Crystal structure study on human S100A13 at 2.0 A resolution

The S100 protein family is the largest group of calcium-binding protein families, which consists of at least 25 members. S100A13, which is widely expressed in a variety of tissues, is a unique member of the S100 protein family. Previous reports showed that S100A13 might be involved in the stress-induced release of some signal peptide-less proteins (such as FGF-1 and IL-1alpha) and also associated with inflammatory functions. It was also reported that S100A13 is a new angiogenesis marker. Here we report the crystal structure of the Ca(2+)-bound form of S100A13 at 2.0 A resolution. S100A13 is a homodimer with four EF-hand motifs in an asymmetric unit, displaying a folding pattern similar to other S100 members. However, S100A13 has the unique structural feature with all alpha-helices being amphiphilic, which was not found in other members of S100s. We propose that this characteristic structure of S100A13 might be related to its ability to mediate the release of FGF-1 and IL-1alpha.     

Three-dimensional solution structure of a unique S100 protein

S100A13 is a homodimeric protein that belongs to the S100 subfamily of EF-hand Ca2+-binding proteins. S100A13 exhibits unique physical and functional properties not observed in other members of the S100 family. S100A13 is crucial for the non-classical export of acidic fibroblast growth factors (FGFs-1), which lack signal peptide at their N-terminal end. In the present study, we report the three-dimensional solution structure of Ca2+-bound S100A13 using a variety of 3D NMR experiments. The structure of S100A13 is globular with four helices and an antiparallel beta-sheet in each subunit. The dimer interface is formed mainly by an antiparallel arrangement of helices H1, H1', H4, and H4'. Isothermal titration calorimetry (ITC) experiments show that S100A13 binds non-cooperatively to four calcium ions. Prominent differences exist between the three-dimensional structures of S100A13 and other S100 proteins. The hydrophobic pocket that largely contributes to protein-protein interactions in other S100 proteins is absent in S100A13. The structure of S100A13 is characterized by a large patch of negatively charged residues flanked by dense cationic clusters contributed largely by the positively charged residues located at the C-terminal end. Results of ITC experiments reveal that S100A13 lacking the C-terminal segment (residues 88-98) fails to bind FGF-1. The three-dimensional structure of S100A13 not only provides useful clues on its role in the non-classical export of signal peptide-less proteins such as FGF-1 but also paves the way for rational design of drugs against FGF-induced tumors.     

Synergistic Ca2+ and Cu2+ requirements of the FGF1-S100A13 interaction measured by quartz crystal microbalance: an initial step in amlexanox-reversible non-classical release of FGF1

It is known that fibroblast growth factor-1 (FGF1) lacking a conventional signal peptide sequence shows non-classical release independent of the endoplasmic reticulum-Golgi system. Recent studies reveal that FGF1 is co-released with S100A13, a Ca2+-binding protein that acts as an extracellular cargo molecule. Although both FGF1 and S100A13 are Cu2+-binding proteins, the role of Cu2+, as well as that of Ca2+, in non-classical release, remains to be clarified. In the present study we examined the requirements of both metal ions for the interaction between these two proteins. The addition of Ca2+ significantly increased the ka value, while decreasing the KD value, for the interaction between Strep-tagII-S100A13 and GST-FGF1; both values were obtained by use of a quartz crystal microbalance, a real-time mass-measuring device. The EC50 of Ca2+ to enhance the interaction was 10.11 microM. Although the addition of Cu2+ alone had no effect, it caused a marked potentiation of the Ca2+-enhanced interaction. The EC50 of Cu2+ for the potentiation was 50.45 nM. On the other hand, the EC50 of Ca2+ and the KD values were decreased from 11.69 to 2.07 microM and 0.75 to 0.38 x 10(-7)M, respectively, by the addition of 200 nM Cu2+. The Cu2+-induced potentiation of this interaction was abolished by amlexanox, which inhibits non-classical release of FGF1. All of these findings suggest that synergistic effects of Ca2+ and Cu2+ play a key role in the interaction between FGF1 and S100A13, which is the initial step in non-classical release of FGF1.     

S100A1 and calmodulin compete for the same binding site on ryanodine receptor

In heart and skeletal muscle an S100 protein family member, S100A1, binds to the ryanodine receptor (RyR) and promotes Ca(2+) release. Using competition binding assays, we further characterized this system in skeletal muscle and showed that Ca(2+)-S100A1 competes with Ca(2+)-calmodulin (CaM) for the same binding site on RyR1. In addition, the NMR structure was determined for Ca(2+)-S100A1 bound to a peptide derived from this CaM/S100A1 binding domain, a region conserved in RyR1 and RyR2 and termed RyRP12 (residues 3616-3627 in human RyR1). Examination of the S100A1-RyRP12 complex revealed residues of the helical RyRP12 peptide (Lys-3616, Trp-3620, Lys-3622, Leu-3623, Leu-3624, and Lys-3626) that are involved in favorable hydrophobic and electrostatic interactions with Ca(2+)-S100A1. These same residues were shown previously to be important for RyR1 binding to Ca(2+)-CaM. A model for regulating muscle contraction is presented in which Ca(2+)-S100A1 and Ca(2+)-CaM compete directly for the same binding site on the ryanodine receptor.     

S100A13-lipid interactions-role in the non-classical release of the acidic fibroblast growth factor

S100A13 is a 98-amino acid, calcium binding protein. It is known to participate in the non-classical secretion of signal peptide-less proteins, such as the acidic fibroblast growth factor. In this study, we investigate the lipid binding properties of S10013 using a number of biophysical techniques, including multidimensional NMR spectroscopy. Isothermal titration calorimetry and steady state fluorescence experiments show that apoS100A13 exhibits preferential binding to small unilamelar vesicles of l-phosphatidyl serine (pS). In comparison, Ca2+-bound S100A13 is observed to bind weakly to unilamelar vesicles (SUVs) of pS. Equilibrium thermal unfolding and limited trypsin digestion analysis reveal that apoS100A13 is significantly destabilized upon binding to SUVs of pS. Results of the far UV circular dichroism and ANS (8-anilino-1-napthalene sufonate) binding experiments indicate a subtle conformational change resulting in the increase in the solvent-accessible hydrophobic surface in the protein. Availability of the solvent-exposed hydrophobic surface(s) in apoS10013 facilitates its interaction with the lipid vesicles. Our data suggest that Ca2+ binding dictates the membrane binding affinity of S100A13. Based on the results of this study, a model describing the sequence of molecular events that possibly can occur during the non-classical secretion of FGF-1 is presented.     

Modulation of sarcoplasmic reticulum Ca2+ release in skeletal muscle expressing ryanodine receptor impaired in regulation by calmodulin and S100A1

In vitro, calmodulin (CaM) and S100A1 activate the skeletal muscle ryanodine receptor ion channel (RyR1) at submicromolar Ca(2+) concentrations, whereas at micromolar Ca(2+) concentrations, CaM inhibits RyR1. One amino acid substitution (RyR1-L3625D) has previously been demonstrated to impair CaM binding and regulation of RyR1. Here we show that the RyR1-L3625D substitution also abolishes S100A1 binding. To determine the physiological relevance of these findings, mutant mice were generated with the RyR1-L3625D substitution in exon 74, which encodes the CaM and S100A1 binding domain of RyR1. Homozygous mutant mice (Ryr1(D/D)) were viable and appeared normal. However, single RyR1 channel recordings from Ryr1(D/D) mice exhibited impaired activation by CaM and S100A1 and impaired CaCaM inhibition. Isolated flexor digitorum brevis muscle fibers from Ryr1(D/D) mice had depressed Ca(2+) transients when stimulated by a single action potential. However, during repetitive stimulation, the mutant fibers demonstrated greater relative summation of the Ca(2+) transients. Consistently, in vivo stimulation of tibialis anterior muscles in Ryr1(D/D) mice demonstrated reduced twitch force in response to a single action potential, but greater summation of force during high-frequency stimulation. During repetitive stimulation, Ryr1(D/D) fibers exhibited slowed inactivation of sarcoplasmic reticulum Ca(2+) release flux, consistent with increased summation of the Ca(2+) transient and contractile force. Peak Ca(2+) release flux was suppressed at all voltages in voltage-clamped Ryr1(D/D) fibers. The results suggest that the RyR1-L3625D mutation removes both an early activating effect of S100A1 and CaM and delayed suppressing effect of CaCaM on RyR1 Ca(2+) release, providing new insights into CaM and S100A1 regulation of skeletal muscle excitation-contraction coupling.     

Prostaglandin synthesis in rat brain astrocytes is under the control of the n-3 docosahexaenoic acid, released by group VIB calcium-independent phospholipase A2

In the current study, we reveal that in astrocytes the VIB Ca(2+)-independent phospholipase A(2) is the enzyme responsible for the release of docosahexaenoic acid (22:6n-3). After pharmacological inhibition and siRNA silencing of VIB Ca(2+)-independent phospholipase A(2), docosahexaenoic acid release was strongly suppressed in astrocytes, which were acutely stimulated (30 min) with ATP and glutamate or after prolonged (6 h) stimulation with the endotoxin lipopolysaccharide. Docosahexaenoic acid release proceeds simultaneously with arachidonic acid (20:4n-6) release and prostaglandin liberation from astrocytes. We found that prostaglandin production is negatively controlled by endogenous docosahexaenoic acid, since pharmacological inhibition and siRNA silencing of VIB Ca(2+)-independent phospholipase A(2) significantly amplified the prostaglandin release by astrocytes stimulated with ATP, glutamate, and lipopolysaccharide. Addition of exogenous docosahexaenoic acid inhibited prostaglandin synthesis, which suggests that the negative control of prostaglandin synthesis observed here is likely due to competitive inhibition of cyclooxygenase-1/2 by free docosahexaenoic acid. Additionally, treatment of astrocytes with docosahexaenoic acid leads to the reduction in cyclooxygenase-1 expression, which also contributes to reduced prostaglandin production observed in lipopolysaccharide-stimulated cells. Thus, we identify a regulatory mechanism important for the brain, in which docosahexaenoic acid released from astrocytes by VIB Ca(2+)-independent phospholipase A(2) negatively controls prostaglandin production.     

Identification of apocalmodulin and Ca2+-calmodulin regulatory domain in skeletal muscle Ca2+ release channel, ryanodine receptor

Fusion proteins and full-length mutants were generated to identify the Ca(2+)-free (apoCaM) and Ca(2+)-bound (CaCaM) calmodulin binding sites of the skeletal muscle Ca(2+) release channel/ryanodine receptor (RyR1). [(35)S]Calmodulin (CaM) overlays of fusion proteins revealed one potential Ca(2+)-dependent (aa 3553-3662) and one Ca(2+)-independent (aa 4302-4430) CaM binding domain. W3620A or L3624D substitutions almost abolished completely, whereas V3619A or L3624A substitutions reduced [(35)S]CaM binding to fusion protein (aa 3553-3662). Three full-length RyR1 single-site mutants (V3619A,W3620A,L3624D) and one deletion mutant (Delta4274-4535) were generated and expressed in human embryonic kidney 293 cells. L3624D exhibited greatly reduced [(35)S]CaM binding affinity as indicated by a lack of noticeable binding of apoCaM and CaCaM (nanomolar) and the requirement of CaCaM (micromolar) for the inhibition of RyR1 activity. W3620A bound CaM (nanomolar) only in the absence of Ca(2+) and did not show inhibition of RyR1 activity by 3 microm CaCaM. V3619A and the deletion mutant bound apoCaM and CaCaM at levels compared with wild type. V3619A activity was inhibited by CaM with IC(50) approximately 200 nm, as compared with IC(50) approximately 50 nm for wild type and the deletion mutant. [(35)S]CaM binding experiments with sarcoplasmic reticulum vesicles suggested that apoCaM and CaCaM bind to the same region of the native RyR1 channel complex. These results indicate that the intact RyR1 has a single CaM binding domain that is shared by apoCaM and CaCaM.     

Voltage-Dependent N-Type Ca2+ Channel Activity Regulates the Interaction Between FGF-1 and S100A13 for Stress-Induced Non-Vesicular Release

Summary 1. The Ca²⁺-mediated regulation of interaction between FGF-1 and S100A13 in NG108-15 cells was studied. When the stress by depriving B27 supplement from the culture was given, cellular levels of both proteins were decreased, while their releases were significantly increased within 3 h. These stress-induced changes were all abolished by amlexanox, an anti-allergic drug.2. These releases were significantly inhibited by the addition of EGTA or BAPTA-AM, cellular or extracellular Ca²⁺-chelating agent, respectively. The addition of ω-conotoxin GVIA, a N-type Ca²⁺-channel blocker caused a complete inhibition of the release, while increased the cytosolic levels of both proteins. However, ω-conotoxin MVIIC, the non-N-type Ca²⁺-channel blocker was ineffective.3. In NG108-15 cells, which had been transfected with Venus-FGF-1 and CFP-S100A13, the supplement-deprivation stress caused several spike-type fluorescence resonance energy transfer (FRET) signals, suggesting that both proteins showing interaction would be immediately released. These spikes were completely abolished by the addition of ω-conotoxin GVIA. However, the addition of amlexanox caused bell-shaped FRET signals without spikes.4. Thus, it is suggested that the interaction between FGF-1 and S100A13 responsible for stress-induced non-vesicular release is dependent of Ca²⁺-influx through N-type Ca²⁺-channels.     

Functional characterization of P2Y1 versus P2X receptors in RBA-2 astrocytes: elucidate the roles of ATP release and protein kinase C

A physiological concentration of extracellular ATP stimulated biphasic Ca(2+) signal, and the Ca(2+) transient was decreased and the Ca(2+) sustain was eliminated immediately after removal of ATP and Ca(2+) in RBA-2 astrocytes. Reintroduction of Ca(2+) induced Ca(2+) sustain. Stimulation of P2Y(1) receptors with 2-methylthioadenosine 5'-diphosphate (2MeSADP) also induced a biphasic Ca(2+) signaling and the Ca(2+) sustains were eliminated using Ca(2+)-free buffer. The 2MeSADP-mediated biphasic Ca(2+) signals were inhibited by phospholipase C (PLC) inhibitor U73122, and completely blocked by P2Y(1) selective antagonist MRS2179 and protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) whereas enhanced by PKC inhibitors GF109203X and Go6979. Inhibition of capacitative Ca(2+) entry (CCE) decreased the Ca(2+)-induced Ca(2+) entry; nevertheless, ATP further enhanced the Ca(2+)-induced Ca(2+) entry in the intracellular Ca(2+) store-emptied and CCE-inhibited cells indicating that ATP stimulated Ca(2+) entry via CCE and ionotropic P2X receptors. Furthermore, the 2MeSADP-induced Ca(2+) sustain was eliminated by apyrase but potentiated by P2X(4) allosteric effector ivermectin (IVM). The agonist ADPbetaS stimulated a lesser P2Y(1)-mediated Ca(2+) signal and caused a two-fold increase in ATP release but that were not affected by IVM whereas inhibited by PMA, PLC inhibitor ET-18-OCH(3) and phospholipase D (PLD) inhibitor D609, and enhanced by removal of intra- or extracellular Ca(2+). Taken together, the P2Y(1)-mediated Ca(2+) sustain was at least in part via P2X receptors activated by the P2Y(1)-induced ATP release, and PKC played a pivotal role in desensitization of P2Y(1) receptors in RBA-2 astrocytes.     

Intercellular calcium signaling in astrocytes via ATP release through connexin hemichannels.

Astrocytes are capable of widespread intercellular communication via propagated increases in intracellular Ca(2+) concentration. We have used patch clamp, dye flux, ATP assay, and Ca(2+) imaging techniques to show that one mechanism for this intercellular Ca(2+) signaling in astrocytes is the release of ATP through connexin channels ("hemichannels") in individual cells. Astrocytes showed low Ca(2+)-activated whole-cell currents consistent with connexin hemichannel currents that were inhibited by the connexin channel inhibitor flufenamic acid (FFA). Astrocytes also showed molecular weight-specific influx and release of dyes, consistent with flux through connexin hemichannels. Transmembrane dye flux evoked by mechanical stimulation was potentiated by low Ca(2+) and was inhibited by FFA and Gd(3+). Mechanical stimulation also evoked release of ATP that was potentiated by low Ca(2+) and inhibited by FFA and Gd(3+). Similar whole-cell currents, transmembrane dye flux, and ATP release were observed in C6 glioma cells expressing connexin43 but were not observed in parent C6 cells. The connexin hemichannel activator quinine evoked ATP release and Ca(2+) signaling in astrocytes and in C6 cells expressing connexin43. The propagation of intercellular Ca(2+) waves in astrocytes was also potentiated by quinine and inhibited by FFA and Gd(3+). Release of ATP through connexin hemichannels represents a novel signaling pathway for intercellular communication in astrocytes and other non-excitable cells.     

Activation of the neurokinin-1 receptor in rat spinal astrocytes induces Ca2+ release from IP3-sensitive Ca2+ stores and extracellular Ca2+ influx through TRPC3

Substance P (SP) plays an important role in pain transmission through the stimulation of the neurokinin (NK) receptors expressed in neurons of the spinal cord, and the subsequent increase in the intracellular Ca(2+) concentration ([Ca(2+)](i)) as a result of this stimulation. Recent studies suggest that spinal astrocytes also contribute to SP-related pain transmission through the activation of NK receptors. However, the mechanisms involved in the SP-stimulated [Ca(2+)](i) increase by spinal astrocytes are unclear. We therefore examined whether (and how) the activation of NK receptors evoked increase in [Ca(2+)](i) in rat cultured spinal astrocytes using a Ca(2+) imaging assay. Both SP and GR73632 (a selective agonist of the NK1 receptor) induced both transient and sustained increases in [Ca(2+)](i) in a dose-dependent manner. The SP-induced increase in [Ca(2+)](i) was significantly attenuated by CP-96345 (an NK1 receptor antagonist). The GR73632-induced increase in [Ca(2+)](i) was completely inhibited by pretreatment with U73122 (a phospholipase C inhibitor) or xestospongin C (an inositol 1,4,5-triphosphate (IP(3)) receptor inhibitor). In the absence of extracellular Ca(2+), GR73632 induced only a transient increase in [Ca(2+)](i). In addition, H89, an inhibitor of protein kinase A (PKA), decreased the GR73632-mediated Ca(2+) release from intracellular Ca(2+) stores, while bisindolylmaleimide I, an inhibitor of protein kinase C (PKC), enhanced the GR73632-induced influx of extracellular Ca(2+). RT-PCR assays revealed that canonical transient receptor potential (TRPC) 1, 2, 3, 4 and 6 mRNA were expressed in spinal astrocytes. Moreover, BTP2 (a general TRPC channel inhibitor) or Pyr3 (a TRPC3 inhibitor) markedly blocked the GR73632-induced sustained increase in [Ca(2+)](i). These findings suggest that the stimulation of the NK-1 receptor in spinal astrocytes induces Ca(2+) release from IP(3-)sensitive intracellular Ca(2+) stores, which is positively modulated by PKA, and subsequent Ca(2+) influx through TRPC3, which is negatively regulated by PKC.     

Copper binding affinity of S100A13, a key component of the FGF-1 nonclassical copper-dependent release complex

S100A13 is a member of the S100 protein family that is involved in the copper-dependent nonclassical secretion of signal peptideless proteins fibroblast growth factor 1 and interleukin 1 lpha. In this study, we investigate the effects of interplay of Cu2+ and Ca2+ on the structure of S100A13 using a variety of biophysical techniques, including multi-dimensional NMR spectroscopy. Results of the isothermal titration calorimetry experiments show that S100A13 can bind independently to both Ca2+ and Cu2+ with almost equal affinity (Kd in the micromolar range). Terbium binding and isothermal titration calorimetry data reveal that two atoms of Cu2+/Ca2+ bind per subunit of S100A13. Results of the thermal denaturation experiments monitored by far-ultraviolet circular dichroism, limited trypsin digestion, and hydrogen-deuterium exchange (using 1H-15N heteronuclear single quantum coherence spectra) reveal that Ca2+ and Cu2+ have opposite effects on the stability of S100A13. Binding of Ca2+ stabilizes the protein, but the stability of the protein is observed to decrease upon binding to Cu2+. 1H-15N chemical shift perturbation experiments indicate that S100A13 can bind simultaneously to both Ca2+ and Cu2+ and the binding of the metal ions is not mutually exclusive. The results of this study suggest that the Cu2+-binding affinity of S100A13 is important for the formation of the FGF-1 homodimer and the subsequent secretion of the signal peptideless growth factor through the nonclassical release pathway.     

Activation and inhibition of skeletal RyR channels by a part of the skeletal DHPR II-III loop: effects of DHPR Ser687 and FKBP12.

Peptides, corresponding to sequences in the N-terminal region of the skeletal muscle dihydropyridine receptor (DHPR) II-III loop, have been tested on sarcoplasmic reticulum (SR) Ca2+ release and ryanodine receptor (RyR) activity. The peptides were: A1, Thr671-Leu690; A2, Thr671-Leu690 with Ser687 Ala substitution; NB, Gly689-Lys708 and A1S, scrambled A1 sequence. The relative rates of peptide-induced Ca2+ release from normal (FKBP12+) SR were A2 > A1 > A1S > NB. Removal of FKBP12 reduced the rate of A1-induced Ca2+ release by approximately 30%. A1 and A2 (but not NB or A1S), in the cytoplasmic (cis) solution, either activated or inhibited single FKBP12+ RyRs. Maximum activation was seen at -40 mV, with 10 microM A1 or 50 nM A2. The greatest A1-induced increase in mean current (sixfold) was seen with 100 nM cis Ca2+. Inhibition by A1 was greatest at +40 mV (or when permeant ions flowed from cytoplasm to SR lumen) with 100 microM cis Ca2+, where channel activity was almost fully inhibited. A1 did not activate FKBP12-stripped RyRs, although peptide-induced inhibition remained. The results show that peptide A activation of RyRs does not require DHPR Ser687, but required FKBP12 binding to RyRs. Peptide A must interact with different sites to activate or inhibit RyRs, because current direction-, voltage-, cis [Ca2+]-, and FKBP12-dependence of activation and inhibition differ.     

Enhanced calcium transients in glial cells in neonatal cerebellar cultures derived from S100B null mice

S100B is the major low-affinity Ca(2+)-binding protein in astrocytes. In order to study the role of S100B in the maintenance of Ca(2+) homeostasis, we generated S100B null mice by a targeted inactivation of the S100B gene. Absence of S100B expression was demonstrated by Northern and Western blotting for S100B mRNA and protein, respectively, and immunoperoxidase staining of sections of various brain regions. S100B null mice were viable, fertile, and exhibited no overt behavioral abnormalities up to 12 months of age. On the basis of light microscopy and immunohistochemical staining, there were no discernable alterations in the distribution and morphology of astrocytes or neurons in sections of adult brains of these mice. Astrocytes in cerebellar cultures derived from 6-day-old S100B null mice exhibited enhanced Ca(2+) transients in response to treatment with KCl or caffeine. On the other hand, granule neurons, in the same cultures, exhibited normal Ca(2+) transients in response to treatment with KCl, caffeine, or N-methyl-d-aspartate. These results demonstrate a specific decrease in Ca(2+)-handling capacity in astrocytes derived from S100B null mice and suggest that S100B plays a role in the maintenance of Ca(2+) homeostasis in astrocytes.     

Calcium-binding properties of wild-type and EF-hand mutants of S100B in the presence and absence of a peptide derived from the C-terminal negative regulatory domain of p53

S100B is a dimeric Ca(2+)-binding protein that undergoes a 90 +/- 3 degrees rotation of helix 3 in the typical EF-hand domain (EF2) upon the addition of calcium. The large reorientation of this helix is a prerequisite for the interaction between each subunit of S100B and target proteins such as the tumor suppressor protein, p53. In this study, Tb(3+) was used as a probe to examine how binding of a 22-residue peptide derived from the C-terminal regulatory domain of p53 affects the rate of Ca(2+) ion dissociation. In competition studies with Tb(3+), the dissociation rates of Ca(2+) (k(off)) from the EF2 domains of S100B in the absence and presence of the p53 peptide was determined to be 60 and 7 s(-)(1), respectively. These data are consistent with a previously reported result, which showed that that target peptide binding to S100B enhances its calcium-binding affinity [Rustandi et al. (1998) Biochemistry 37, 1951-1960]. The corresponding Ca(2+) association rate constants for S100B, k(on), for the EF2 domains in the absence and presence of the p53 peptide are 1.1 x 10(6) and 3.5 x 10(5) M(-)(1) s(-)(1), respectively. These two association rate constants are significantly below the diffusion control ( approximately 10(9) M(-)(1) s(-)(1)) and likely involve both Ca(2+) ion association and a Ca(2+)-dependent structural rearrangement, which is slightly different when the target peptide is present. EF-hand calcium-binding mutants of S100B were engineered at the -Z position (EF-hand 1, E31A; EF-hand 2, E72A; both EF-hands, E31A + E72A) and examined to further understand how specific residues contribute to calcium binding in S100B in the absence and presence of the p53 peptide.     

Mechanism of endothelin-1-induced cytosolic Ca(2+) mobility in cultured H9c2 myocardiac ventricular cells

The effect of endothelin-1 (ET-1) on the intracellular free Ca(2+) ([Ca(2+)](i)) mobility in cultured H9c2 myocardiac ventricular cells was studied after loading with fura-2-AM. In Ca(2+)-containing buffer, ET-1 induced [Ca(2+)](i) rise from 10(-7) to 10(-9) M. ET-1 induced [Ca(2+)](i), which was composed of a first small peak and a secondary persistent plateau. In Ca(2+)-free buffer, pretreatment with 10(-7) M ET-1 inhibited the thapsigargin and carbonylcyanide m-chlorophenylhydrazone (CCCP)-induced [Ca(2+)](i) increase. Meanwhile, pretreatment with thapsigargin and CCCP also inhibited ET-1-induced [Ca(2+)](i) rise. In Ca(2+)-containing buffer, the ET(A) receptor antagonist (BQ123) completely abolished the secondary rising peak and plateau. Conversely, the ET(B) receptor antagonist (BQ788) completely inhibited the first small peak and secondary peak plateau. Nifedipine and La(3+) also abolished the 10(-7) M ET-1-induced [Ca(2+)](i) in the first rising peak. The internal Ca(2+) release induced by ET-1 was inhibited by U73122 (phospholipase C inhibitor), propranolol (phospholipase D inhibitor) and aristolochic acid (phospholipase A2 inhibitor). After incubation of 10(-7) M ET-1 in Ca(2+)-free buffer, the addition of 5 mM CaCl(2) increased Ca(2+) influx, implying that release of Ca(2+) from internal stores further induces capacitative Ca(2+) entry. Taken together, these results suggest that both ET(A) and ET(B) receptors are involved in ET-1-induced [Ca(2+)](i) rise in H9c2 myocardiac ventricular cells. Whereas ET(B) receptor seems to mediate the initial Ca(2+) influx via L-type Ca(2+) channel, ET(A) receptor appears to be involved in the subsequent Ca(2+) release from endoplasmic reticulum and mitochondria Ca(2+) stores.     

Calcium mobilization by nicotinic acid adenine dinucleotide phosphate (NAADP) in rat astrocytes

Nicotinic acid adenine dinucleotide phosphate (NAADP) has been shown to release intracellular Ca(2+) in several types of cells. We have used Ca(2+)-sensitive fluorescent dyes (Fura-2, Fluo-4) to measure intracellular Ca(2+) in astrocytes in culture and in situ. Bath-applied NAADP elicited a reversible and concentration-dependent Ca(2+) rise in up to 90% of astrocytes in culture (EC(50)=7 microM). The NAADP-evoked Ca(2+) rise was maintained in the absence of extracellular Ca(2+), but was suppressed after depleting the Ca(2+) stores of the ER with ATP (20 microM), with cyclopiazonic acid (10 microM) or with ionomycin (5 microM). P(2) receptor antagonist pyridoxalphosphate-6-azophenyl-2'4'-disulfonic acid (PPADS, 100 microM), IP(3) receptor blocker 2-aminoethoxydiphenyl borate (2-APB, 100 microM) and PLC inhibitor U73122 (10 microM) also reduced or suppressed the NAADP-evoked Ca(2+) rise. NAADP still evoked a Ca(2+) response after application of glycyl-l-phenylalanine-beta-naphthylamide (GPN, 200 microM), which permeabilizes lysosomes, or preincubation with H(+)-ATPase inhibitor bafilomycin A1 (4 microM) and of p-trifluoromethoxy carbonyl cyanide phenylhydrazone (FCCP, 2 microM), that impairs mitochondrial Ca(2+) handling. In acute brain slices, NAADP (10 microM) evoked Ca(2+) transients in cerebellar Bergmann glial cells and in hippocampal astrocytes. Our results suggest that NAADP recruits Ca(2+) from inositol 1,4,5-trisphosphate-sensitive Ca(2+) stores in mammalian astrocytes, at least partly by activating metabotropic P(2)Y receptors.     

Functional consequences of phosphomimetic mutations at key cAMP-dependent protein kinase phosphorylation sites in the type 1 inositol 1,4,5-trisphosphate receptor

Regulation of Ca(2+) release through inositol 1,4,5-trisphosphate receptors (InsP(3)R) has important consequences for defining the particular spatio-temporal properties of intracellular Ca(2+) signals. In this study, regulation of Ca(2+) release by phosphorylation of type 1 InsP(3)R (InsP(3)R-1) was investigated by constructing "phosphomimetic" charge mutations in the functionally important phosphorylation sites of both the S2+ and S2- InsP(3)R-1 splice variants. Ca(2+) release was investigated following expression in Dt-40 3ko cells devoid of endogenous InsP(3)R. In cells expressing either the S1755E S2+ or S1589E/S1755E S2- InsP(3)R-1, InsP(3)-induced Ca(2+) release was markedly enhanced compared with nonphosphorylatable S2+ S1755A and S2- S1589A/S1755A mutants. Ca(2+) release through the S2- S1589E/S1755E InsP(3)R-1 was enhanced approximately 8-fold over wild type and approximately 50-fold when compared with the nonphosphorylatable S2- S1589A/S1755A mutant. In cells expressing S2- InsP(3)R-1 with single mutations in either S1589E or S1755E, the sensitivity of Ca(2+) release was enhanced approximately 3-fold; sensitivity was midway between the wild type and the double glutamate mutation. Paradoxically, forskolin treatment of cells expressing either single Ser/Glu mutation failed to further enhance Ca(2+) release. The sensitivity of Ca(2+) release in cells expressing S2+ S1755E InsP(3)R-1 was comparable with the sensitivity of S2- S1589E/S1755E InsP(3)R-1. In contrast, mutation of S2+ S1589E InsP(3)R-1 resulted in a receptor with comparable sensitivity to wild type cells. Expression of S2- S1589E/S1755E InsP(3)R-1 resulted in robust Ca(2+) oscillations when cells were stimulated with concentrations of alpha-IgM antibody that were threshold for stimulation in S2- wild type InsP(3)R-1-expressing cells. However, at higher concentrations of alpha-IgM antibody, Ca(2+) oscillations of a similar period and magnitude were initiated in cells expressing either wild type or S2- phosphomimetic mutations. Thus, regulation by phosphorylation of the functional sensitivity of InsP(3)R-1 appears to define the threshold at which oscillations are initiated but not the frequency or amplitude of the signal when established.