Formation of the Stable Structural Analog of ADP-sensitive Phosphoenzyme of Ca2+-ATPase with Occluded Ca2+ by Beryllium Fluoride: STRUCTURAL CHANGES DURING PHOSPHORYLATION AND ISOMERIZATION*

As a stable analog for ADP-sensitive phosphorylated intermediate of sarcoplasmic reticulum Ca²⁺-ATPase E1PCa₂·Mg, a complex of E1Ca₂·BeF_x, was successfully developed by addition of beryllium fluoride and Mg²⁺ to the Ca²⁺-bound state, E1Ca₂. In E1Ca₂·BeF_x, most probably E1Ca₂·BeF₃⁻, two Ca²⁺ are occluded at high affinity transport sites, its formation required Mg²⁺ binding at the catalytic site, and ADP decomposed it to E1Ca₂, as in E1PCa₂·Mg. Organization of cytoplasmic domains in E1Ca₂·BeF_x was revealed to be intermediate between those in E1Ca₂·AlF₄⁻ ADP (transition state of E1PCa₂ formation) and E2·BeF₃⁻·(ADP-insensitive phosphorylated intermediate E2P·Mg). Trinitrophenyl-AMP (TNP-AMP) formed a very fluorescent (superfluorescent) complex with E1Ca₂·BeF_x in contrast to no superfluorescence of TNP-AMP bound to E1Ca₂·AlF_x. E1Ca₂·BeF_x with bound TNP-AMP slowly decayed to E1Ca₂, being distinct from the superfluorescent complex of TNP-AMP with E2·BeF₃⁻, which was stable. Tryptophan fluorescence revealed that the transmembrane structure of E1Ca₂·BeF_x mimics E1PCa₂·Mg, and between those of E1Ca₂·AlF₄⁻·ADP and E2·BeF₃⁻. E1Ca₂·BeF_x at low 50–100 μm Ca²⁺ was converted slowly to E2·BeF₃⁻ releasing Ca²⁺, mimicking E1PCa₂·Mg → E2P·Mg + 2Ca²⁺. Ca²⁺ replacement of Mg²⁺ at the catalytic site at approximately millimolar high Ca²⁺ decomposed E1Ca₂·BeF_x to E1Ca₂. Notably, E1Ca₂·BeF_x was perfectly stabilized for at least 12 days by 0.7 mm lumenal Ca²⁺ with 15 mm Mg²⁺. Also, stable E1Ca₂·BeF_x was produced from E2·BeF₃⁻ at 0.7 mm lumenal Ca²⁺ by binding two Ca²⁺ to lumenally oriented low affinity transport sites, as mimicking the reverse conversion E2P· Mg + 2Ca²⁺ → E1PCa₂·Mg.Sarcoplasmic reticulum Ca²⁺-ATPase (SERCA1a),² a representative member of the P-type ion transporting ATPases, catalyze Ca²⁺ transport coupled with ATP hydrolysis (Fig. 1) (1–9). The enzyme forms phosphorylated intermediates from ATP or P_i in the presence of Mg²⁺ (10–13). In the transport cycle, the enzyme is first activated by cooperative binding of two Ca²⁺ ions at high affinity transport sites (E2 to E1Ca₂, steps 1–2) (14) and autophosphorylated at Asp³⁵¹ with MgATP to form the ADP-sensitive phosphoenzyme (E1P, step 3), which reacts with ADP to regenerate ATP in the reverse reaction. Upon this E1P formation, the two bound Ca²⁺ are occluded in the transport sites (E1PCa₂). Subsequent isomeric transition to the ADP-insensitive form (E2PCa₂), i.e. loss of ADP sensitivity at the catalytic site, results in rearrangement of the Ca²⁺ binding sites to deocclude Ca²⁺, reduce the affinity, and open the lumenal gate, thus releasing Ca²⁺ into the lumen (E2P, steps 4–5). Finally Asp³⁵¹-acylphosphate in E2P is hydrolyzed to form the Ca²⁺-unbound inactive E2 state (steps 6 and 7). Mg²⁺ bound at the catalytic site is required as a physiological catalytic cofactor in phosphorylation and dephosphorylation and thus for the transport cycle. The cycle is totally reversible, e.g. E2P can be formed from P_i in the presence of Mg²⁺ and absence of Ca²⁺, and subsequent Ca²⁺ binding at lumenally oriented low affinity transport sites of E2P reverses the Ca²⁺-releasing step and produces E1PCa₂, which is then decomposed to E1Ca₂ by ADP.Open in a separate windowFIGURE 1.Ca²⁺ transport cycle of Ca²⁺-ATPase.Various intermediate structural states in the transport cycle were fixed as their structural analogs produced by appropriate ligands such as AMP-PCP (non-hydrolyzable ATP analog) or metal fluoride compounds (phosphate analogs), and their crystal structures were solved at the atomic level (15–22). The three cytoplasmic domains, N, P, and A, largely move and change their organization state during the transport cycle, and the changes are coupled with changes in the transport sites. Most remarkably, in the change from E1Ca₂·AlF₄⁻·ADP (the transition state for E1PCa₂ formation, E1PCa₂·ADP·Mg^‡) to E2·BeF₃⁻ (the ground state E2P·Mg) (23–25), the A domain largely rotates by more than 90° approximately parallel to the membrane plane and associates with the P domain, thereby destroying the Ca²⁺ binding sites, and opening the lumenal gate, thus releasing Ca²⁺ into the lumen (see Fig. 2). E1PCa₂·Ca·AMP-PN formed by CaAMP-PNP without Mg²⁺ is nearly the same as E1Ca₂·AlF₄⁻·ADP and E1Ca₂·CaAMP-PCP in their crystal structures (17, 18, 22).Open in a separate windowFIGURE 2.Structure of SERCA1a and its change during processing of phosphorylated intermediate. E1Ca₂·AlF₄⁻·ADP (the transition state analog for phosphorylation E1PCa₂·ADP·Mg^‡) and E2·BeF₃⁻ (the ground state E2P analog (25)) were obtained from the Protein Data Bank (PDB accession code 1T5T (17) and 2ZBE (21), respectively). Cytoplasmic domains N (nucleotide binding), P (phosphorylation), and A (actuator), and 10 transmembrane helices (M1–M10) are indicated. The arrows on the domains, M1′ and M2 (Tyr¹²²) in E1Ca₂·AlF₄⁻·ADP, indicate their approximate motions predicted for E1PCa₂·ADP·Mg^‡ → E2P·Mg. The phosphorylation site Asp³⁵¹, TGES¹⁸⁴ of the A domain, Arg¹⁹⁸ (tryptic T2 site) on the Val²⁰⁰ loop (DPR¹⁹⁸AV²⁰⁰NQD) of the A domain, and Thr²⁴² (proteinase K site) on the A/M3-linker are shown. Seven hydrophobic residues gather in the E2P state to form the Tyr¹²²-hydrophobic cluster (Y122-HC); Tyr¹²²/Leu¹¹⁹ on the top part of M2, Ile¹⁷⁹/Leu¹⁸⁰/Ile²³² of the A domain, and Val⁷⁰⁵/Val⁷²⁶ of the P domain. The overall structure of E1Ca₂·AlF₄⁻·ADP is virtually the same as those of E1Ca₂·CaAMP-PCP and E1PCa₂·Ca·AMP-PN (17, 18, 22).Despite these atomic structures, yet unsolved is the structure of E1PCa₂·Mg, the genuine physiological intermediate E1PCa₂ with bound Mg²⁺ at the catalytic site without the nucleotide. Its stable structural analog has yet to be developed. E1PCa₂·Mg is the major intermediate accumulating almost exclusively at steady state under physiological conditions. Its rate-limiting isomerization results in Ca²⁺ deocclusion/release producing E2P·Mg as a key event for Ca²⁺ transport. In E1Ca₂·CaAMP-PCP, E1Ca₂·AlF₄⁻·ADP, and E1PCa₂·Ca·AMP-PN, the N and P domains are cross-linked and strongly stabilized by the bound nucleotide and/or Ca²⁺ at the catalytic site, thus they are crystallized (17, 18, 22). Kinetically, E1PCa₂·Ca formed with CaATP is markedly stabilized due to Ca²⁺ binding at the catalytic Mg²⁺ site, and its isomerization to E2P is strongly retarded in contrast to E1PCa₂·Mg (26, 27). Thus, the bound Ca²⁺ at the catalytic Mg²⁺ site likely produces a significantly different structural state from that with bound Mg²⁺.Therefore, it is now essential to develop a genuine E1PCa₂·Mg analog without bound nucleotide and thereby gain further insight into the structural mechanism in the Ca²⁺ transport process. It is also crucial to further clarify the structural importance of Mg²⁺ as the physiological catalytic cation. In this study, we successfully developed the complex E1Ca₂·BeF_x, most probably E1Ca₂·BeF₃⁻, as the E1PCa₂·Mg analog by adding beryllium fluoride (BeF_x) to the E1Ca₂ state without any nucleotides. For its formation, Mg²⁺ binding at the catalytic site was required and Ca²⁺ substitution for Mg²⁺ was absolutely unfavorable, revealing a likely structural reason for its preference as the physiological cofactor. In E1Ca₂·BeF₃⁻, two Ca²⁺ ions bound at the high affinity transport sites are occluded. It was also produced from E2·BeF₃⁻ by lumenal Ca²⁺ binding at the lumenally oriented low affinity transport sites, mimicking E2P·Mg + 2Ca²⁺ → E1PCa₂·Mg. All properties of the newly developed E1Ca₂·BeF₃⁻ fulfilled the requirements as the E1PCa₂·Mg analog, and hence we were able to uncover the hitherto unknown nature of E1PCa₂·Mg as well as structural events occurring in the phosphorylation and isomerization processes. Also, we successfully found the conditions that perfectly stabilize the E1Ca₂·BeF₃⁻ complex.     

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.     

Structural Insights into Ca2+-dependent Regulation of Inositol 1,4,5-Trisphosphate Receptors by CaBP1

Calcium-binding protein 1 (CaBP1), a neuron-specific member of the calmodulin (CaM) superfamily, modulates Ca²⁺-dependent activity of inositol 1,4,5-trisphosphate receptors (InsP₃Rs). Here we present NMR structures of CaBP1 in both Mg²⁺-bound and Ca²⁺-bound states and their structural interaction with InsP₃Rs. CaBP1 contains four EF-hands in two separate domains. The N-domain consists of EF1 and EF2 in a closed conformation with Mg²⁺ bound at EF1. The C-domain binds Ca²⁺ at EF3 and EF4, and exhibits a Ca²⁺-induced closed to open transition like that of CaM. The Ca²⁺-bound C-domain contains exposed hydrophobic residues (Leu¹³², His¹³⁴, Ile¹⁴¹, Ile¹⁴⁴, and Val¹⁴⁸) that may account for selective binding to InsP₃Rs. Isothermal titration calorimetry analysis reveals a Ca²⁺-induced binding of the CaBP1 C-domain to the N-terminal region of InsP₃R (residues 1-587), whereas CaM and the CaBP1 N-domain did not show appreciable binding. CaBP1 binding to InsP₃Rs requires both the suppressor and ligand-binding core domains, but has no effect on InsP₃ binding to the receptor. We propose that CaBP1 may regulate Ca²⁺-dependent activity of InsP₃Rs by promoting structural contacts between the suppressor and core domains.Calcium ion (Ca²⁺) in the cell functions as an important messenger that controls neurotransmitter release, gene expression, muscle contraction, apoptosis, and disease processes (1). Receptor stimulation in neurons promotes large increases in intracellular Ca²⁺ levels controlled by Ca²⁺ release from intracellular stores through InsP₃Rs (2). The neuronal type-1 receptor (InsP₃R1)² is positively and negatively regulated by cytosolic Ca²⁺ (3-6), important for the generation of repetitive Ca²⁺ transients known as Ca²⁺ spikes and waves (1). Ca²⁺-dependent activation of InsP₃R1 contributes to the fast rising phase of Ca²⁺ signaling known as Ca²⁺-induced Ca²⁺ release (7). Ca²⁺-induced inhibition of InsP₃R1, triggered at higher cytosolic Ca²⁺ levels, coordinates the temporal decay of Ca²⁺ transients (6). The mechanism of Ca²⁺-dependent regulation of InsP₃Rs is complex (8, 9), and involves direct Ca²⁺ binding sites (5, 10) as well as remote sensing by extrinsic Ca²⁺-binding proteins such as CaM (11, 12), CaBP1 (13, 14), CIB1 (15), and NCS-1 (16).Neuronal Ca²⁺-binding proteins (CaBP1-5 (17)) represent a new sub-branch of the CaM superfamily (18) that regulate various Ca²⁺ channel targets. Multiple splice variants and isoforms of CaBPs are localized in different neuronal cell types (19-21) and perform specialized roles in signal transduction. CaBP1, also termed caldendrin (22), has been shown to modulate the Ca²⁺-sensitive activity of InsP₃Rs (13, 14). CaBP1 also regulates P/Q-type voltage-gated Ca²⁺ channels (23), L-type channels (24), and the transient receptor potential channel, TRPC5 (25). CaBP4 regulates Ca²⁺-dependent inhibition of L-type channels in the retina and may be genetically linked to retinal degeneration (26). Thus, the CaBP proteins are receiving increased attention as a family of Ca²⁺ sensors that control a variety of Ca²⁺ channel targets implicated in neuronal degenerative diseases.CaBP proteins contain four EF-hands, similar in sequence to those found in CaM and troponin C (18) (Fig. 1). By analogy to CaM (27), the four EF-hands are grouped into two domains connected by a central linker that is four residues longer in CaBPs than in CaM. In contrast to CaM, the CaBPs contain non-conserved amino acids within the N-terminal region that may confer target specificity. Another distinguishing property of CaBPs is that the second EF-hand lacks critical residues required for high affinity Ca²⁺ binding (17). CaBP1 binds Ca²⁺ only at EF3 and EF4, whereas it binds Mg²⁺ at EF1 that may serve a functional role (28). Indeed, changes in cytosolic Mg²⁺ levels have been detected in cortical neurons after treatment with neurotransmitter (29). Other neuronal Ca²⁺-binding proteins such as DREAM (30), CIB1 (31), and NCS-1 (32) also bind Mg²⁺ and exhibit Mg²⁺-induced physiological effects. Mg²⁺ binding in each of these proteins helps stabilize their Ca²⁺-free state to interact with signaling targets.Open in a separate windowFIGURE 1.Amino acid sequence alignment of human CaBP1 with CaM. Secondary structural elements (α-helices and β-strands) were derived from NMR analysis. The four EF-hands (EF1, EF2, EF3, and EF4) are highlighted green, red, cyan, and yellow. Residues in the 12-residue Ca²⁺-binding loops are underlined and chelating residues are highlighted bold. Non-conserved residues in the hydrophobic patch are colored red.Despite extensive studies on CaBP1, little is known about its structure and target binding properties, and regulation of InsP₃Rs by CaBP1 is somewhat controversial and not well understood. Here, we present the NMR solution structures of both Mg²⁺-bound and Ca²⁺-bound conformational states of CaBP1 and their structural interactions with InsP₃R1. These CaBP1 structures reveal important Ca²⁺-induced structural changes that control its binding to InsP₃R1. Our target binding analysis demonstrates that the C-domain of CaBP1 exhibits Ca²⁺-induced binding to the N-terminal cytosolic region of InsP₃R1. We propose that CaBP1 may regulate Ca²⁺-dependent channel activity in InsP₃Rs by promoting a structural interaction between the N-terminal suppressor and ligand-binding core domains that modulates Ca²⁺-dependent channel gating (8, 33, 34).     

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.     

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).     

Focus Issue on Calcium Signaling: Analyses of a Gravistimulation-Specific Ca2+ Signature in Arabidopsis using Parabolic Flights

Gravity is a critical environmental factor affecting the morphology and functions of organisms on the Earth. Plants sense changes in the gravity vector (gravistimulation) and regulate their growth direction accordingly. In Arabidopsis (Arabidopsis thaliana) seedlings, gravistimulation, achieved by rotating the specimens under the ambient 1g of the Earth, is known to induce a biphasic (transient and sustained) increase in cytoplasmic calcium concentration ([Ca2+]_c). However, the [Ca2+]_c increase genuinely caused by gravistimulation has not been identified because gravistimulation is generally accompanied by rotation of specimens on the ground (1g), adding an additional mechanical signal to the treatment. Here, we demonstrate a gravistimulation-specific Ca²⁺ response in Arabidopsis seedlings by separating rotation from gravistimulation by using the microgravity (less than 10⁻⁴g) conditions provided by parabolic flights. Gravistimulation without rotating the specimen caused a sustained [Ca2+]_c increase, which corresponds closely to the second sustained [Ca2+]_c increase observed in ground experiments. The [Ca2+]_c increases were analyzed under a variety of gravity intensities (e.g. 0.5g, 1.5g, or 2g) combined with rapid switching between hypergravity and microgravity, demonstrating that Arabidopsis seedlings possess a very rapid gravity-sensing mechanism linearly transducing a wide range of gravitational changes (0.5g–2g) into Ca²⁺ signals on a subsecond time scale.Calcium ion (Ca²⁺) functions as an intracellular second messenger in many signaling pathways in plants (White and Broadley, 2003; Hetherington and Brownlee, 2004; McAinsh and Pittman, 2009; Spalding and Harper, 2011). Endogenous and exogenous signals are spatiotemporally encoded by changing the free cytoplasmic concentration of Ca²⁺ ([Ca2+]_c), which in turn triggers [Ca2+]_c-dependent downstream signaling (Sanders et al., 2002; Dodd et al., 2010). A variety of [Ca2+]_c increases induced by diverse environmental and developmental stimuli are reported, such as phytohormones (Allen et al., 2000), temperature (Plieth et al., 1999; Dodd et al., 2006), and touch (Knight et al., 1991; Monshausen et al., 2009). The [Ca2+]_c increase couples each stimulus and appropriate physiological responses. In the Ca²⁺ signaling pathways, the stimulus-specific [Ca2+]_c pattern (e.g. amplitude and oscillation) provide the critical information for cellular signaling (Scrase-Field and Knight, 2003; Dodd et al., 2010). Therefore, identification of the stimulus-specific [Ca2+]_c signature is crucial for an understanding of the intracellular signaling pathways and physiological responses triggered by each stimulus, as shown in the case of cold acclimation (Knight et al., 1996; Knight and Knight, 2000).Plants often exhibit biphasic [Ca2+]_c increases in response to environmental stimuli. Thus, slow cooling causes a fast [Ca2+]_c transient followed by a second, extended [Ca2+]_c increase in Arabidopsis (Arabidopsis thaliana; Plieth et al., 1999; Knight and Knight, 2000). The Ca²⁺ channel blocker lanthanum (La³⁺) attenuated the fast transient but not the following increase (Knight and Knight, 2000), suggesting that these two [Ca2+]_c peaks have different origins. Similarly, hypoosmotic shock caused a biphasic [Ca2+]_c increase in tobacco (Nicotiana tabacum) suspension-culture cells (Takahashi et al., 1997; Cessna et al., 1998). The first [Ca2+]_c peak was inhibited by gadolinium (Gd³⁺), La³⁺, and the Ca²⁺ chelator EGTA (Takahashi et al., 1997; Cessna et al., 1998), whereas the second [Ca2+]_c increase was inhibited by the intracellular Ca²⁺ store-depleting agent caffeine but not by EGTA (Cessna et al., 1998). The amplitude of the first [Ca2+]_c peak affected the amplitude of the second increase and vice versa (Cessna et al., 1998). These results suggest that even though the two [Ca2+]_c peaks originate from different Ca²⁺ fluxes (e.g. Ca²⁺ influx through the plasma membrane and Ca²⁺ release from subcellular stores, respectively), they are closely interrelated, showing the importance of the kinetic and pharmacological analyses of these [Ca2+]_c increases.Changes in the gravity vector (gravistimulation) could work as crucial environmental stimuli in plants and are generally achieved by rotating the specimens (e.g. +180°) in ground experiments. Use of Arabidopsis seedlings expressing apoaequorin, a Ca²⁺-reporting photoprotein (Plieth and Trewavas, 2002; Toyota et al., 2008a), has revealed that gravistimulation induces a biphasic [Ca2+]_c increase that may be involved in the sensory pathway for gravity perception/response (Pickard, 2007; Toyota and Gilroy, 2013) and the intracellular distribution of auxin transporters (Benjamins et al., 2003; Zhang et al., 2011). These two Ca²⁺ changes have different characteristics. The first transient [Ca2+]_c increase depends on the rotational velocity but not angle, whereas the second sustained [Ca2+]_c increase depends on the rotational angle but not velocity. The first [Ca2+]_c transient was inhibited by Gd³⁺, La³⁺, and the Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid but not by ruthenium red (RR), whereas the second sustained [Ca2+]_c increase was inhibited by all these chemicals. These results suggest that the first transient and second sustained [Ca2+]_c increases are related to the rotational stimulation and the gravistimulation, respectively, and are mediated by distinct molecular mechanisms (Toyota et al., 2008a). However, it has not been demonstrated directly that the second sustained [Ca2+]_c increase is induced solely by gravistimulation; it could be influenced by other factors, such as an interaction with the first transient [Ca2+]_c increase (Cessna et al., 1998), vibration, and/or deformation of plants during the rotation.To elucidate the genuine Ca²⁺ signature in response to gravistimulation in plants, we separated rotation and gravistimulation under microgravity (μg; less than 10⁻⁴g) conditions provided by parabolic flight (PF). Using this approach, we were able to apply rotation and gravistimulation to plants separately (Fig. 1). When Arabidopsis seedlings were rotated +180° under μg conditions, the [Ca2+]_c response to the rotation was transient and almost totally attenuated in a few seconds. Gravistimulation (transition from μg to 1.5g) was then applied to these prerotated specimens at the terminating phase of the PF. This gravistimulation without simultaneous rotation induced a sustained [Ca2+]_c increase. The kinetic properties of this sustained [Ca2+]_c increase were examined under different gravity intensities (0.5g–2g) and sequences of gravity intensity changes (Fig. 2A). This analysis revealed that gravistimulation-specific Ca²⁺ response has an almost linear dependency on gravitational acceleration (0.5g–2g) and an extremely rapid responsiveness of less than 1 s.Open in a separate windowFigure 1.Diagram of the experimental procedures for applying separately rotation and gravistimulation to Arabidopsis seedlings. Rotatory stimulation (green arrow) was applied by rotating the seedlings 180° under μg conditions, and 1.5g 180° rotation gravistimulation (blue arrow) was applied to the prerotated seedlings after μg.Open in a separate windowFigure 2.Acceleration, temperature, humidity, and pressure in an aircraft during flight experiments. A, Accelerations along x, y, and z axes in the aircraft during PF. The direction of flight (FWD) and coordinates (x, y, and z) are indicated in the bottom graph. The inset shows an enlargement of the acceleration along the z axis (gravitational acceleration) during μg conditions lasting for approximately 20 s. B, Temperature, humidity, and pressure in the aircraft during PF. Shaded areas in graphs denote the μg condition.     

Cyclopiazonic Acid Is Complexed to a Divalent Metal Ion When Bound to the Sarcoplasmic Reticulum Ca2+-ATPase

We have determined the structure of the sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA) in an E2·P_i-like form stabilized as a complex with , an ATP analog, adenosine 5′-(β,γ-methylene)triphosphate (AMPPCP), and cyclopiazonic acid (CPA). The structure determined at 2.5Å resolution leads to a significantly revised model of CPA binding when compared with earlier reports. It shows that a divalent metal ion is required for CPA binding through coordination of the tetramic acid moiety at a characteristic kink of the M1 helix found in all P-type ATPase structures, which is expected to be part of the cytoplasmic cation access pathway. Our model is consistent with the biochemical data on CPA function and provides new measures in structure-based drug design targeting Ca²⁺-ATPases, e.g. from pathogens. We also present an extended structural basis of ATP modulation pinpointing key residues at or near the ATP binding site. A structural comparison to the Na⁺,K⁺-ATPase reveals that the Phe⁹³ side chain occupies the equivalent binding pocket of the CPA site in SERCA, suggesting an important role of this residue in stabilization of the potassium-occluded E2 state of Na⁺,K⁺-ATPase.The Ca²⁺-ATPase from sarco(endo)plasmic reticulum of rabbit skeletal muscle (SERCA,⁵ isoform 1a) is a thoroughly studied member of the P-type ATPase family (1). SERCA possesses 10 transmembrane helices (M1 through M10) with both the N terminus and the C terminus facing the cytoplasmic side and three cytoplasmic domains, inserted in loops between M2 and M3 (A-domain) and between M4 and M5 (P- and N-domain) (2). The enzyme mediates the uptake of Ca²⁺ ions into the lumen of the sarcoplasmic reticulum (SR) after their release into the cytoplasm through calcium release channels during muscle contraction (3). SERCA, plasma membrane Ca²⁺-ATPase, and a third, Golgi-located secretory pathway Ca²⁺-ATPase are important factors in calcium and manganese homeostasis, transport, signaling, and regulation (4, 5).Crystal structures of all major states in the reaction cycle of SERCA have been determined. These include the Ca₂E₁·ATP state (6, 7) with high affinity Ca²⁺ binding sites accessible from the cytoplasmic side of the SR membrane, the calcium-occluded transition state (6), the open E2P state with luminal facing ion binding sites that have low affinity for Ca²⁺ and high affinity for protons (8) and the proton-occluded H_2–3E2[ATP] state with a bound modulatory ATP (9). This considerable amount of structural information has turned the Ca²⁺-ATPase into a valuable model system for studies on structural rearrangements that take place during the catalytic cycle of P-type ATPases. SERCA is considered a promising drug target in medical research, with a particular focus on prostate cancer and infectious diseases. Several compounds have already been shown to bind and inhibit SERCA by stabilizing the enzyme in a particular conformational state. Thapsigargin (TG), cyclopiazonic acid (CPA), and 2,5-di-(tert-butyl) hydroquinone (BHQ) stabilize an E2-like state, and 1,3-dibromo-2,4,6-tri (methylisothiouronium)benzene stabilizes an E1-P-like conformation (10–13). CPA is a toxic indole tetramic acid first isolated from Penicillium cyclopium (14) and later found to be produced by Aspergillus versicolor and Aspergillus flavus. Like TG, CPA specifically binds to and inhibits SERCA with nanomolar affinity (15). Indeed, CPA is widely used in biochemical and physiological studies on Ca²⁺ signaling and muscle function, where it causes Ca²⁺ store depletion due to specific inhibition of Ca²⁺ reuptake by SERCA. CPA and TG were originally proposed to bind to similar sites on SERCA (16), but recent crystal structures have shown a distinct site of interaction (17, 18). Despite these structural insights, a previously demonstrated magnesium dependence of CPA binding (19) remained unexplained, and opposing CPA binding modes were observed (see below).Tetramic acids are synthesized naturally, and more than 150 natural derivatives have been isolated from bacterial and fungal species (reviewed in Ref. 20). Tetramic acids possessing a 3-acyl group have the ability to chelate divalent metal ions. For instance, tenuazonic acid from the fungus Phoma sorghina has been shown to form complexes with Ca²⁺ and Mg²⁺ (21), as well as heavier metals such as Cu(II), Ni(II), and Fe(III) (22).Previously published crystallographic structures of the SERCA·CPA complex (PDB ID 2O9J and 2EAS) demonstrated that CPA binds within the proposed calcium access channel of SERCA. However, the structures did not reveal a role for magnesium, and the orientation of CPA within this binding site differed in the two studies (17, 18). To address these ambiguities, we have determined the crystal structure of SERCA in complex with , AMPPCP (an ATP analog), and Mn²⁺·CPA. The structure reveals novel insight into CPA binding, which we find to be mediated by a divalent cation, as demonstrated by means of the anomalous scattering properties of Mn²⁺. Further and improved refinement using previously deposited data (PDB ID 2O9J and 2OA0), in light of our new findings, also revealed a strong plausibility for a magnesium ion bound at this site. Furthermore, we find a new configuration of the bound AMPPCP nucleotide, addressing the modulatory role of ATP binding to the E2·P_i occluded conformation of SERCA.     

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.     

Calmodulin has the Potential to Function as a Ca2+-Dependent Adaptor Protein

Calmodulin (CaM) is a versatile Ca²⁺-binding protein that regulates the activity of numerous effector proteins in response to Ca²⁺ signals. Several CaM-dependent regulatory mechanisms have been identified, including autoinhibitory domain displacement, sequestration of a ligand-binding site, active site reorganization, and target protein dimerization. We recently showed that the N- and C-lobes of animal and plant CaM isoforms could independently and sequentially bind to target peptides derived from the CaM-binding domain of Nicotiana tabacum mitogen-activated protein kinase phosphatase (NtMKP1), to form a 2:1 peptide:CaM complex. This suggests that CaM might facilitate the dimerization of NtMKP1, although the dimerization mechanism is distinct from the previously described simultaneous binding of other target peptides to CaM. The independent and sequential binding of the NtMKP1 peptides to CaM also suggests an alternative plausible scenario in which the C-lobe of CaM remains tethered to NtMKP1, and the N-lobe is free to recruit a second target protein to the complex, such as an NtMKP1 target. Thus, we hypothesize that CaM may be capable of functioning as a Ca²⁺-dependent adaptor or recruiter protein.Key Words: calmodulin, calcium, EF-hand, adaptor protein, mitogen-activated protein kinase phosphataseCalcium (Ca²⁺) is a dynamic secondary messenger that regulates many signaling events in both plant and animal cells. Intracellular Ca²⁺ transients and oscillations (Ca²⁺ signals) are decoded by a large superfamily of calcium-binding proteins, the most important of which is calmodulin (CaM).^1–3 The prototypical CaM protein consists of four tandem helix-loop-helix “EF-hand” Ca²⁺-binding motifs that are divided into distinct N- and C-terminal globular lobes connected by a flexible linker. CaM proteins from all species including the single mammalian CaM and the many different plant CaM isoforms each undergo similar Ca²⁺-induced conformational changes involving a rearrangement of the position of its α-helices that opens distinct hydrophobic target protein-binding patches on the surface of each lobe; known as the “open” conformation (Fig. 1B). These hydrophobic patches can interact with numerous different target proteins including protein kinases, protein phosphatases, cytoskeletal proteins and other cell signaling enzymes, to regulate their activity. The closed or semi-open conformations adopted by the N- and C-lobes of Ca²⁺-free CaM (apo-CaM) (Fig. 1A) can also interact with another subset of proteins, to target CaM to certain cellular locations or facilitate Ca²⁺-independent regulatory events.^1–3Open in a separate windowFigure 1Structures of CaM and CaM-target complexes. (A) apo-CaM (PDB:1DMo), (B) Ca²⁺-CaM (PDB:1CLL). Complexes of CaM bound to (C) CaMBD of smooth muscle myosin light chain kinase (PDB:1CDL), (D) partial CaMBD of plasma membrane Ca²⁺-pump C20W (PDB:1CFF), (E) the adenylyl cyclase protein from Bacillus anthracis (PDB:1K93), (F) 2 glutamate decarboxylase CaMBD''s (PDB:1NWD), (G) 2 CaM proteins bound to 2 small conductance Ca²⁺-activated potassium channel (SK channel) CaMBD''s (PDB:1G4Y), (H) 2 apo-CaM proteins bound to 2 tandem IQ motifs from murine myosin V (PDB:2IX7). In each panel CaM is shown in ivory, the target molecule is shown in blue and the Ca²⁺ ions bound to the N- and/or C-lobes of CaM are represented by red spheres.The CaM-dependent regulation of target proteins can occur through numerous different mechanisms. For example, Ca²⁺-CaM can relieve autoinhibition by binding to a short (20–25 residue) calmodulin-binding domain (CaMBD) sequence that is adjacent to or within an autoinhibitory region of the enzyme (Fig. 2A).³ Numerous structures of these Ca²⁺-CaM-CaMBD complexes have been reported, which reveal a characteristic “wrap-around” binding mode (Fig. 1C). Typically the CaM C-lobe binds with high affinity to a Trp residue within the N-terminal part of the target sequence, and the flexible central linker allows the N-lobe to pivot and bind to a second bulky hydrophobic “anchor” residue within the C-terminal part of the target sequence.³ Truncation of this second anchor residue can lead to binding of only one CaM domain and an extended CaM conformation (Fig. 1D).^4,5 Studies with plant CaM isoforms having mutations to non-CaMBD-coordinating residues have also suggested that a secondary binding interface exists on the opposite surface of the CaM protein which also contributes to the activation of some of these target enzymes.^6,7Open in a separate windowFigure 2Schematic model for the various mechanisms of CaM-dependent target regulation. (A) autoinhibitory domain displacement, (B) sequestering of a ligand binding site, (C) active-site reorganization, (D) CaM-induced target protein dimerization (1:2 complex), (E) CaM-induced target protein dimerization (2:2 complex), (F) hypothesized model for CaM acting as an adaptor/recruiter protein. In each panel CaM is shown as a red dumbbell shaped molecule with Ca²⁺ ions represented by yellow circles, and the target proteins are shown in various colors. See the text for details on each model.Another regulatory mechanism involving Ca²⁺-CaM-binding to a single contiguous CaMBD sequence may occur with the potato kinesin-like CaM-binding protein (KCBP)⁸ as well as some plant cyclic-nucleotide gated channels (CNGC''s).⁹ In both cases the Ca²⁺-CaM binding site on the target protein overlaps with the respective ligand binding site, and thus the binding of KCBP to microtubules or the binding of cyclic nucleotide monophosphates to CNGC''s may be prevented by interaction with Ca²⁺-CaM (Fig. 2B). In a variation on this mechanism, CaM can bind to the cytoplasmic juxtamembrane region of the human epidermal growth factor receptor and sequester a threonine residue which is a specific phosphorylation target of protein kinase C (PKC). CaM-binding inhibits PKC phosphorylation of this threonine, and PKC phosphorylation inhibits CaM-binding.¹⁰There are also several examples of CaM-target interactions where the N- and C-lobes bind to noncontiguous target protein regions, and play distinct roles in target regulation. The structures of a CaM-activated adenylyl cyclase from Bacillus anthracis with and without bound CaM shows how the N- and C-lobes of CaM can bind two distant regions of the adenylyl cyclase enzyme and induce a conformation reorganization that creates the enzyme''s active site (Figs. 1E and ​and2C2C).¹¹ An interesting feature of this interaction is that the CaM N-lobe remains Ca²⁺-free and in a closed conformation, while the C-lobe is in a canonical Ca²⁺-bound open conformation. Indeed, Ca²⁺-binding to the C-lobe but not N-lobe is required for activation of the adenylyl cyclase.¹²The N- and C-lobes of Ca²⁺-CaM can also each simultaneously bind to identical peptides derived from the petunia glutamate decarboxylase (GAD) enzyme to form a 1:2 Ca²⁺-CaM:GAD complex (Fig. 1F).^13,14 This suggests that Ca²⁺-CaM-induced target protein dimerization may be another way in which CaM can regulate target proteins (Fig. 2D). CaM-dependent dimerization has also been shown to regulate the activity of small conductance Ca²⁺-activated K⁺ channels (SK channel), although in this case a novel 2:2 CaM:SK channel complex is formed (Figs. 1G and ​and2E2E).¹⁵ This structure is also unique because Ca²⁺ is bound to the “lower affinity” N-lobe EF-hands, but not to the “higher affinity” C-lobe EF-hands of CaM.In addition to the SK channel, CaM can regulate voltage-gated sodium channels, voltage-gated calcium channels, as well as ryanodine-sensitive calcium release channels.¹⁶ With these channels CaM typically binds in complex Ca²⁺-dependent and Ca²⁺-independent ways to several noncontiguous target sequences in the same protein, and often to so-called IQ motifs (IQXXXRGXXXR). IQ motifs are generally thought to be constitutive apo-CaM binding sites which retain CaM under resting (low [Ca²⁺]) cellular conditions to ensure a rapid response to Ca²⁺-stimuli.¹⁷ However many IQ motifs can also bind specifically to Ca²⁺-CaM or to both apo-CaM and Ca²⁺-CaM. Structures of some Ca²⁺-CaM-IQ domain complexes have revealed wrap-around binding modes, albeit with differences in lobe and peptide orientation compared to other complexes.^18–20 For a discussion about the mechanisms of CaM-dependent ion channel regulation (see ref. ¹⁶). A very recent crystal structure of apo-CaM bound to an IQ domain from myosin V (Fig. 1H) has also revealed yet another variation on the wrap-around binding mode, where the apo-C-lobe of CaM adopts a semi-open conformation and forms numerous interactions with the target sequence, while the apo-N-lobe adopts a closed conformation and forms weaker interactions with the IQ domain.²¹Using several biophysical techniques we recently characterized the interaction between CaM isoforms (mammalian CaM, soybean CaM isoforms SCaM-1 and SCaM-4) and a novel CaMBD derived from the Nicotiana tabacum mitogen-activated protein kinase phosphatase (NtMKP1).²² The NtMKP1 protein was initially identified as a CaM-binding protein by Ohashi and coworkers,²³ and the same group recently showed that CaM-binding NtMKP1 homologs are also present in other plant species as well.²⁴ We found that each CaM isoform was capable of binding to the NtMKP1 CaMBD in the absence of Ca²⁺ using only the apo-C-lobe, with the primary binding site consisting of NtMKP1 residues N438 - S449, and additional C-terminal residues G450 - K460 enhancing the overall binding affinity (K_d ∼10⁻⁵ M). In the presence of Ca²⁺, a 1:1 complex could be formed with the CaM C-lobe having significantly increased affinity for the N438 - S449 region of NtMKP1 (K_d 10⁻⁷ − 10⁻¹⁰ M). However, the Ca²⁺-loaded CaM N-lobe interacted only very weakly with the C-terminal NtMKP1 sequence in this 1:1 complex, despite an abundance of seemingly suitable hydrophobic “anchor” residues in this region. Interestingly, the addition of more peptide triggered the independent binding of a second NtMKP1 peptide to the Ca²⁺-CaM N-lobe (Kd 10⁻⁵ − 10⁻⁶ M) to form a 1:2 Ca²⁺-CaM:NtMKP1 complex. As with GAD, these results suggest that CaM is capable of facilitating the dimerization of NtMKP1, although the independent and sequential NtMKP1 peptide binding to the C- and N-lobes markedly distinguishes the CaM-NtMKP1 interaction from the simultaneous high-affinity binding of 2 GAD CaMBD''s to CaM.While our NtMKP1 study was ongoing, Ohashi and coworkers reported that CaM is incapable of stimulating the phosphatase activity of the NtMKP1 enzyme, thereby implying that the CaM-NtMKP1 interaction is necessary for something other than direct enzyme regulation.²⁵ The independent and sequential binding of the NtMKP1 fragments to the Ca²⁺-saturated C- and then N-lobes of CaM observed in our study suggests a plausible situation in which the C-lobe of CaM is tightly bound to NtMKP1, leaving the N-lobe free to recruit a different target protein to the complex, for example, a NtMKP1 protein substrate. Therefore, CaM may be capable of acting as an adaptor or recruiter protein, which would add yet another mechanism of target regulation to CaM''s repertoire (Fig. 2F). In addition to NtMKP1 peptides, the isolated N-lobe of CaM is capable of binding to other CaMBD peptides^26,27 as well as intact target proteins,²⁸ increasing the likelihood that the N-lobe could serve as a recruiter domain. The pre-association of the apo-C-lobe of CaM with NtMKP1 under resting conditions would also ensure a rapid response response to Ca²⁺-stimuli, since CaM would only need to recruit one rather than both protein targets.Although the ability of CaM to act as an adaptor protein in vivo has not yet been demonstrated, there are examples of related EF-hand proteins acting as adaptor proteins, including centrin²⁹ and calcium- and integrin-binding protein 1.³⁰ With the abundance of poorly characterized CaM-binding proteins in plants, many of which have CaMBD''s with little sequence resemblance to the better characterized motifs in animals¹ it seems likely that sequences will be identified which bind preferentially to the CaM N-lobe. Considering the incredible assortment of known CaM interaction modes and regulatory mechanisms, many of which have only been identified within the last decade, it is likely only a matter of time before CaM is proven to function as an adaptor protein in vivo.     

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.     

Purinergic P2X7 Receptors Mediate ATP-induced Saliva Secretion by the Mouse Submandibular Gland

Salivary glands express multiple isoforms of P2X and P2Y nucleotide receptors, but their in vivo physiological roles are unclear. P2 receptor agonists induced salivation in an ex vivo submandibular gland preparation. The nucleotide selectivity sequence of the secretion response was BzATP ≫ ATP > ADP ≫ UTP, and removal of external Ca²⁺ dramatically suppressed the initial ATP-induced fluid secretion (∼85%). Together, these results suggested that P2X receptors are the major purinergic receptor subfamily involved in the fluid secretion process. Mice with targeted disruption of the P2X₇ gene were used to evaluate the role of the P2X₇ receptor in nucleotide-evoked fluid secretion. P2X₇ receptor protein and BzATP-activated inward cation currents were absent, and importantly, purinergic receptor agonist-stimulated salivation was suppressed by more than 70% in submandibular glands from P2X₇-null mice. Consistent with these observations, the ATP-induced increases in [Ca²⁺]_i were nearly abolished in P2X₇^–/– submandibular acinar and duct cells. ATP appeared to also act through the P2X₇ receptor to inhibit muscarinic-induced fluid secretion. These results demonstrate that the ATP-sensitive P2X₇ receptor regulates fluid secretion in the mouse submandibular gland.Salivation is a Ca²⁺-dependent process (1, 2) primarily associated with the neurotransmitters norepinephrine and acetylcholine, release of which stimulates α-adrenergic and muscarinic receptors, respectively. Both types of receptors are coupled to G proteins that activate phospholipase Cβ (PLCβ) during salivary gland stimulation. PLCβ activation cleaves phosphatidylinositol 1,4-bisphosphate resulting in diacylglycerol and inositol 1,4,5-trisphosphate (InsP₃) production. Activation of Ca²⁺-selective InsP₃ receptor channels localized to the endoplasmic reticulum of salivary acinar cells increases the intracellular free calcium concentration ([Ca²⁺]_i).⁴ Depletion of the endoplasmic reticulum Ca²⁺ pool triggers extracellular Ca²⁺ influx and a sustained elevation in [Ca²⁺]_i. This increase in [Ca²⁺]_i activates Ca²⁺-dependent K⁺ and Cl^– channels promoting Cl^– secretion across the apical membrane and a lumen negative, electrochemical gradient that supports Na⁺ efflux into the lumen. The accumulation of NaCl creates an osmotic gradient which drives water movement into the lumen, thus generating isotonic primary saliva. This primary fluid is then modified by the ductal system, which reabsorbs NaCl and secretes KHCO₃ producing a final saliva that is hypotonic (1, 2).Salivation also has a non-cholinergic, non-adrenergic component, the origin of which is unclear (3). In addition to muscarinic and α-adrenergic receptors, salivary acinar cells express other receptors that are coupled to an increase in [Ca²⁺]_i such as purinergic P2 and substance P receptors. Like muscarinic and α-adrenergic receptors, P2 receptor activation leads to a sustained increase in [Ca²⁺]_i in salivary acinar cells (4). In contrast, substance P receptor activation rapidly desensitizes and therefore generates only a relatively transient increase in [Ca²⁺]_i (5) that is unlikely to appreciably contribute to fluid secretion. The purinergic P2 receptor family is comprised of G protein-coupled P2Y and ionotropic P2X receptors activated by extracellular di- and triphosphate nucleotides. Activation of both subfamilies of P2 receptors causes an increase in [Ca²⁺]_i. P2Y receptors increase [Ca²⁺]_i via InsP₃-induced Ca²⁺ mobilization from intracellular stores (similar to α-adrenergic and muscarinic receptors) while P2X receptors act as ligand-gated, non-selective cation channels that mediate extracellular Ca²⁺ influx (6). Salivary gland tissues express at least four isoforms of P2X (P2X₄ and P2X₇) and P2Y (P2Y₁ and P2Y₂) subtypes; however, their in vivo physiological significance has yet to be characterized (7–11).Our results revealed that ATP acts in isolation to stimulate fluid secretion from the mouse submandibular gland, but secretion is inhibited when ATP is simultaneously presented with a muscarinic receptor agonist. Ablation of the P2X₇ gene had no effect on the salivary flow rate evoked by muscarinic receptor activation, but markedly reduced ATP-mediated fluid secretion and rescued the inhibitory effects of ATP on muscarinic receptor activation. Submandibular gland acinar cells from P2X₇^–/– animals had dramatically impaired ATP-activated Ca²⁺ signaling, consistent with this being the mechanism responsible for the reduction in ATP-mediated fluid secretion in these mice. Together, these results demonstrated that ATP regulates salivation, acting mainly through the P2X₇ receptor. Activation of the P2X₇ receptor may play a major role in non-adrenergic, non-cholinergic stimulated fluid secretion.     

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.     

Structure and Functional Analysis of a Ca2+ Sensor Mutant of the Na+/Ca2+ Exchanger

The mammalian Na⁺/Ca²⁺ exchanger, NCX1.1, serves as the main mechanism for Ca²⁺ efflux across the sarcolemma following cardiac contraction. In addition to transporting Ca²⁺, NCX1.1 activity is also strongly regulated by Ca²⁺ binding to two intracellular regulatory domains, CBD1 and CBD2. The structures of both of these domains have been solved by NMR spectroscopy and x-ray crystallography, greatly enhancing our understanding of Ca²⁺ regulation. Nevertheless, the mechanisms by which Ca²⁺ regulates the exchanger remain incompletely understood. The initial NMR study showed that the first regulatory domain, CBD1, unfolds in the absence of regulatory Ca²⁺. It was further demonstrated that a mutation of an acidic residue involved in Ca²⁺ binding, E454K, prevents this structural unfolding. A contradictory result was recently obtained in a second NMR study in which Ca²⁺ removal merely triggered local rearrangements of CBD1. To address this issue, we solved the crystal structure of the E454K-CBD1 mutant and performed electrophysiological analyses of the full-length exchanger with mutations at position 454. We show that the lysine substitution replaces the Ca²⁺ ion at position 1 of the CBD1 Ca²⁺ binding site and participates in a charge compensation mechanism. Electrophysiological analyses show that mutations of residue Glu-454 have no impact on Ca²⁺ regulation of NCX1.1. Together, structural and mutational analyses indicate that only two of the four Ca²⁺ ions that bind to CBD1 are important for regulating exchanger activity.Cardiac contraction/relaxation relies upon Ca²⁺ fluxes across the plasma membrane (sarcolemma) of cardiomyocytes. Rapid Ca²⁺ influx (primarily through L-type Ca²⁺ channels) triggers the release of additional Ca²⁺ from the sarcoplasmic reticulum (SR),⁴ resulting in cardiomyocyte contraction. Removal of cytosolic Ca²⁺ by reuptake into the SR (through the SR Ca²⁺-ATPase) and expulsion from the cell (primarily through the Na⁺/Ca²⁺ exchanger, NCX1.1) results in relaxation (1). Altered Ca²⁺ cycling is observed in a number of pathophysiological situations including ischemia, hypertrophy, and heart failure (2). Understanding the function and regulation of NCX1.1 is thus of fundamental importance to understand cardiac physiology.NCX1.1 utilizes the electrochemical potential of the Na⁺ gradient to extrude Ca²⁺ in a ratio of three Na⁺ ions to one Ca²⁺ ion (3). In addition to transporting both Na⁺ and Ca²⁺, NCX1.1 is also strongly regulated by these two ions. Intracellular Na⁺ can induce NCX1.1 to enter an inactivated state, whereas Ca²⁺ bound to regulatory sites removes Na⁺-dependent inactivation and also activates Na⁺/Ca²⁺ exchange (3). These regulatory sites are located on a large cytoplasmic loop (∼500 residues located between transmembrane helices V and VI) containing two calcium binding domains (CBD1 and CBD2), which sense cytosolic Ca²⁺ levels. We have previously shown that Ca²⁺ binding to the primary site in CBD2 is required for full exchange regulation (4); CBD1, however, is a site of higher affinity and appears to dominate the activation of exchange activity by Ca²⁺.Both CBDs have an immunoglobulin fold formed from two antiparallel β sheets generating a β sandwich with a differing number of Ca²⁺ ions coordinated at the tip of the domain (4, 5). CBD1 binds four Ca²⁺ ions, whereas CBD2 binds only two Ca²⁺ ions. An initial NMR study revealed a local unfolding of the upper portion of CBD1 upon release of Ca²⁺ (6). In contrast, CBD2 did not display an unfolding response upon Ca²⁺ removal. A comparative analysis between CBDs revealed a difference in charge at residues in equivalent positions near the Ca²⁺ coordination site; Glu-454 in CBD1 is replaced by Lys-585 in CBD2. The unstructuring of CBD1 upon Ca²⁺ removal was alleviated by reversing the charge of the acidic residue (E454K) involved in Ca²⁺ coordination (6). Previously, we solved the structures of the Ca²⁺-bound and -free conformations of CBD2 and revealed a charge compensation mechanism involving Lys-585 (4). The positively charged lysine residue assumes the position of one of the Ca²⁺ ions upon Ca²⁺ depletion, permitting CBD2 to retain its overall fold (4). A similar phenomenon is predicted to take place in E454K-CBD1 mutant. In addition, Hilge et al. (6) showed that the E454K mutation of CBD1 decreases Ca²⁺ affinity to a level similar to that of CBD2 and suggested that the E454K mutation would cause the loss of primary regulation of NCX1.1 by CBD1.The significance of some of these observations is unclear as a recent NMR study (7) of CBD1 under more physiologically relevant conditions revealed no significant alteration in tertiary structure in the absence of Ca²⁺. It was hypothesized that Ca²⁺ binding induces localized conformational and dynamic changes involving several of the binding site residues. To clarify this issue, we solved the crystal structure of the E454K-CBD1 mutant and examined the functional effects of different CBD1 mutations in the full-length NCX1.1. The results indicate that charge compensation is indeed provided by the residue Lys-454 to replace one Ca²⁺, whereas the overall E454K-CBD1 structure is only slightly perturbed. The charge compensation, however, has no impact on Ca²⁺ regulation of NCX1.1.