Signaling Processes for Initiating Smooth Muscle Contraction upon Neural Stimulation

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

NHERF3 (PDZK1) Contributes to Basal and Calcium Inhibition of NHE3 Activity in Caco-2BBe Cells

Elevated intracellular Ca²⁺ ([Ca²⁺]_i) inhibition of NHE3 is reconstituted by NHERF2, but not NHERF1, by a mechanism involving the formation of multiprotein signaling complexes. To further evaluate the specificity of the NHERF family in calcium regulation of NHE3 activity, the current study determined whether NHERF3 reconstitutes elevated [Ca²⁺]_i regulation of NHE3. In vitro, NHERF3 bound the NHE3 C terminus between amino acids 588 and 667. In vivo, NHE3 and NHERF3 associate under basal conditions as indicated by co-immunoprecipitation, confocal microscopy, and fluorescence resonance energy transfer. Treatment of PS120/NHE3/NHERF3 cells, but not PS120/NHE3 cells, with the Ca²⁺ ionophore, 4-bromo-{"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 (0.5 μm): 1) inhibited NHE3 V_max activity; 2) decreased NHE3 surface amount; 3) dissociated NHE3 and NHERF3 at the plasma membrane by confocal immunofluorescence and fluorescence resonance energy transfer. Similarly, in Caco-2BBe cells, NHERF3 and NHE3 colocalized in the BB under basal conditions but after elevation of [Ca²⁺]_i by carbachol, this overlap was abolished. NHERF3 short hairpin RNA knockdown (>50%) in Caco-2BBe cells significantly reduced basal NHE3 activity by decreasing BB NHE3 amount. Also, carbachol-mediated inhibition of NHE3 activity was abolished in Caco-2BBe cells in which NHERF3 protein expression was significantly reduced. In summary: 1) NHERF3 colocalizes and directly binds NHE3 at the plasma membrane under basal conditions; 2) NHERF3 reconstitutes [Ca²⁺]_i inhibition of NHE3 activity and dissociates from NHE3 in fibroblasts and polarized intestinal epithelial cells with elevated [Ca²⁺]_i; 3) NHERF3 short hairpin RNA significantly reduced NHE3 basal activity and brush border expression in Caco-2BBe cells. These results demonstrate that NHERF3 reconstitutes calcium inhibition of NHE3 activity by anchoring NHE3 basally and releasing it with elevated Ca²⁺.In normal digestive physiology, the brush border (BB)² Na⁺/H⁺ exchanger, NHE3, mediates the majority of the NaCl and NaHCO₃ absorption in the ileum (1). Sequential inhibition and stimulation of NHE3 occur as part of digestive physiology. Short-term regulation of NHE3 activity is achieved through a variety of factors that affect NHE3 turnover number and/or surface expression and often involve a role for the cytoskeleton and accessory proteins, including the multi-PDZ domain containing proteins, NHERF1 and NHERF2 (1, 2). However, many details of this regulation are not understood.The NHERF (Na⁺/H⁺ exchanger regulatory factor) family of multi-PDZ domain containing proteins consists of four evolutionarily related members, all of which are expressed in epithelial cells of the mammalian small intestine (2). NHERF1 and NHERF2 have been previously shown to contribute to acute NHE3 stimulation and inhibition (3–10). Recently, two additional PDZ domain containing proteins, termed NHERF3/PDZK1 and NHERF4/PDZK2/IKEPP, have been demonstrated to possess sequence homology with NHERF1 and NHERF2 (11–14). However, unlike NHERF1 and NHERF2, which are comprised of two tandem PDZ domains flanked by a C-terminal ezrin/radixin/moesin binding domain, NHERF3 and NHERF4 consist of four PDZ domains but no other protein-protein interacting domains (12).NHERF3 was initially identified by a yeast two-hybrid screen from a human kidney cDNA library using the membrane-associated protein MAP17, as bait (12). NHERF3 is expressed in the brush border of epithelial cells of the kidney proximal tubule and the small intestine (12). NHERF3 associates with and, in a few cases, has been shown to regulate the activity of multiple apical membrane ion transporters including the cystic fibrosis transmembrane regulator (CFTR), urate anion exchanger 1 (URAT1), sodium-phosphate cotransporter type IIa (NaP_iIIa), proton-coupled peptide transporter (PEPT2), and organic cation/carnitine cotransporter (OCTN2) (15–19). Furthermore, NHERF3 directly binds the C terminus of NHE3 (20). Recent studies have begun evaluating the effect of NHERF3 on mouse intestinal Na⁺ and Cl⁻ transport. Basal electroneutral sodium absorption was decreased by >40% in the NHERF3 null mouse jejunum (21) and by >80% in the colon (22). In addition, Cinar et al. (22) demonstrated that cAMP and [Ca²⁺]_i inhibition of NHE3 activity was abolished in the NHERF3 null mouse colon. However, the mechanism by which NHERF3 regulates NHE3 activity was not resolved.Several physiological and pathophysiological agonists, acting through [Ca²⁺]_i-induced second messenger systems, are known to inhibit electroneutral NaCl absorption in the small intestine (1, 23). Elevation of [Ca²⁺]_i has previously been demonstrated to inhibit NHE3 activity in a NHERF2-, but not NHERF1-dependent manner (5). NHERF2 regulation of NHE3 involves the formation of multiprotein complexes at the plasma membrane that include NHE3, NHERF2, α-actinin-4, and PKCα, which induce endocytic removal of NHE3 from the plasma membrane by a PKC-dependent mechanism (5, 24). Because multiple PDZ proteins exist in the apical pole of epithelial cells (2), the current study was designed to determine whether NHERF3 could reconstitute Ca²⁺ regulation of NHE3 activity and to define how that occurred.     

Dynamics of ATP-induced Calcium Signaling in Single Mouse Thymocytes

Extracellular ATP (ATP_o) elicits a robust change in the concentration of intracellular Ca²⁺ ([Ca²⁺]_i) in fura-2–loaded mouse thymocytes. Most thymocytes (60%) exposed to ATP_o exhibited a biphasic rise in [Ca²⁺]_i; [Ca²⁺]_i rose slowly at first to a mean value of 260 nM after 163 s and then increased rapidly to a peak level of 735 nM. In many cells, a declining plateau, which lasted for more than 10 min, followed the crest in [Ca²⁺]_i. Experiments performed in the absence of extracellular [Ca²⁺]_o abolished the rise in thymocyte [Ca²⁺]_i, indicating that Ca²⁺ influx, rather than the release of stored Ca²⁺, is stimulated by ATP_o. ATP_o- mediated Ca²⁺ influx was potentiated as the [Mg²⁺]_o was reduced, confirming that ATP⁴⁻ is the active agonist form. In the absence of Mg²⁺ _o, 3′-O-(4-benzoyl)benzoyl-ATP (BzATP) proved to be the most effective agonist of those tested. The rank order of potency for adenine nucleotides was BzATP⁴⁻>ATP⁴⁻>MgATP²⁻>ADP³⁻, suggesting purinoreceptors of the P2X₇/P2Z class mediate the ATP_o response. Phenotyping experiments illustrate that both immature (CD4⁻CD8⁻, CD4⁺CD8⁺) and mature (CD4⁺CD8⁻, CD4⁻CD8⁺) thymocyte populations respond to ATP. Further separation of the double-positive population by size revealed that the ATP_o-mediated [Ca²⁺]_i response was much more pronounced in large (actively dividing) than in small (terminally differentiated) CD4⁺CD8⁺ thymocytes. We conclude that thymocytes vary in sensitivity to ATP_o depending upon the degree of maturation and suggest that ATP_o may be involved in processes that control cellular differentiation within the thymus.Extracellular ATP (ATP_o)¹ and its metabolic products evoke physiological responses in virtually all tissues and cell types from central nervous to peripheral organ systems (for review see Dubyak and El-Moatassim, 1993; Harden et al., 1995). Tissues and isolated cells vary in sensitivity to purine agonists. Nucleotides (ATP, ADP, and AMP) and adenosine, the nucleoside product of ATP catabolism, elicit distinct responses in target cells by triggering P2 and P1 purinergic receptors, respectively (Burnstock, 1978). P2 purinoceptors can be further separated into two broad categories. The first group, divided into P2Y and P2U subtypes, couples nucleotide binding to effector molecules via G proteins. The second P2 category is comprised of nucleotide-sensitive ion channels and pores. ATP-gated P2 purinoceptors, designated P2X₁ through P2X₆ (cation channels) and P2X₇ (a dual function cation channel/pore), display extensive sequence identity (North, 1996) but disparate tissue distribution, biophysical properties, agonist profiles, and pharmacology (P2X₁, Valera et al., 1994; P2X₂-P2X₆, Collo et al., 1996; P2X₇, Surprenant et al., 1996). Moreover, P2X receptors functionally resemble acetylcholine- and serotonin-gated channels with respect to gating and ionic permeability but are structurally unique. Thus, nucleotides, together with acetylcholine, glutamate, GABA, glycine, and serotonin, are included in a small group of compounds that function as agonists for a structurally diverse set of ligand-gated ion channels and pores, as well as G protein-coupled receptors.ATP_o elicits a broad spectrum of physiological changes in cells of the immune system. In mast cells, ATP release has been shown to mediate cell-to-cell signaling (Osipchuk and Cahalan, 1992). In lymphocytes, ATP_o triggers cellular depolarization, greater permeability to small organic molecules (<400 D; Wiley et al., 1993; Chused et al., 1996), and a rise in the concentration of intracellular Ca²⁺ ([Ca²⁺]_i; El-Moatassim et al., 1987; Wiley and Dubyak, 1989). The ATP_o-mediated rise in [Ca²⁺]_i modifies the functional properties of thymocytes via DNA synthesis (Gregory and Kern, 1978, 1981; Ikehara et al., 1981) and blastogenesis (El-Moatassim et al., 1987). Moreover, an increase in [Ca²⁺]_i has been linked to programmed cell death in thymocyte populations; Ca²⁺ release from intracellular stores evoked by thapsigargin, a microsomal Ca²⁺-ATPase inhibitor, triggers the DNA fragmentation correlated with thymocyte apoptosis (Jiang et al., 1994; Zhivotovsky et al., 1994).Based upon a sensitivity profile for purine agonists and pharmacological agents, lymphocytes are not believed to possess G protein-linked purinoceptors (El-Moatassim et al., 1989b ). Rather, lymphocytes and related cell lines express purinoceptors of the ion channel/pore subtype (P2X₇). This ATP-gated pathway, originally termed P2Z (Gordon, 1986), has been characterized in mast cells (Cockcroft and Gomperts, 1979a ; Tatham and Lindau, 1990), transformed 3T3 fibroblasts (Heppel et al., 1985), macrophages (Buisman et al., 1988), parotid acinar cells (Soltoff et al., 1992), and phagocytic cells of the thymic reticulum (Coutinho-Silva et al., 1996). During whole cell patch–clamp experiments, putative P2Z channels in human B lymphocytes (Bretschneider et al., 1995) and rat peritoneal macrophages (Naumov et al., 1995) exhibit rapid activation kinetics when exposed to ATP_o. The ATP_o response depends critically upon extracellular divalent cations (Mg²⁺ and Ca²⁺), such that cellular depolarization and membrane permeability are greatest in divalent-free media. The ability of Mg²⁺- and Ca²⁺–ATP complexes to reduce receptor occupancy by lowering the concentration of ATP⁴⁻, the effective form of the nucleotide agonist, is a hallmark of P2X₇/P2Z purinoceptor physiology (Cockcroft and Gomperts, 1979b ).In this study, we examined the dynamics of [Ca²⁺]_i changes elicited by ATP_o at the single-cell level in fura-2– loaded thymocytes. To our surprise, we found that the ATP_o-mediated [Ca²⁺]_i increase varies significantly between individual cells. Moreover, the kinetics of the rise in [Ca²⁺]_i at the single-cell level is characterized by a biphasic time course that is not detectable in average profiles. To correlate stages of thymocyte development with the degree of sensitivity to ATP_o, we measured the surface expression of specific T-lymphocyte markers, CD4 and CD8, before performing Ca²⁺-imaging experiments. Our data illustrate that thymocytes vary in sensitivity to ATP_o depending upon level of maturation and degree of blastogenesis. Small, terminally differentiated, CD4⁺CD8⁺ thymocytes were least sensitive to ATP_o, while 90% of the single-positive (CD4⁺CD8⁻ or CD4⁻CD8⁺) cells, believed to be the immediate precursors of mature peripheral T-lymphocytes, exhibited a robust, ATP_o-dependent rise in [Ca²⁺]_i. The in vitro data we have gathered suggest that ATP_o may drive thymocyte differentiation in the intact thymus.     

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.     

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.     

Time-dependent Autoinactivation of Phospho-Thr286-��Ca2+/Calmodulin-dependent Protein Kinase II

Ca²⁺/calmodulin-dependent protein kinase II (αCaMKII) is thought to exert its role in memory formation by autonomous Ca²⁺-independent persistent activity conferred by Thr²⁸⁶ autophosphorylation, allowing the enzyme to remain active even when intracellular [Ca²⁺] has returned to resting levels. Ca²⁺ sequestration-induced inhibition, caused by a burst of Thr^305/306 autophosphorylation via calmodulin (CaM) dissociation from the Thr^305/306 sites, is in conflict with this view. The processes of CaM binding, autophosphorylation, and inactivation are dissected to resolve this conflict. Upon Ca²⁺ withdrawal, CaM sequential domain dissociation is observed, starting with the rapid release of the first (presumed N-terminal) CaM lobe, thought to be bound at the Thr^305/306 sites. The time courses of Thr^305/306 autophosphorylation and inactivation, however, correlate with the slow dissociation of the second (presumed C-terminal) CaM lobe. Exposure of the Thr^305/306 sites is thus not sufficient for their autophosphorylation. Moreover, Thr^305/306 autophosphorylation and autoinactivation are shown to occur in the continuous presence of Ca²⁺ and bound Ca²⁺/CaM by time courses similar to those seen following Ca²⁺ sequestration. Our investigation of the activity and mechanisms of phospho-Thr₂₈₆-αCaMKII thus shows time-dependent autoinactivation, irrespective of the continued presence of Ca²⁺ and CaM, allowing a very short, if any, time window for Ca²⁺/CaM-free phospho-Thr²⁸⁶-αCaMKII activity. Physiologically, the time-dependent autoinactivation mechanisms of phospho-Thr²⁸⁶-αCaMKII (t½ of ∼50 s at 37 °C) suggest a transient kinase activity of ∼1 min duration in the induction of long term potentiation and thus memory formation.Ca²⁺/calmodulin-dependent protein kinase II (αCaMKII)² is essential in hippocampal learning and N-methyl-d-aspartate receptor-dependent synaptic plasticity, causing long term potentiation (1, 2). The exact mechanisms of αCaMKII in memory functions have not yet been identified.αCaMKII is a broad specificity Ser/Thr protein kinase, which catalyzes the phosphorylation of over 100 protein and peptide substrates in vitro (3). Uniquely, the CaMKII family possesses two distinct kinase mechanisms. The first mechanism is a “canonical” intrasubunit phosphorylation, commonly found in monomeric kinases, in which the phosphorylatable residue of the substrate bound to the helical subdomain of the catalytic domain at the active site is lined up with the terminal phosphate of ATP (4). Although there is a large number of potential “canonical” substrates for αCaMKII at the synapse (5), so far AMPA receptors have been shown to be possible physiological substrates of αCaMKII (6). For the purpose of this study, syntide 2, a commonly used peptide substrate derived from phosphorylation site 2 of glycogen synthase (7), was chosen.The second mechanism, intersubunit autophosphorylation, takes advantage of the oligomeric organization of CaMKII (8). The most important autophosphorylation site in the α isoform is Thr²⁸⁶, which resides in the vicinity of the autoinhibitory domain (9). Peptide substrates with homologous sequences to this region have been reported to be phosphorylated by αCaMKII. This, however, occurs with a low V_max, and these substrates show properties of a non-competitive inhibitor with respect to phosphorylation of “canonical” substrates (10) and of Thr²⁸⁶ autophosphorylation itself (11). Examples of such substrates include autocamtide, a peptide substrate derived from the autoinhibitory region (12) and the NR2B subunit of the N-methyl-d-aspartate receptor, which has been identified as a potential physiological target of phospho-Thr²⁸⁶-αCaMKII at the postsynaptic membrane (13). The possible physiological significance of NR2B phosphorylation is not yet known. There is evidence to suggest that Thr²⁸⁶ autophosphorylation is required to achieve full activity of the enzyme, since the unphosphorylatable T286A mutant enzyme has much diminished activity compared with wild type enzyme (14, 15).Thr²⁸⁶ autophosphorylation causes CaM “trapping,” a >10⁴-fold increase in the affinity of αCaMKII for Ca²⁺/CaM (16–18). At the same time, Thr²⁸⁶ autophosphorylation is also attributed to confer Ca²⁺- and CaM-independent persistent “autonomous” kinase activity to αCaMKII. However, due to the extremely high affinity of phospho-Thr₂₈₆-αCaMKII for Ca²⁺/CaM, [Ca²⁺] of <10 nm is required to achieve full dissociation of Ca²⁺/CaM, since CaM trapping occurs by virtue of Ca²⁺ trapping (19). Partial activity measured upon partial Ca²⁺ withdrawal therefore may not always reflect Ca²⁺/CaM-free enzyme (9). Furthermore, the physiological resting [Ca²⁺] range is 50–100 nm; therefore, phospho-Thr²⁸⁶-αCaMKII is likely always to have residual Ca²⁺/CaM bound. This may be partially Ca²⁺-saturated CaM (19).Persistent autonomous activity conferred by Thr²⁸⁶ autophosphorylation is thought to enable αCaMKII to function as a memory molecule (20, 21). In contrast, however, following the development of chemical long term potentiation, rapid inactivation has also been reported (22). The extent of an autonomous activity is further obscured by the finding that Ca²⁺ sequestration induces a burst of autophosphorylation at residues Thr^305/306, followed by a loss of activity (23). Moreover, when examined across a broad range of [Ca²⁺], the Ca²⁺/CaM dependence of phospho-Thr²⁸⁶-αCaMKII activity is apparent (19). It is thus vital to establish the mechanisms of activation and inactivation of αCaMKII at the molecular level in order to understand how it may function physiologically in learning and memory. To this end, it is necessary to dissect the mechanisms of Ca²⁺/CaM dissociation, Thr^305/306 autophosphorylation, and inactivation of phospho-Thr²⁸⁶-αCaMKII and to establish the time window for autonomous Ca²⁺/CaM-independent activity.     

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

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

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.     

Intestinal Anion Exchanger Down-regulated in Adenoma (DRA) Is Inhibited by Intracellular Calcium

The Na/H exchanger 3 (NHE3) and the Cl/HCO₃ exchanger down-regulated in adenoma (DRA) together facilitate intestinal electroneutral NaCl absorption. Elevated Ca²⁺_i inhibits NHE3 through mechanisms involving the PDZ domain proteins NHE3 kinase A regulatory protein (E3KARP) or PDZ kidney 1 (PDZK1). DRA also possesses a PDZ-binding motif, but the roles of interactions with E3KARP or PDZK1 and Ca²⁺_i in DRA regulation are unknown. Wild type DRA and a mutant lacking the PDZ interaction motif (DRA-ETKFminus) were expressed constitutively in human embryonic kidney (HEK) and inducibly in Caco-2/BBE cells. DRA-mediated Cl/HCO₃ exchange was measured as intracellular pH changes. Ca²⁺_i was assessed fluorometrically. DRA was induced 8–16-fold and was delivered to the apical surface of polarized Caco-2 cells. Putative anion transporter 1 and cystic fibrosis transmembrane regulator did not contribute to Cl/HCO₃ exchange in transfected Caco-2 cells. The calcium ionophore 4Br-{"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 inhibited DRA and DRA-ETKFminus in HEK cells, but only full-length DRA was inhibited in Caco-2 cells. In contrast, 100 μm UTP, which increased Ca²⁺_i, inhibited full-length DRA but not DRA-ETKFminus in Caco-2 and HEK cells. In HEK cells, which express little PDZK1, additional transfection of PDZK1 was required for UTP to inhibit DRA. As HEK cells do not express cystic fibrosis transmembrane regulator or NHE3, the data indicate that Ca²⁺_i-dependent DRA inhibition is not because of modulation of other transport activities. In polarized epithelium, this inhibition requires interaction of DRA with PDZK1. Together with data from PDZK1^−/− mice, these data underscore the prominent role of PDZK1 in Ca²⁺_i-mediated inhibition of colonic NaCl absorption.In the gastrointestinal tract electroneutral NaCl absorption occurs in the distal ileum and proximal colon by parallel Na/H exchange and Cl/HCO₃ exchange. Studies in knock-out mice have confirmed that NHE3² (Na/H exchanger, isoform 3; SLC9A3) and DRA (down-regulated in adenoma; SLC26A3) are the primary transporters responsible for these events (1, 2). The importance of the latter is emphasized by the human genetic disorder congenital chloride diarrhea (3), in which a nonfunctional DRA leads to life-threatening diarrhea. DRA is also expressed in the duodenum (in the lower villus) and in the pancreas (4–6), where it is involved in chloride and bicarbonate secretion together with CFTR (4–7). All three transport proteins, NHE3, DRA, and CFTR, have PDZ interaction motifs that facilitate binding to several members of the NHERF class of PDZ adapter proteins (8, 9).Electroneutral NaCl absorption is regulated largely in parallel but reciprocally with electrogenic chloride secretion (10). In different systems NHE3 is acutely regulated by cAMP, cGMP, intracellular calcium, lysophosphatidic acid, and epidermal growth factor (11) as well as by tumor necrosis factor-α (12). Notably, some of these regulatory processes are mediated through the interaction of NHE3 with several members of the NHERF class of PDZ adapter proteins (8, 11).Relatively little is known about the acute regulation of DRA. In the context of chloride and bicarbonate secretion, DRA is activated by cAMP, if it is expressed in a complex with CFTR and PDZ adapter proteins (PDZK1, also known as CAP70, and/or NHERF) (6, 7, 13). It is expected that DRA is inhibited in vivo in parallel with NHE3 during NaCl absorption, and in Caco-2/BBE cells transfected with NHE3 and DRA, this coupled inhibition has recently been shown (14). In Xenopus oocytes DRA is refractory to regulation by the calmodulin antagonist calmidazolium (10 μm), the PP2A inhibitor calyculin A (100 nm), or actin-modifying agents (13). Other data suggest that direct phosphorylation may regulate DRA, as mutation of tyrosine 756 (Y756F) increases DRA activity, although no signaling pathway has been suggested (13). Thus the regulation of DRA remains poorly understood. Moreover, no data address whether DRA regulation can occur independently or is always dependent on regulation of partner transporters, i.e. CFTR or NHE3, to which it is functionally and structurally coupled.Here we show that DRA activity is inhibited by elevations of Ca²⁺_i, that this regulation is independent of CFTR and NHE3, and that regulation requires interactions between DRA and the PDZ adapter protein PDZK1.     

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.     

Dependence of STIM1/Orai1-mediated Calcium Entry on Plasma Membrane Phosphoinositides

Recent studies identified two main components of store-operated calcium entry (SOCE): the endoplasmic reticulum-localized Ca²⁺ sensor protein, STIM1, and the plasma membrane (PM)-localized Ca²⁺ channel, Orai1/CRACM1. In the present study, we investigated the phosphoinositide dependence of Orai1 channel activation in the PM and of STIM1 movements from the tubular to PM-adjacent endoplasmic reticulum regions during Ca²⁺ store depletion. Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂) levels were changed either with agonist stimulation or by chemically induced recruitment of a phosphoinositide 5-phosphatase domain to the PM, whereas PtdIns4P levels were decreased by inhibition or down-regulation of phosphatidylinositol 4-kinases (PI4Ks). Agonist-induced phospholipase C activation and PI4K inhibition, but not isolated PtdIns(4,5)P₂ depletion, substantially reduced endogenous or STIM1/Orai1-mediated SOCE without preventing STIM1 movements toward the PM upon Ca²⁺ store depletion. Patch clamp analysis of cells overexpressing STIM1 and Orai1 proteins confirmed that phospholipase C activation or PI4K inhibition greatly reduced I_CRAC currents. These results suggest an inositide requirement of Orai1 activation but not STIM1 movements and indicate that PtdIns4P rather than PtdIns(4,5)P₂ is a likely determinant of Orai1 channel activity.Store-operated Ca²⁺ entry (SOCE)³ is a ubiquitous Ca²⁺ entry pathway that is regulated by the Ca²⁺ content of the endoplasmic reticulum (ER) (1). SOCE has been identified as the major route of Ca²⁺ entry during activation of cells of the immune system such as T cells and mast cells (2, 3), and it is also present and functionally important in other cells such as platelets (4) and developing myotubes (5). The long awaited mechanism of how the ER luminal Ca²⁺ content is sensed and the information transferred to the plasma membrane (PM) has been clarified recently after identification of the ER Ca²⁺ sensor proteins STIM1 and -2 (6, 7) and the PM Ca²⁺ channels Orai1, -2, and -3 (8–10). According to current views, a decrease in the ER Ca²⁺ concentration is sensed by the luminal EF-hand of the single-transmembrane STIM proteins causing their multimerization. This oligomerization occurs in the tubular ER, where it promotes the interaction of the cytoplasmic C termini of STIM with PM components and association with the PM-localized Orai channels, causing both their clustering and activation in the PM (reviewed recently in Refs. 11–13). Analysis of the interacting domains within the STIM1 and Orai1 proteins suggests that the cytoplasmic domain of STIM1 is necessary and sufficient to activate Orai1 (14), whereas the latter requires its C-terminal membrane-adjacent cytoplasmic tail to be fully activated by the STIM proteins (15, 16). Both STIM1 and -2 contain a polybasic segment in their C termini, and such regions are often responsible for the PM localization of proteins (mostly of the small GTP-binding protein class) via interaction with anionic phospholipids such as phosphatidylserine or PtdIns(4,5)P₂ (17). However, the role of this domain in STIM1 function(s) remains controversial. Deletion of the polybasic tail is reported to prevent PM association but not clustering of STIM1 upon ER store depletion (18). In other studies, truncated STIM1 lacking the polybasic domain shows only slightly altered activation (15) or inactivation (19) kinetics without major defects in supporting Orai1-mediated Ca²⁺ influx. The most recent studies identify the minimal Orai1 activation domain in STIM1 (20, 21) and find that the polybasic domain is not essential for this function but makes electrostatic interaction with classical transient receptor potential channels (22).PM phosphoinositides have been widely reported as regulators of the activity of several ion channels and transporters (23). However, only a few studies have addressed the inositide requirement of SOCE and none specifically that of the Orai1-mediated Ca²⁺ entry process. Sensitivity of SOCE to phosphatidylinositol 3-kinases (PI3K) inhibitors has been reported, but this required concentrations that suggested inhibition of targets other than PI3Ks, possibly myosin light chain kinase or the type-III PI4Ks (4, 24–26). Here we have described studies addressing the role of PM phosphoinositides in STIM1 movements as well as in Orai1 channel gating. Our results show that phosphoinositides do not have a major role in the prominent reorganization of STIM1 after Ca²⁺ store depletion but suggest a function of PtdIns4P rather than PtdIns(4,5)P₂ in supporting the Orai1-mediated Ca²⁺ entry process.     

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

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

Fluorescence Resonance Energy Transfer-Sensitized Emission of Yellow Cameleon 3.60 Reveals Root Zone-Specific Calcium Signatures in Arabidopsis in Response to Aluminum and Other Trivalent Cations

Fluorescence resonance energy transfer-sensitized emission of the yellow cameleon 3.60 was used to study the dynamics of cytoplasmic calcium ([Ca²⁺]_cyt) in different zones of living Arabidopsis (Arabidopsis thaliana) roots. Transient elevations of [Ca²⁺]_cyt were observed in response to glutamic acid (Glu), ATP, and aluminum (Al³⁺). Each chemical induced a [Ca²⁺]_cyt signature that differed among the three treatments in regard to the onset, duration, and shape of the response. Glu and ATP triggered patterns of [Ca²⁺]_cyt increases that were similar among the different root zones, whereas Al³⁺ evoked [Ca²⁺]_cyt transients that had monophasic and biphasic shapes, most notably in the root transition zone. The Al³⁺-induced [Ca²⁺]_cyt increases generally started in the maturation zone and propagated toward the cap, while the earliest [Ca²⁺]_cyt response after Glu or ATP treatment occurred in an area that encompassed the meristem and elongation zone. The biphasic [Ca²⁺]_cyt signature resulting from Al³⁺ treatment originated mostly from cortical cells located at 300 to 500 μ m from the root tip, which could be triggered in part through ligand-gated Glu receptors. Lanthanum and gadolinium, cations commonly used as Ca²⁺ channel blockers, elicited [Ca²⁺]_cyt responses similar to those induced by Al³⁺. The trivalent ion-induced [Ca²⁺]_cyt signatures in roots of an Al³⁺-resistant and an Al³⁺-sensitive mutant were similar to those of wild-type plants, indicating that the early [Ca²⁺]_cyt changes we report here may not be tightly linked to Al³⁺ toxicity but rather to a general response to trivalent cations.The role of calcium ions (Ca²⁺) as a ubiquitous cellular messenger in animal and plant cells is well established (Berridge et al., 2000; Sanders et al., 2002; Ng and McAinsh, 2003). Cellular signal transduction pathways are elicited as a result of fluctuations of free Ca²⁺ in the cytoplasm ([Ca²⁺]_cyt) in response to external and intracellular signals. These changes in [Ca²⁺]_cyt influence numerous cellular processes, including vesicle trafficking, cell metabolism, cell proliferation and elongation, stomatal opening and closure, seed and pollen grain germination, fertilization, ion transport, and cytoskeletal organization (Hepler, 2005). [Ca²⁺]_cyt fluctuations occur because cells have a Ca²⁺ signaling “toolkit” (Berridge et al., 2000) composed of on/off switches and a multitude of Ca²⁺-binding proteins. The on switches depend on membrane-localized Ca²⁺ channels that control the entry of Ca²⁺ into the cytosol (Piñeros and Tester, 1995, 1997; Thion et al., 1998; Kiegle et al., 2000a; White et al., 2000; Demidchik et al., 2002; Miedema et al., 2008). On the other hand, the off switches consist of a family of Ca²⁺-ATPases and Ca²⁺/H⁺ exchangers in the plasma membrane or endomembrane that remove Ca²⁺ from the cytosol, bringing the [Ca²⁺]_cyt down to the initial resting level (Lee et al., 2007; Li et al., 2008).The numerous cellular processes regulated by Ca²⁺ have led investigators to ask how specificity in Ca²⁺ signaling is maintained. It has been proposed that specificity in Ca²⁺ signaling is achieved because a particular stimulus elicits a distinct Ca²⁺ signature, which is defined by the timing, magnitude, and frequency of [Ca²⁺]_cyt changes. For instance, tip-growing plant cells such as root hairs and pollen tubes exhibit oscillatory elevations in [Ca²⁺]_cyt that partly mirror the oscillatory nature of growth in these cell types (Cárdenas et al., 2008; Monshausen et al., 2008). Another example is nuclear Ca²⁺ spiking in root hairs of legumes exposed to NOD factors (Oldroyd and Downie, 2006; Peiter et al., 2007). Recently, it was shown that mechanical forces applied to an Arabidopsis (Arabidopsis thaliana) root can trigger a stimulus-specific [Ca²⁺]_cyt response (Monshausen et al., 2009). Translating the Ca²⁺ signature into a defined cellular response is governed by a number of Ca²⁺-binding proteins such as calreticulin that act as [Ca²⁺]_cyt buffers, which shape both the amplitude and duration of the Ca²⁺ signal or Ca²⁺ sensors such as calmodulin that impact other downstream cellular effectors (Berridge et al., 2000; White and Broadley, 2003; Hepler, 2005).A deeper understanding of Ca²⁺ signaling mechanisms in plants has been driven in large part by our ability to monitor dynamic changes in [Ca²⁺]_cyt in the cell. Such measurements have been conducted using Ca²⁺-sensitive fluorescent indicator dyes (e.g. Indo and Fura), the luminescent protein aequorin (Knight et al., 1991, 1996; Legué et al., 1997; Wymer et al., 1997; Cárdenas et al., 2008), and more recently the yellow cameleon (YC) Ca²⁺ sensor, a chimeric protein that relies on fluorescence resonance energy transfer (FRET) as an indicator of [Ca²⁺]_cyt changes in the cell (Allen et al., 1999; Miwa et al., 2006; Qi et al., 2006; Tang et al., 2007; Haruta et al., 2008). The YC reporter is composed of cyan fluorescent protein (CFP), the C terminus of calmodulin (CaM), a Gly-Gly linker, the CaM-binding domain of myosin light chain kinase (M13), and a yellow fluorescent protein (YFP; Miyawaki et al., 1997, 1999). The increased interaction between M13 and CaM upon binding of Ca²⁺ to CaM triggers a conformational change in the protein that brings the CFP and YFP in close proximity, resulting in enhanced FRET efficiency between the two fluorophores (Miyawaki, 2003). Thus, changes in FRET efficiency between CFP and YFP in the cameleon reporter are correlated with changes in [Ca²⁺]_cyt.Since it was first introduced, improved versions of the cameleon reporter have been selected to more accurately report [Ca²⁺]_cyt levels in the cell. For instance, the YC3.60 version was selected because of its resistance to cytoplasmic acidification and its higher dynamic range compared with the earlier cameleons. The higher dynamic range of YC3.60 is due to the use of a circularly permutated YFP called Venus (cpVenus) that is capable of absorbing a greater amount of energy from CFP (Nagai et al., 2004). Recently, the utility of YC3.60 for monitoring [Ca²⁺]_cyt was demonstrated in Arabidopsis roots and pollen tubes using ratiometric imaging approaches (Monshausen et al., 2007, 2008, 2009; Haruta et al., 2008; Iwano et al., 2009). Here, we further evaluated YC3.60 as a [Ca²⁺]_cyt sensor in plants using confocal microscopy and FRET-sensitized emission imaging. Unlike the direct ratiometric measurement of cpVenus and CFP reported in previous studies using YC3.60-expressing plants (Monshausen et al., 2008, 2009), the sensitized FRET approach we describe here involves the use of donor-only (CFP) and acceptor-only (YFP) controls, allowing us to correct for bleed-through and background signals from the FRET specimen (van Rheenen et al., 2004; Feige et al., 2005).For this study, we focused on monitoring [Ca²⁺]_cyt changes in Arabidopsis seedling roots after aluminum (Al³⁺) exposure. Although Ca²⁺ signaling has long been implicated in mediating Al³⁺ responses in plants (Rengel and Zhang, 2003), the [Ca²⁺]_cyt changes evoked by Al³⁺ reported in the literature have been inconsistent, and as such, the significance of these [Ca²⁺]_cyt responses to mechanisms of Al³⁺ toxicity are not very clear. For instance, some studies reported that Al³⁺ caused a decrease in [Ca²⁺]_cyt in plants (Jones et al., 1998b; Kawano et al., 2004), and others demonstrated elevated [Ca²⁺]_cyt upon Al³⁺ treatment (Nichol and Oliveira, 1995; Lindberg and Strid, 1997; Jones et al., 1998a; Zhang and Rengel, 1999; Ma et al., 2002; Bhuja et al., 2004).Here, we report that Arabidopsis roots expressing the YC3.60 reporter exhibited transient elevations in [Ca²⁺]_cyt within seconds of Al³⁺ exposure. The general pattern of [Ca²⁺]_cyt changes observed after Al³⁺ treatment were distinct from those elicited by ATP or Glu, reinforcing the concept of specificity in [Ca²⁺]_cyt signaling. We also observed root zone-dependent variations in the [Ca²⁺]_cyt signatures evoked by Al³⁺ in regard to the shape, duration, and timing of the [Ca²⁺]_cyt response. Other trivalent ions such as lanthanum (La³⁺) and gadolinium (Ga³⁺), which have been widely used as Ca²⁺ channel blockers (Monshausen et al., 2009), also induced a rapid rise in [Ca²⁺]_cyt in root cells that were similar to those elicited by Al³⁺. Al³⁺, La³⁺, and Gd³⁺ elicited similar [Ca²⁺]_cyt signatures in the Al³⁺-tolerant mutant alr104 (Larsen et al., 1998) and the Al³⁺-sensitive mutant als3-1 (Larsen et al., 2005), indicating that the early [Ca²⁺]_cyt increases we report here may not be tightly linked to mechanisms of Al³⁺ toxicity but rather to a general trivalent cation response. Our study further shows that FRET-sensitized emission imaging of Arabidopsis roots expressing YC3.60 provides a robust method for documenting [Ca²⁺]_cyt signatures in different root developmental zones that should be useful for future studies on Ca²⁺ signaling mechanisms in plants.