P analog structures (29) (see Fig. 2). The Darier disease-causing mutations of Ser¹⁸⁶ of SERCA2b, S186P and S186F also alter the kinetics of the EP processing and its importance as the residue in the immediate vicinity of TGES¹⁸⁴ has been pointed out (30, 31). Notably also, Glu⁴³⁹ is situated near the adenine binding pocket and its importance in the ATP binding and ATP-induced structural change have been shown (32, 33). In the structure E2(TG)AMPPCP (E2·ATP), Glu⁴³⁹ interacts with the modulatory ATP binding via Mg²⁺, and is involved in the acceleration of the Ca²⁺-ATPase cycle (16).Considering these critical findings on each of Glu⁴³⁹ and Ser¹⁸⁶, it is crucial to reveal the role of the Ser¹⁸⁶-Glu⁴³⁹ hydrogen-bonding interaction between the A and N domains in the EP processing and its ATP modulation (i.e. regulatory ATP-induced acceleration). We therefore made a series of mutants on both Ser¹⁸⁶ and Glu⁴³⁹ including the swap substitution mutant, S186A, E439A, S186A/E439A, S186E, E439S, S186E/E439S, and explored their kinetic properties. Results showed that the Ser¹⁸⁶-Glu⁴³⁹ hydrogen bond is critical for the stabilization of the E2P ground state structure, and possibly functioning as to make the E2P resident time long enough for Ca²⁺ release (E2PCa₂ → E2P + 2Ca²⁺) thus to avoid its hydrolysis without Ca²⁺ release. Results also revealed that the side-chain configurations of Ser¹⁸⁶ and Glu⁴³⁹ are fixed by their hydrogen bond, thereby conferring the proper modulatory ATP binding to occur at the cellular ATP level to accelerate the rate-limiting EP isomerization.     

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.     

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.     

Loss of TMEM16A Causes a Defect in Epithelial Ca2+-dependent Chloride Transport

Molecular identification of the Ca²⁺-dependent chloride channel TMEM16A (ANO1) provided a fundamental step in understanding Ca²⁺-dependent Cl⁻ secretion in epithelia. TMEM16A is an intrinsic constituent of Ca²⁺-dependent Cl⁻ channels in cultured epithelia and may control salivary output, but its physiological role in native epithelial tissues remains largely obscure. Here, we demonstrate that Cl⁻ secretion in native epithelia activated by Ca²⁺-dependent agonists is missing in mice lacking expression of TMEM16A. Ca²⁺-dependent Cl⁻ transport was missing or largely reduced in isolated tracheal and colonic epithelia, as well as hepatocytes and acinar cells from pancreatic and submandibular glands of TMEM16A^−/− animals. Measurement of particle transport on the surface of tracheas ex vivo indicated largely reduced mucociliary clearance in TMEM16A^−/− mice. These results clearly demonstrate the broad physiological role of TMEM16A^−/− for Ca²⁺-dependent Cl⁻ secretion and provide the basis for novel treatments in cystic fibrosis, infectious diarrhea, and Sjöegren syndrome.Electrolyte secretion in epithelial tissues is based on the major second messenger pathways cAMP and Ca²⁺, which activate the cystic fibrosis transmembrane conductance regulator (CFTR)² Cl⁻ channels and Ca²⁺-dependent Cl⁻ channels, respectively (1–3). CFTR conducts Cl⁻ in epithelial cells of airways, intestine, and the ducts of pancreas and sweat gland, while Ca²⁺-dependent Cl⁻ channels secrete Cl⁻ in pancreatic acini and salivary and sweat glands (4–6). Controversy exists as to the contribution of these channels to Cl⁻ secretion in submucosal glands of airways and the relevance for cystic fibrosis (7–9). While cAMP-dependent Cl⁻ secretion by CFTR is well examined, detailed analysis of epithelial Ca²⁺-dependent Cl⁻ secretion is hampered by the lack of a molecular counterpart. Although bestrophins may form Ca²⁺-dependent Cl⁻ channels and facilitate Ca²⁺-dependent Cl⁻ secretion in epithelial tissues (10, 11), they are unlikely to form secretory Cl⁻ channels in the apical cell membrane, because Ca²⁺-dependent Cl⁻ secretion is still present in epithelia of mice lacking expression of bestrophin (12). Bestrophins may rather have an intracellular function by facilitating receptor mediated Ca²⁺ signaling and activation of membrane localized channels (13). With the discovery that TMEM16A produces Ca²⁺-activated Cl⁻ currents with biophysical and pharmacological properties close to those in native epithelial tissues, these proteins are now very likely candidates for endogenous Ca²⁺-dependent Cl⁻ channels (14–17). In cultured airway epithelial cells, small interfering RNA knockdown of endogenous TMEM16A largely reduced calcium-dependent chloride secretion (16). However, apart from preliminary studies of airways and salivary glands, the physiological significance of TMEM16A in native epithelia, particularly in glands, is unclear (14, 17).     

Kinase-dependent Regulation of Inositol 1,4,5-Trisphosphate-dependent Ca2+ Release during Oocyte Maturation

Fertilization induces a species-specific Ca²⁺ transient with specialized spatial and temporal dynamics, which are essential to temporally encode egg activation events such as the block to polyspermy and resumption of meiosis. Eggs acquire the competence to produce the fertilization-specific Ca²⁺ transient during oocyte maturation, which encompasses dramatic potentiation of inositol 1,4,5-trisphosphate (IP₃)-dependent Ca²⁺ release. Here we show that increased IP₃ receptor (IP₃R) sensitivity is initiated at the germinal vesicle breakdown stage of maturation, which correlates with maturation promoting factor (MPF) activation. Extensive phosphopeptide mapping of the IP₃R resulted in ∼70% coverage and identified three residues, Thr-931, Thr-1136, and Ser-114, which are specifically phos pho ryl a ted during maturation. Phospho-specific antibody analyses show that Thr-1136 phos pho ryl a tion requires MPF activation. Activation of either MPF or the mitogen-activated protein kinase cascade independently, functionally sensitizes IP₃-dependent Ca²⁺ release. Collectively, these data argue that the kinase cascades driving meiotic maturation potentiates IP₃-dependent Ca²⁺ release, possibly trough direct phos pho ryl a tion of the IP₃R.Egg activation refers to the cellular and molecular events that take place immediately following fertilization, transitioning the zygote into embryogenesis. In vertebrates, egg activation encompasses the block to polyspermy and the completion of oocyte meiosis, which is coupled to the extrusion of the second polar body. Interestingly, in all sexually reproducing organisms tested to date the cellular events associated with egg activation are Ca²⁺-dependent (1). Importantly the Ca²⁺ signal at fertilization encodes the progression of these cellular events in a defined temporal sequence that ensures a functional egg-to-embryo transition (2, 3). The first order of business for the fertilized egg is to block polyspermy, which could be lethal to the embryo. This presents a particularly difficult problem for the large Xenopus oocyte. Therefore, this species employs a fast and slow blocks to polyspermy, both of which are Ca²⁺-dependent (4). In addition, the Ca²⁺ release wave at fertilization releases the metaphase II cytostatic factor-dependent arrest in Xenopus oocytes. As is the case in other vertebrates, Xenopus eggs arrest at metaphase of meiosis II, an event that marks the completion of maturation.Therefore, Ca²⁺ dynamics at fertilization initiate and temporally encode critical cellular events for the egg-to-embryo transition. Specificity in Ca²⁺ signaling is encoded to a large extent in the spatial, temporal, and amplitude features of the Ca²⁺ signal. This endows Ca²⁺ signaling with its versatility and specificity, where in the same cell Ca²⁺ signals can mediate distinct cellular responses (5, 6).Ca²⁺ signaling pathways and intracellular organelles remodel during oocyte maturation, a complex cellular differentiation that prepares the egg for fertilization and egg activation (7, 8). In Xenopus the activity and distribution of multiple essential Ca²⁺-transporting proteins is modulated dramatically during oocyte maturation (8). Functional studies and mathematical modeling support the conclusion that the two critical determinants of Ca²⁺ signaling remodeling during Xenopus oocyte maturation are the internalization of the plasma-membrane Ca²⁺-ATPase, and the sensitization of inositol 1,4,5-trisphosphate (IP₃)²-dependent Ca²⁺ release (9–11). Indeed Ca²⁺ release from intracellular stores through the IP₃ receptor (IP₃R) represents the primary source for the initial Ca²⁺ rise at fertilization in vertebrates (12–14). The sensitivity of IP₃-dependent Ca²⁺ release is enhanced during maturation (10, 15). The IP₃R physically clusters during maturation (9, 16), and this is associated with functional clustering of elementary Ca²⁺ release events (10). IP₃R clustering is important for the slow and continuous nature of Ca²⁺ wave propagation in Xenopus eggs (10). In fact the potentiation of IP₃-dependent Ca²⁺ release is a hallmark of Ca²⁺ signaling differentiation during oocyte maturation in several vertebrate and invertebrate species (17–19). However, the mechanisms underlying enhanced IP₃-dependent Ca²⁺ release are not well understood.An attractive mechanism to explain increased IP₃R sensitivity during oocyte maturation is phosphorylation, given the critical role kinase cascades play in the initiation and progression of the meiotic cell cycle. Furthermore, the affinity of the IP₃R increases during mitosis apparently due to direct phosphorylation by maturation-promoting factor (MPF) (20, 21). In contrast, in starfish eggs, although the increase in Ca²⁺ release was dependent on MPF activation, MPF does not directly phosphorylate the IP₃R, but rather it appears to mediate its effect through the actin cytoskeleton (22, 23). More recently, the MAPK cascade has been shown to be important for shaping Ca²⁺ dynamics in mouse eggs (24). Together, these results argue that phosphorylation plays an important role in the sensitization of IP₃-dependent Ca²⁺ release during M-phase.Xenopus oocyte maturation is initiated by steroids that appear to act on a cell surface receptor (25). An important kinase cascade activated during maturation is the MAPK cascade that is initiated through the accumulation of Mos (Fig. 1A). This cascade culminates in the inhibition of Myt1, which phosphorylates and inhibits MPF. MPF is the key regulator of entry into M-phase and is composed of a Ser/Thr kinase subunit (cdk1) and cyclin B as a regulatory subunit. In addition, activation of Cdc25C is essential for oocyte maturation, because it represents the rate-limiting step in MPF activation (26). Cdc25C is phosphorylated by polo-like kinase through unknown upstream steps. In this work we analyze the functional regulation and phosphorylation pattern of the IP₃R during oocyte maturation to better understand the role of cell cycle kinases in modulating IP₃-dependent Ca²⁺ release.Open in a separate windowFIGURE 1.IP₃-dependent Ca²⁺ release dynamics during maturation. A, kinase cascades driving Xenopus oocyte maturation. B, oocytes were injected with caged-IP₃ and Oregan Green 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis 1 before imaging. Maturation was induced with progesterone, and cells were collected at different time points as indicated. Cells were imaged in line scan mode on a Zeiss LSM510 with the near UV 450 nm laser continuously on, at low intensity to produce a slow gradual IP₃ rise. After imaging each cell was lysed and analyzed individually for the activation state of MAPK and MPF. MPF was assayed using an anti-phospho-Tyr-15-cdk1 antibody (arrow). Dephosphorylation is indicative of MPF activation. MAPK activation was detected using a phospho-specific MAPK antibody (arrowhead). Tubulin was the loading control (dash). C, percent of cells at each time point that either exhibit no release for the duration of the line scan (No Rel., black), puffs only (puffs, green), puffs followed by a wave (Puff-Wave, blue), or only a Ca²⁺ wave (Wave, red). For each time point n = 11–23 cells. D, amplitude of the first peak during the line scan as compared with the maximal Ca²⁺ signal. Mean ± S.E. (n = 9–18). E, latency until the first Ca²⁺ signal (Time to first peak) as compared with the time required to reach maximal signal (Time to Max). Mean ± S.E. (n = 9–18). For C–E: oocytes (Ooc); cells treated with progesterone that have not undergone GVBD at 2 or more hours after progesterone (p > 2); cells at GVBD and up to 0.5 h after GVBD (GVBD 0–0.5); cells from 0.5 to 2.5 h after GVBD (GVBD 0.5–2.5); fully mature eggs at 3 or more hours after GVBD (>3 egg).     

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.     

Evidence for a Slow-Turnover Form of the Ca2+-Independent Phosphoenolpyruvate Carboxylase Kinase in the Aleurone-Endosperm Tissue of Germinating Barley Seeds

Phosphoenolpyruvate carboxylase (PEPC) activity was detected in aleurone-endosperm extracts of barley (Hordeum vulgare) seeds during germination, and specific anti-sorghum (Sorghum bicolor) C₄ PEPC polyclonal antibodies immunodecorated constitutive 103-kD and inducible 108-kD PEPC polypeptides in western analysis. The 103- and 108-kD polypeptides were radiolabeled in situ after imbibition for up to 1.5 d in ³²P-labeled inorganic phosphate. In vitro phosphorylation by a Ca²⁺-independent PEPC protein kinase (PK) in crude extracts enhanced the enzyme''s velocity and decreased its sensitivity to l-malate at suboptimal pH and [PEP]. Isolated aleurone cell protoplasts contained both phosphorylated PEPC and a Ca²⁺-independent PEPC-PK that was partially purified by affinity chromatography on blue dextran-agarose. This PK activity was present in dry seeds, and PEPC phosphorylation in situ during imbibition was not affected by the cytosolic protein-synthesis inhibitor cycloheximide, by weak acids, or by various pharmacological reagents that had proven to be effective blockers of the light signal transduction chain and PEPC phosphorylation in C₄ mesophyll protoplasts. These collective data support the hypothesis that this Ca²⁺-independent PEPC-PK was formed during maturation of barley seeds and that its presumed underlying signaling elements were no longer operative during germination.Higher-plant PEPC (EC 4.1.1.31) is subject to in vivo phosphorylation of a regulatory Ser located in the N-terminal domain of the protein. In vitro phosphorylation by a Ca²⁺-independent, low-molecular-mass (30–39 kD) PEPC-PK modulates PEPC regulation interactively by opposing metabolite effectors (e.g. allosteric activation by Glc-6-P and feedback inhibition by l-malate; Andreo et al., 1987), decreasing significantly the extent of malate inhibition of the leaf enzyme (Carter et al., 1991; Chollet et al., 1996; Vidal et al., 1996; Vidal and Chollet, 1997). These metabolites control the rate of phosphorylation of PEPC via an indirect target-protein effect (Wang and Chollet, 1993; Echevarría et al., 1994; Vidal and Chollet, 1997).Several lines of evidence support the view that this protein-Ser/Thr kinase is the physiologically relevant PEPC-PK (Li and Chollet, 1993; Chollet et al., 1996; Vidal et al., 1996; Vidal and Chollet, 1997). The presence and inducible nature of leaf PEPC-PK have been established further in various C₃, C₄, and CAM plant species (Chollet et al., 1996). In all cases, CHX proved to be a potent inhibitor of this up-regulation process so that apparent changes in the turnover rate of PEPC-PK itself or another, as yet unknown, protein factor were invoked to account for this observation (Carter et al., 1991; Jiao et al., 1991; Chollet et al., 1996). Consistent with this proposal are recent findings about PEPC-PK from leaves of C₃, C₄, and CAM plants that determined activity levels of the enzyme to depend on changes in the level of the corresponding translatable mRNA (Hartwell et al., 1996).Using a cellular approach we previously showed in sorghum (Sorghum bicolor) and hairy crabgrass (Digitaria sanguinalis) that PEPC-PK is up-regulated in C₄ mesophyll cell protoplasts following illumination in the presence of a weak base (NH₄Cl or methylamine; Pierre et al., 1992; Giglioli-Guivarc''h et al., 1996), with a time course (1–2 h) similar to that of the intact, illuminated sorghum (Bakrim et al., 1992) or maize leaf (Echevarría et al., 1990). This light- and weak-base-dependent process via a complex transduction chain is likely to involve sequentially an increase in pHc, inositol trisphosphate-gated Ca²⁺ channels of the tonoplast, an increase in cytosolic Ca²⁺, a Ca²⁺-dependent PK, and PEPC-PK.Considerably less is known about the up-regulation of PEPC-PK and PEPC phosphorylation in nongreen tissues. A sorghum root PEPC-PK purified on BDA was shown to phosphorylate in vitro both recombinant C₄ PEPC and the root C₃-like isoform, thereby decreasing the enzyme''s malate sensitivity (Pacquit et al., 1993). PEPC from soybean root nodules was phosphorylated in vitro and in vivo by an endogenous PK (Schuller and Werner, 1993; Zhang et al., 1995; Zhang and Chollet, 1997). A Ca²⁺-independent nodule PEPC-PK containing two active polypeptides (32–37 kD) catalyzed the incorporation of phosphate on a Ser residue of the target enzyme and was modulated by photosynthate transported from the shoots (Zhang and Chollet, 1997). Regulatory seryl phosphorylation of a heterotetrameric (α₂β₂) banana fruit PEPC by a copurifying, Ca²⁺-independent PEPC-PK was shown to occur in vitro (Law and Plaxton, 1997). Although phosphorylation was also detected in vivo and found to concern primarily the α-subunit, PEPC exists mainly in the dephosphorylated form in preclimacteric, climacteric, and postclimacteric fruit.In a previous study we showed that PEPC undergoes regulatory phosphorylation in aleurone-endosperm tissue during germination of wheat seeds (Osuna et al., 1996). Here we report on PEPC and the requisite PEPC-PK in germinating barley (Hordeum vulgare) seeds. PEPC was highly phosphorylated by a Ca²⁺-independent Ser/Thr PEPC-PK similar to that found in other plant systems studied previously (Chollet et al., 1996); however, the PK was already present in the dry seed and its activity did not require protein synthesis during imbibition.     

Borate-Rhamnogalacturonan II Bonding Reinforced by Ca2+ Retains Pectic Polysaccharides in Higher-Plant Cell Walls

The extent of in vitro formation of the borate-dimeric-rhamnogalacturonan II (RG-II) complex was stimulated by Ca²⁺. The complex formed in the presence of Ca²⁺ was more stable than that without Ca²⁺. A naturally occurring boron (B)-RG-II complex isolated from radish (Raphanus sativus L. cv Aokubi-daikon) root contained equimolar amounts of Ca²⁺ and B. Removal of the Ca²⁺ by trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid induced cleavage of the complex into monomeric RG-II. These data suggest that Ca²⁺ is a normal component of the B-RG-II complex. Washing the crude cell walls of radish roots with a 1.5% (w/v) sodium dodecyl sulfate solution, pH 6.5, released 98% of the tissue Ca²⁺ but only 13% of the B and 22% of the pectic polysaccharides. The remaining Ca²⁺ was associated with RG-II. Extraction of the sodium dodecyl sulfate-washed cell walls with 50 mm trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid, pH 6.5, removed the remaining Ca²⁺_, 78% of B, and 49% of pectic polysaccharides. These results suggest that not only Ca²⁺ but also borate and Ca²⁺ cross-linking in the RG-II region retain so-called chelator-soluble pectic polysaccharides in cell walls.Boron (B) is an essential element for higher plant growth, although its primary function is not known (Loomis and Durst, 1992). Determining the sites of B in cells is required to identify its function. In cultured tobacco cells more than 80% of cellular B is in the cell wall (Matoh et al., 1993), whereas the membrane fraction (Kobayashi et al., 1997) and protoplasts (Matoh et al., 1992) do not contain a significant amount of B. In radish (Raphanus sativus L. cv Aokubi-daikon) root cell walls, B cross-links two RG-II regions of pectic polysaccharides through a borate-diol ester (Kobayashi et al., 1995, 1996). The association of B with RG-II has been confirmed in sugar beet (Ishii and Matsunaga, 1996), bamboo (Kaneko et al., 1997), sycamore and pea (O''Neill et al., 1996), and red wine (Pellerin et al., 1996). In cultured tobacco cells the B associated with RG-II accounts for about 80% of the cell wall B (Kobayashi et al., 1997) and RG-II may be the exclusive carrier of B in higher plant cell walls (Matoh et al., 1996). Germanic acid, which partly substitutes for B in the growth of the B-deprived plants (Skok, 1957), also cross-links two RG-II chains (Kobayashi et al., 1997). These results suggest that the physiological role of B is to cross-link cell wall pectic polysaccharides in the RG-II region and thereby form a pectic network.It is believed that in the cell wall pectic polysaccharides are cross-linked with Ca²⁺, which binds to carboxyl groups of the polygalacturonic acid regions (Jarvis, 1984). Thus, the ability of B and Ca²⁺ to cross-link cell wall pectic polysaccharides needs to be evaluated. In this report we describe the B-RG-II complex of radish root and the role of B-RG-II and Ca²⁺ in the formation of a pectic network.     

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.     

G-protein-coupled Receptor Kinase-interacting Proteins Inhibit Apoptosis by Inositol 1,4,5-Triphosphate Receptor-mediated Ca2+ Signal Regulation

The inositol 1,4,5-trisphosphate (IP₃) receptor (IP₃R) is an intracellular IP₃-gated calcium (Ca²⁺) release channel and plays important roles in regulation of numerous Ca²⁺-dependent cellular responses. Many intracellular modulators and IP₃R-binding proteins regulate the IP₃R channel function. Here we identified G-protein-coupled receptor kinase-interacting proteins (GIT), GIT1 and GIT2, as novel IP₃R-binding proteins. We found that both GIT1 and GIT2 directly bind to all three subtypes of IP₃R. The interaction was favored by the cytosolic Ca²⁺ concentration and it functionally inhibited IP₃R activity. Knockdown of GIT induced and accelerated caspase-dependent apoptosis in both unstimulated and staurosporine-treated cells, which was attenuated by wild-type GIT1 overexpression or pharmacological inhibitors of IP₃R, but not by a mutant form of GIT1 that abrogates the interaction. Thus, we conclude that GIT inhibits apoptosis by modulating the IP₃R-mediated Ca²⁺ signal through a direct interaction with IP₃R in a cytosolic Ca²⁺-dependent manner.The inositol 1,4,5-trisphosphate (IP₃)³ receptor (IP₃R) consisting of three subtypes, IP₃R1, IP₃R2, and IP₃R3, is a tetrameric intracellular IP₃-gated calcium (Ca²⁺) release channel localized at the endoplasmic reticulum (ER) with its NH₂ terminus and COOH-terminal tail (CTT) exposed to the cytoplasm (1, 2; see Fig. 1A). IP₃Rs are composed of five functional domains. The long NH₂-terminal cytoplasmic region contains three domains, a coupling/suppressor domain, an IP₃-binding core domain, and an internal coupling domain. The COOH-terminal region has a six-membrane spanning channel domain and a short cytoplasmic CTT “gatekeeper domain” that is critical for IP₃R channel opening (2, 3). Ca²⁺ release activity of the IP₃R channel is regulated by many intracellular modulators (ATP, calmodulin, and Ca²⁺), protein kinases, and IP₃R-binding proteins (2, 4), and the tight regulation of IP₃R channel activity by these factors generates various spatial and temporal intracellular Ca²⁺ patterns such as Ca²⁺ spikes and Ca²⁺ oscillations, leading to numerous cellular responses (1, 2, 5, 6).Open in a separate windowFIGURE 1.GIT1 and GIT2 bind to all three subtypes of IP₃R. A, schematic of ER residential IP₃R. The CTT of IP₃R1 is used as bait in a yeast two-hybrid screen. B, schematic representation of GIT1, GIT2, and two GIT1 fragments identified from the yeast two-hybrid screen. Functional domains are indicated. ARF-GAP, ARF-specific GTPase-activating protein domain; ANK-REP, ankyrin repeats; CC, coiled-coil domains; SHD, the Spa2-homology domain; EF, EF-hand; IQ, IQ-like motifs; aa, amino acid. C, GIT1 binds to IP₃R1 in vitro. GST and GST-IP₃R1/CTT were incubated with mouse brain lysate for a pull-down assay. The input and pulled-down samples were probed with α-GIT1. D and E, GIT1 binds to IP₃R1 in vivo. Mouse brain lysates were processed to control IgG and α-IP₃R1 (D) or α-GIT1 (E) for IP. The input and IP samples were probed with α-GIT1 and α-IP₃R1. F and G, both GIT1 and GIT2 bind to all three IP₃R subtypes. HeLa cells coexpressing GFP-fused IP₃R1, IP₃R2, or IP₃R3 and mRFP-fused GIT1 (F) or GIT2 (G) were processed for IP using α-RFP. The input and IP samples were blotted with α-GFP (top) and α-RFP (bottom).One of the physiological roles of IP₃R-mediated Ca²⁺ signaling is a pro-apoptotic regulator during apoptosis. Ca²⁺ released from ER can stimulate several key enzymes activated during apoptosis such as endonucleases (7) and calpain (8). In addition, the close proximity of ER to mitochondria may facilitate the mitochondrial overload of Ca²⁺ released from the IP₃Rs with certain apoptotic stimuli, triggering the opening of the mitochondrial permeability transition pore and the release of apoptotic signaling molecules, such as cytochrome c and apoptosis-inducing factor, which leads to the activation of caspases (5, 6). Moreover, several key components of apoptotic cascades, such as cytochrome c (9) and anti-apoptosis proteins Bcl-2 (10, 11) and Bcl-X_L (12), have been reported to interact with the internal coupling domain and/or the CTT of IP₃R and enhance the Ca²⁺-release activity of IP₃Rs during apoptosis. In this study, we identified the ubiquitously expressed G-protein-coupled receptor kinase-interacting proteins (GIT) (13), GIT1 and GIT2, as novel IP₃R-binding proteins that bind to the CTT of IP₃R and inhibit apoptosis by regulation of IP₃R-mediated Ca²⁺ signal.     

T-type Ca2+ Channels Promote Oxygenation-induced Closure of the Rat Ductus Arteriosus Not Only by Vasoconstriction but Also by Neointima Formation

The ductus arteriosus (DA), an essential vascular shunt for fetal circulation, begins to close immediately after birth. Although Ca²⁺ influx through several membrane Ca²⁺ channels is known to regulate vasoconstriction of the DA, the role of the T-type voltage-dependent Ca²⁺ channel (VDCC) in DA closure remains unclear. Here we found that the expression of α1G, a T-type isoform that is known to exhibit a tissue-restricted expression pattern in the rat neonatal DA, was significantly up-regulated in oxygenated rat DA tissues and smooth muscle cells (SMCs). Immunohistological analysis revealed that α1G was localized predominantly in the central core of neonatal DA at birth. DA SMC migration was significantly increased by α1G overexpression. Moreover, it was decreased by adding α1G-specific small interfering RNAs or using R(−)-efonidipine, a highly selective T-type VDCC blocker. Furthermore, an oxygenation-mediated increase in an intracellular Ca²⁺ concentration of DA SMCs was significantly decreased by adding α1G-specific siRNAs or using R(−)-efonidipine. Although a prostaglandin E receptor EP4 agonist potently promoted intimal thickening of the DA explants, R(−)-efonidipine (10⁻⁶ m) significantly inhibited EP4-promoted intimal thickening by 40% using DA tissues at preterm in organ culture. Moreover, R(−)-efonidipine (10⁻⁶ m) significantly attenuated oxygenation-induced vasoconstriction by ∼27% using a vascular ring of fetal DA at term. Finally, R(−)-efonidipine significantly delayed the closure of in vivo DA in neonatal rats. These results indicate that T-type VDCC, especially α1G, which is predominantly expressed in neonatal DA, plays a unique role in DA closure, implying that T-type VDCC is an alternative therapeutic target to regulate the patency of DA.The ductus arteriosus (DA)² is an essential vascular shunt between the aortic arch and the pulmonary trunk during a fetal period (1). After birth, the DA closes immediately in accordance with its smooth muscle contraction and vascular remodeling, whereas the connecting vessels such as the aorta and pulmonary arteries remain open. When the DA fails to close after birth, the condition is known as patent DA, which is a common form of congenital heart defect. Patent DA is also a frequent problem with significant morbidity and mortality in premature infants. Investigating the molecular mechanism of DA closure is important not only for vascular biology but also for clinical problems in pediatrics.Voltage-dependent Ca²⁺ channels (VDCCs) consist of multiple subtypes, named L-, N-, P/Q-, R-, and T-type. L-type VDCCs are known to play a primary role in regulating Ca²⁺ influx and thus vascular tone in the development of arterial smooth muscle including the DA (2–4). Our previous study demonstrated that all T-type VDCCs were expressed in the rat DA (5). α1G subunit, especially, was the most dominant isoform among T-type VDCCs. The abundant expression of α1G subunit suggests that it plays a role in the vasoconstriction and vascular remodeling of the DA. In this regard, Nakanishi et al. (6) demonstrated that 0.5 mm nickel, which blocks T-type VDCC, inhibited oxygen-induced vasoconstriction of the rabbit DA. On the other hand, Tristani-Firouzi et al. (7) demonstrated that T-type VDCCs exhibited little effect on oxygen-sensitive vasoconstriction of the rabbit DA. Thus, the role of T-type VDCCs in DA vasoconstriction has remained controversial.In addition to their role in determining the contractile state, a growing body of evidence has demonstrated that T-type VDCCs play an important role in regulating differentiation (8, 9), proliferation (10–12), migration (13, 14), and gene expression (15) in vascular smooth muscle cells (SMCs). Hollenbeck et al. (16) and Patel et al. (17) demonstrated that nickel inhibited platelet-derived growth factor-BB-induced SMC migration. Rodman et al. (18) demonstrated that α1G promoted SMC proliferation in the pulmonary artery. The DA dramatically changes its morphology during development. Intimal cushion formation, a characteristic feature of vascular remodeling of the DA (19–21), involves many cellular processes: an increase in SMC migration and proliferation, production of hyaluronic acid under the endothelial layer, impaired elastin fiber assembly, and so on (1, 19, 21–23). Although our previous study demonstrated that T-type VDCCs are involved in smooth muscle cell proliferation in the DA (5), the role of T-type VDCCs in vascular remodeling of the DA has remained poorly understood.In the present study, we hypothesized that T-type VDCCs, especially α1G subunit, associate with vascular remodeling and vasoconstriction in the DA. To test our hypothesis, we took full advantage of recent molecular and pharmacological developments. We chose the recently developed, highly selective T-type VDCC blocker R(−)-efonidipine instead of low dose nickel for our study. Selective inhibition or activation of α1G subunit was also obtained using small interfering RNA (siRNA) technology or by overexpression of the α1G subunit gene, respectively. We found that Ca²⁺ influx through T-type VDCCs promoted oxygenation-induced DA closure through SMC migration and vasoconstriction.     

Plasma Membrane-associated Annexin A6 Reduces Ca2+ Entry by Stabilizing the Cortical Actin Cytoskeleton

The annexins are a family of Ca²⁺- and phospholipid-binding proteins, which interact with membranes upon increase of [Ca²⁺]_i or during cytoplasmic acidification. The transient nature of the membrane binding of annexins complicates the study of their influence on intracellular processes. To address the function of annexins at the plasma membrane (PM), we fused fluorescent protein-tagged annexins A6, A1, and A2 with H- and K-Ras membrane anchors. Stable PM localization of membrane-anchored annexin A6 significantly decreased the store-operated Ca²⁺ entry (SOCE), but did not influence the rates of Ca²⁺ extrusion. This attenuation was specific for annexin A6 because PM-anchored annexins A1 and A2 did not alter SOCE. Membrane association of annexin A6 was necessary for a measurable decrease of SOCE, because cytoplasmic annexin A6 had no effect on Ca²⁺ entry as long as [Ca²⁺]_i was below the threshold of annexin A6-membrane translocation. However, when [Ca²⁺]_i reached the levels necessary for the Ca²⁺-dependent PM association of ectopically expressed wild-type annexin A6, SOCE was also inhibited. Conversely, knockdown of the endogenous annexin A6 in HEK293 cells resulted in an elevated Ca²⁺ entry. Constitutive PM localization of annexin A6 caused a rearrangement and accumulation of F-actin at the PM, indicating a stabilized cortical cytoskeleton. Consistent with these findings, disruption of the actin cytoskeleton using latrunculin A abolished the inhibitory effect of PM-anchored annexin A6 on SOCE. In agreement with the inhibitory effect of annexin A6 on SOCE, constitutive PM localization of annexin A6 inhibited cell proliferation. Taken together, our results implicate annexin A6 in the actin-dependent regulation of Ca²⁺ entry, with consequences for the rates of cell proliferation.Calcium entry into cells either through voltage- or receptor-operated channels, or following the depletion of intracellular stores is a major factor in maintaining intracellular Ca²⁺ homeostasis. Resting [Ca²⁺]_i is low (∼100 nm compared with extracellular [Ca²⁺]_ex of 1.2 mm) and can be rapidly increased by inositol triphosphate-mediated release from the intracellular Ca²⁺ stores (mostly endoplasmic reticulum (ER)³), or by channel-mediated influx across the plasma membrane (PM). Store-operated calcium entry (SOCE) has been proposed as the main process controlling Ca²⁺ entry in non-excitable cells (1), and the recent discovery of Orai1 and STIM provided the missing link between the Ca²⁺-release activated current (I_CRAC) and the ER Ca²⁺ sensor (2–4). Translocation of STIM within the ER, accumulation in punctae at the sites of contact with PM and activation of Ca²⁺ channels have been proposed as a model of its regulation of Orai1 activity (5, 6). However, many details of the functional STIM-Orai1 protein complex and its regulation remain to be elucidated. The actin cytoskeleton plays a major role in the regulation of SOCE, possibly by influencing the function of ion channels or by interfering with the interaction between STIM and Orai1 (7–9). However, the proteins connecting the actin cytoskeleton and SOCE activity at the PM have yet to be identified.The annexins are a multigene family of Ca²⁺- and phospholipid-binding proteins, which have been implicated in many Ca²⁺-regulated processes. Their C-terminal core is evolutionarily conserved and contains Ca²⁺-binding sites, their N-terminal tails are unique and enable the protein to interact with distinct cytoplasmic partners. At low [Ca²⁺]_i, annexins are diffusely distributed throughout the cytosol, however, after stimulation resulting in the increase of [Ca²⁺]_i, annexins are targeted to distinct subcellular membrane locations, such as the PM, endosomes, or secretory vesicles (10). Annexins are involved in the processes of vesicle trafficking, cell division, apoptosis, calcium signaling, and growth regulation (11), and frequent changes in expression levels of annexins are observed in disease (12, 13). Previously, using biochemical methods and imaging of fluorescent protein-tagged annexins in live cells, we demonstrated that annexins A1, A2, A4, and A6 interacted with the PM as well as with internal membrane systems in a highly coordinated manner (10, 14). In addition, there is evidence of Ca²⁺-independent membrane association of several annexins, including annexin A6 (15–19); some of which point to the existence of pH-dependent binding mechanisms (20–22). Given the fact that several annexins are present within any one cell, it is likely that they form a [Ca²⁺] and pH sensing system, with a regulatory influence on other signaling pathways.The role of annexins as regulators of ion channel activity has been addressed previously (23–25). In particular, annexin A6 has been implicated in regulation of the sarcoplasmic reticulum ryanodine-sensitive Ca²⁺ channel (25), the neuronal K⁺ and Ca²⁺ channels (26), and the cardiac Na⁺/Ca²⁺ exchanger (27). Cardiac-specific overexpression of annexin A6 resulted in lower basal [Ca²⁺], a depression of [Ca²⁺]_i transients and impaired cardiomyocyte contractility (28). In contrast, the cardiomyocytes from the annexin A6 null-mutant mice showed increased contractility and accelerated Ca²⁺ clearance (29). Consistent with its role in mediating the intracellular Ca²⁺ signals, especially Ca²⁺ influx, ectopic overexpression of annexin A6 in A431 cells, which lack endogenous annexin A6, resulted in inhibition of EGF-dependent Ca²⁺ entry (30).The difficulty of investigating the influence of annexins on signaling events occurring at the PM lies in the transient and reversible nature of their Ca²⁺ and pH-dependent lipid binding. Although the intracellular Ca²⁺ increase following receptor activation or Ca²⁺ influx promotes the association of the Ca²⁺-sensitive annexins A2 and A6 with the PM, the proteins quickly resume their cytoplasmic localization upon restoration of the basal [Ca²⁺]_i (14). Therefore, to investigate the effects of membrane-associated annexins on Ca²⁺ homeostasis and the cell signaling machinery, we aimed to develop a model system allowing for a constitutive membrane association of annexins. Here we used the PM-anchoring sequences of the H- and K-Ras proteins to target annexins A6 and A1 to the PM independently of [Ca²⁺]. The Ras GTPases are resident at the inner leaflet of the PM and function as molecular switches (31). The C-terminal 9 amino acids of H- and N-Ras and the C-terminal 14 amino acids of K-Ras comprise the signal sequences for membrane anchoring of Ras isoforms (32). Although the palmitoylation and farnesylation of the C terminus of H-Ras (tH) serves as a targeting signal for predominantly cholesterol-rich membrane microdomains at the PM (lipid rafts/caveolae) (33), the polybasic group and the lipid anchor of K-Ras (tK) ensures the association of K-Ras with cholesterol-poor PM membrane domains. Importantly, these minimal C-terminal amino acid sequences are sufficient to target heterologous proteins, for example GFP, to different microdomains at the PM and influence their trafficking (34).In the present study we fused annexins A6, A2, and A1 with fluorescent proteins and introduced the PM-anchoring sequences of either H-Ras (annexin-tH) or K-Ras (annexin-tK) at the C termini of the fusion constructs. We demonstrate that the constitutive PM localization of annexin A6 results in down-regulation of store-operated Ca²⁺ entry. Expression of membrane-anchored annexin A6 causes an accumulation of the cortical F-actin, and cytoskeletal destabilization with latrunculin A abolishes the inhibitory effect of PM-anchored annexin A6 on SOCE. Taken together, our results implicate annexin A6 in the maintenance of intracellular Ca²⁺ homeostasis via actin-dependent regulation of Ca²⁺ entry.     

Localizing the Membrane Binding Region of Group VIA Ca2+-independent Phospholipase A2 Using Peptide Amide Hydrogen/Deuterium Exchange Mass Spectrometry

The Group VIA-2 Ca²⁺-independent phospholipase A₂ (GVIA-2 iPLA₂) is composed of seven consecutive N-terminal ankyrin repeats, a linker region, and a C-terminal phospholipase catalytic domain. No structural information exists for this enzyme, and no information is known about the membrane binding surface. We carried out deuterium exchange experiments with the GVIA-2 iPLA₂ in the presence of both phospholipid substrate and the covalent inhibitor methyl arachidonoyl fluorophosphonate and located regions in the protein that change upon lipid binding. No changes were seen in the presence of only methyl arachidonoyl fluorophosphonate. The region with the greatest change upon lipid binding was region 708–730, which showed a >70% decrease in deuteration levels at numerous time points. No decreases in exchange due to phospholipid binding were seen in the ankyrin repeat domain of the protein. To locate regions with changes in exchange on the enzyme, we constructed a computational homology model based on homologous structures. This model was validated by comparing the deuterium exchange results with the predicted structure. Our model combined with the deuterium exchange results in the presence of lipid substrate have allowed us to propose the first structural model of GVIA-2 iPLA₂ as well as the interfacial lipid binding region.The Group VIA phospholipase A₂ is a member of the phospholipase A₂ superfamily that cleaves fatty acids from the sn-2 position of phospholipids (1, 2). The human Group VIA PLA₂³ gene yields multiple splice variants, including GVIA-1, GVIA-2, GVIA-3 PLA₂, GVIA Ankyrin-1, and GVIA Ankyrin-2 (3, 4). At least two isoforms, GVIA-1 and GVIA-2 iPLA₂, are active. Our laboratory purified and characterized the first mammalian iPLA₂, the 85-kDa GVIA-2 iPLA₂ (5), which became the first cloned iPLA₂ (6). This enzyme can hydrolyze the sn-2 fatty acyl bond of phospholipids and also has potent lysophospholipase and transacylase activity (7). GVIA iPLA₂ is involved in cell proliferation (8), apoptosis (9–11), bone formation (12), sperm development (13), and glucose-induced insulin secretion (14, 15), so its function may vary by cell and tissue.The human GVIA-2 iPLA₂ (806 amino acids), the form of the enzyme studied here, contains seven ankyrin repeats (residues 152–382), a linker region (residues 383–474) with the eighth repeat disrupted by a 54-amino acid insert (16), and a catalytic domain (residues 475–806). The active site serine of the GVIA iPLA₂ lies within a lipase consensus sequence (Gly-X-Ser⁵¹⁹-X-Gly) (1). The activity of GVIA iPLA₂ has been reported to be regulated through several mechanisms. A caspase-3 cleavage site at the N terminus of the enzyme has been identified that is clipped in vitro (17). This truncated form of the enzyme was hyperactive and reduced cell viability when overexpressed in HEK293 cells (17). Another possible control mechanism is through ATP binding on the ⁴⁸⁵GXGXXG motif (18).The activity of phospholipases depends critically on the interaction of the protein with phospholipid membranes. In vitro, GVIA iPLA₂ does not have any specificity for the fatty acid in the sn-2 position of substrate phospholipids (5). GVIA-2 iPLA₂ was found to be membrane-associated when overexpressed in COS-7 cells, and this was further confirmed in rat vascular smooth muscle cells (4, 19). The other active splice variant, GVIA-1, is cytosolic and not specific in targeting membrane surfaces (4, 19), indicating two different regulatory mechanisms between these two splice variants. The 54-residue insertion in the eighth ankyrin repeat alters the property of GVIA-2 iPLA₂ for membrane association. The ankyrin repeats have been reported to be involved in protein-protein interactions, such as 53BP2-p53, GA-binding protein α-GA-binding protein β, p16^INK4a-CDK6, and IκBα-NFκB (16). The ankyrin repeats of GVIA iPLA₂ may directly or indirectly assist membrane association because the catalytic domain by itself does not have activity (3). Determining the regions of the protein that interact with the membrane surface will allow for a more in-depth analysis of the regulatory mechanisms of the enzyme.There is an increasing interest in GVIA iPLA₂ because of its various newly discovered functions in vivo and in vitro. However, there is no published crystal or NMR structure to facilitate analysis on the molecular level. Amide hydrogen/deuterium exchange coupled with mass spectrometry (DXMS) has been widely used to analyze the interface of protein-protein interactions (20), protein conformational changes (21, 22), and protein dynamics (23), and we have now introduced it to study protein-phospholipid interactions (24, 25). There are also reports of using DXMS with homology modeling to validate enzymes where structural information does not exist (26). We used deuterium exchange along with homology modeling to generate models of the ankyrin repeats based on the Ankyrin-R (Protein Data Bank code 1N11) and of the catalytic domain based on patatin (Protein Data Bank code 1OXW). To study the interfacial activation of GVIA iPLA₂, we generated 1-palmitoyl-2-arachidonoyl-sn-phosphatidylcholine (PAPC) vesicles containing the methyl arachidonyl fluorophosphonate (MAFP) inhibitor, which binds to the active site and irreversibly inhibits GVIA iPLA₂ (7). By applying DXMS to the iPLA₂ and using our structural model, we were now able to monitor how GVIA iPLA₂ associates with phospholipid membranes.     

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.     

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.