A Family with Atypical Hailey Hailey Disease- Is There More to the Underlying Genetics than ATP2C1?

The autosomal dominant Hailey Hailey disease (HHD) is caused by mutations in the ATP2C1 gene encoding for human secretory pathway Ca2+/Mn2+ ATPase protein (hSPCA1) in the Golgi apparatus. Clinically, HHD presents with erosions and hyperkeratosis predominantly in the intertrigines. Here we report an exome next generation sequencing (NGS) based analysis of ATPase genes in a Greek family with 3 HHD patients presenting with clinically atypical lesions mainly localized on the neck and shoulders. By NGS of one HHD-patient and in silico SNP calling and SNP filtering we identified a SNP in the expected ATP2C1 gene and SNPs in further ATPase genes. Verification in all 3 affected family members revealed a heterozygous frameshift deletion at position 2355_2358 in exon 24 of ATP2C1 in all three patients. 7 additional SNPs in 4 ATPase genes (ATP9B, ATP11A, ATP2B3 and ATP13A5) were identified. The SNPs rs138177421 in the ATP9B gene and rs2280268 in the ATP13A5 gene were detected in all 3 affected, but not in 2 non affected family members. The SNPs in the ATP2B3 and ATP11A gene as well as further SNPs in the ATP13A5 gene could not be confirmed in all affected family members. One may speculate that besides the level of functional hSPCA1 protein, levels of other ATPase proteins may influence expressivity of the disease and might also contribute, as in this case, to atypical presentations.     

Effect of Hailey-Hailey Disease mutations on the function of a new variant of human secretory pathway Ca2+/Mn2+-ATPase (hSPCA1)

ATP2C1, encoding the human secretory pathway Ca2+/Mn2+ ATPase (hSPCA1), was recently identified as the defective gene in Hailey-Hailey Disease (HHD), an autosomal dominant skin disorder characterized by persistent blisters and erosions. To investigate the underlying cause of HHD, we have analyzed the changes in expression level and function of hSPCA1 caused by mutations found in HHD patients. Mutations were introduced into hSPCA1d, a novel splice variant expressed in keratinocytes, described here for the first time. Encoded by the full-length of optional exons 27 and 28, hSPCA1d was longer than previously identified splice variants. The protein competitively transported Ca2+ and Mn2+ with equally high affinity into the Golgi of COS-1 cells. Ca2+- and Mn2+-dependent phosphoenzyme intermediate formation in forward (ATP-fuelled) and reverse (Pi-fuelled) directions was also demonstrated. HHD mutant proteins L341P, C344Y, C411R, T570I, and G789R showed low levels of expression, despite normal levels of mRNA and correct targeting to the Golgi, suggesting instability or abnormal folding of the mutated hSPCA1 polypeptides. P201L had little effect on the enzymatic cycle, whereas I580V caused a block in the E1 approximately P --> E2-P conformational transition. D742Y and G309C were devoid of Ca2+- and Mn2+-dependent phosphoenzyme formation from ATP. The capacity to phosphorylate from Pi was retained in these mutants but with a loss of sensitivity to both Ca2+ and Mn2+ in D742Y and a preferential loss of sensitivity to Mn2+ in G309C. These results highlight the crucial role played by Asp-742 in the architecture of the hSPCA1 ion-binding site and reveal a role for Gly-309 in Mn2+ transport selectivity.     

Structural and functional interactions between the Ca2+-, ATP-, and caffeine-binding sites of skeletal muscle ryanodine receptor (RyR1)

Ryanodine receptor type 1 (RyR1) releases Ca²⁺ ions from the sarcoplasmic reticulum of skeletal muscle cells to initiate muscle contraction. Multiple endogenous and exogenous effectors regulate RyR1, such as ATP, Ca²⁺, caffeine (Caf), and ryanodine. Cryo-EM identified binding sites for the three coactivators Ca²⁺, ATP, and Caf. However, the mechanism of coregulation and synergy between these activators remains to be determined. Here, we used [³H]ryanodine ligand-binding assays and molecular dynamics simulations to test the hypothesis that both the ATP- and Caf-binding sites communicate with the Ca²⁺-binding site to sensitize RyR1 to Ca²⁺. We report that either phosphomethylphosphonic acid adenylate ester (AMPPCP), a nonhydrolyzable ATP analog, or Caf can activate RyR1 in the absence or the presence of Ca²⁺. However, enhanced RyR1 activation occurred in the presence of Ca²⁺, AMPPCP, and Caf. In the absence of Ca²⁺, Na⁺ inhibited [³H]ryanodine binding without impairing RyR1 activation by AMPPCP and Caf. Computational analysis suggested that Ca²⁺-, ATP-, and Caf-binding sites modulate RyR1 protein stability through interactions with the carboxyterminal domain and other domains in the activation core. In the presence of ATP and Caf but the absence of Ca²⁺, Na⁺ is predicted to inhibit RyR1 by interacting with the Ca²⁺-binding site. Our data suggested that ATP and Caf binding affected the conformation of the Ca²⁺-binding site, and conversely, Ca²⁺ binding affected the conformation of the ATP- and Caf-binding sites. We conclude that Ca²⁺, ATP, and Caf regulate RyR1 through a network of allosteric interactions involving the Ca²⁺-, ATP-, and Caf-binding sites.     

ATP-Activated Nonselective Cation Current in NG108-15 Cells

Abstract: ATP (1 mM) induced a biphasic increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i), i.e., an initial transient increase decayed to a level of sustained increase, in NG108-15 cells. The transient increase was inhibited by a phospholipase C inhibitor, 1-[6-[[17β-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122), whereas the sustained increase was abolished by removal of external Ca²⁺. We examined the mechanism of the ATP-elicited sustained [Ca²⁺]_i increase using the fura-2 fluorescent method and the whole-cell patch clamp technique. ATP (1 mM) induced a membrane current with the reversal potential of 12.5 ± 0.8 mV (n = 10) in Tyrode external solution. The EC₅₀ of ATP was ～0.75 mM. The permeability ratio of various cations carrying this current was Na⁺ (defined as 1) > Li⁺ (0.92 ± 0.01; n = 5) > K⁺ (0.89 ± 0.03; n = 6) > Rb⁺ (0.55 ± 0.02; n = 6) > Cs⁺ (0.51 ± 0.01; n = 5) > Ca²⁺ (0.22 ± 0.03; n = 3) > N-methyl-d -glucamine (0.13 ± 0.01; n = 5), suggesting that ATP activated a nonselective cation current. The ATP-induced current was larger at lower concentrations of external Mg²⁺. ATP analogues that induced the current were 2-methylthio-ATP (2MeSATP), benzoylbenzoic-ATP, adenosine 5′-thiotriphosphate (ATPγS), and adenosine 5′-O-(2-thiodiphosphate), but not adenosine, ADP, α,β-methylene-ATP (AMPCPP), β,γ-methylene-ATP (AMPPCP), or UTP. Concomitant with the current data, 2MeSATP and ATPγS, but not AMPCPP or AMPPCP, increased the sustained [Ca²⁺]_i increase. We conclude that ATP activates a class of Ca²⁺-permeable nonselective cation channels via the P_2z receptor in NG108-15 cells.     

Studies on the mechanism of regulation of the red-cell Ca2+ pump by calmodulin and ATP

(1) The effects of calmodulin binding on the rates of Ca²⁺-dependent phosphorylation and dephosphorylation of the red-cell Ca²⁺ pump, have been tested in membranes stripped of endogenous calmodulin or recombined with purified calmodulin. (2) In Mg²⁺-containing media, phosphorylation and dephosphorylation rates are accelerated by a large factor (at 0°C), but the steady-state level of phosphoenzyme is unaffected by calmodulin binding (at 0°C and 37°C). In Mg²⁺-free media, slower rates of phosphoenzyme formation and hydrolysis are observed, but both rates and the steady-state phosphoenzyme level are raised following calmodulin binding. (3) At 37°C and 0°C, the rate of (Ca²⁺ + Mg²⁺)-ATPase activity is stimulated maximally by 6–7-fold, following calmodulin binding. At 37°C the apparent Ca²⁺ affinity for sustaining ATP hydrolysis is raised at least 20-fold, K_m(Ca) ? 10 μM (—calmodulin) and K_m(Ca) < 0.5 μM (+ calmodulin), but at 0°C the apparent Ca²⁺ affinity is very high in calmodulin-stripped membranes and little or no effect of calmodulin is observed (K_m(Ca) ? 3–4 · 10^-8 M). (Ca²⁺ + Mg²⁺)-ATPase activity in calmodulin activated membranes and at saturating ATP levels, is sharply inhibited by addition of calcium in the range 50–2000 μM. (4) A systematic study of the effects of the nucleotide species MgATP, CaATP and free ATP on (Ca²⁺ + Mg²⁺)-ATPase activity in calmodulin-activated membranes reveals: (a) In the 1–10 μmolar concentration range MgATP, CaATP and free ATP appear to sustain (Ca²⁺ + Mg²⁺)-ATPase activity equally effectively. (b) In the range 100–2000 μM, MgATP accelerates ATP hydrolysis (K_m(MgATP) ? 360 μM), and CaATP is an inhibitor (K_i(CaATP) ? 165 μM), probably competing with MgATP fo the regulatory site. (5) The results suggest that calmodulin binding alters the conformational state of the Ca²⁺- pump active site, producing a high (Ca²⁺ + Mg²⁺)-ATPase activity, high Ca²⁺ affinity and regulation of activity by MgATP.     

Identification and characterization of a multispecific monoclonal antibody G2 against chicken prion protein

We previously generated a monoclonal antibody (mAb), G2, by immunizing mice with Residues 174–247 of the chicken prion protein (ChPrP^C). In this study, we found that G2 possessed an extremely unusual characteristic for a mAb; in particular, it could react with at least three proteins other than ChPrP^C, the original antigenic protein. We immunoscreened a complementary DNA library from chicken brain DNA and found three proteins (SEPT3, ATP6V1C1, and C6H10orf76) that reacts with G2. There were no regions of amino acid sequence similarity between ChPrP^C and SEPT3, ATP6V1C1, or C6H10orf76. We selected ATP6V1C1 as a representative of the three proteins and identified the epitope within ATP6V1C1 that reacts with G2. The amino acid sequence of the G2 epitope within ATP6V1C1 (Pep8) was not related to the G2 epitope within ChPrP^C (Pep18mer). However, enzyme-linked immunosorbent assay, surface plasmon resonance (SPR), and isothermal titration calorimetry (ITC) experiments indicated that these two peptides have similar binding affinity for G2. The apparent K_D values of Pep18mer and Pep8 obtained from SPR experiments were 2.9 × 10⁻⁸ and 1.6 × 10⁻⁸ M, respectively. Antibody inhibition test using each peptide indicated that the binding sites of the two different peptides overlapped each other. We observed that these two peptides substantially differed in several binding characteristics. Based on the SPR experiments, the association and dissociation rate constants of Pep18mer were higher than those of Pep8. A clear difference was also observed in ITC experiments. These differences may be explained by G2 adopting different binding conformations and undergoing different binding pathways.     

Calcium pool size modulates the sensitivity of the ryanodine receptor channel and calcium-dependent ATPase of heavy sarcoplasmic reticulum to extravesicular free calcium concentration

We have examined calcium cycling and associated ATP consumption by isolated heavy sarcoplasmic reticulum (HSR) vesicles incubated in conditions believed to exist in resting muscle. Our goals were to estimate the magnitude of calcium cycling under those conditions and identify the main mechanisms involved in its regulation. The integrity of the HSR vesicles was documented by the retention of [¹⁴C]-sucrose and electron microscopy. HSR actively exchanged Ca²⁺ with the medium through a partially open ryanodine-binding channel (RyR), as evidenced by the rapid attainment of a steady-state gradient between HSR and medium, which was promptly increased by the closure of the channel with ruthenium red (RR) or collapsed by its opening with caffeine. The ATP dependency was evidenced by the sustained ATP consumption after the steady state was attained and by the abrogation of the gradient following inhibition of the pump with thapsigargin (Tg) or the omission of ATP. When HSR vesicles were incubated in a comparatively large pool of calcium (≈1 μmol/mg HSR protein), ATP consumption was 1–1.5 μmol × [min × mg protein]⁻¹ at 0.1 μM free Ca²⁺. Under such conditions, the main regulator of the sarcoplasmic Ca²⁺-dependent ATPase (SERCA) was extravesicular-free Ca²⁺ concentration, with a four- to fivefold increase between 0.1 and 2 μM Ca²⁺, whereas RyR channel activity and the replenishment of the HSR vesicles had only a modest effect on ATP consumption. When calcium pool size was reduced to 0.1 μmol/mg HSR protein, a steady state was established at a lower level of HSR calcium. In spite of a slightly lower free extravesicular Ca²⁺ at equilibrium (≈0.07 μM following an initial concentration of 0.1 μM), both ATP consumption and the open probability of the RyR channel were increased by a factor of three to five. Compared to the large calcium pool, the sensitivity of both RyR channel and SERCA to extravesicular free Ca²⁺ concentration as well as to caffeine and RR was markedly enhanced. Conclusions: (1) In conditions present in resting muscle, HSR calcium is in dynamic equilibrium with the medium through a partially open RyR channel, which requires continuous ATP hydrolysis. (2) The availability of calcium is a major determinant of the sensitivity of both RyR channel and SERCA to free extravesicular Ca²⁺ and possibly other stimuli. (3) These observations are consistent with the concept that calcium cycling in resting muscle may account for a significant fraction of muscle energy demands and further suggest that restricting calcium availability may enhance the energetic demands of this process. J. Cell. Physiol. 175:283–294, 1998. © 1998 Wiley-Liss, Inc.     

Modulation of the kinetic characteristics of the sarcoplasmic reticulum ATPase by membrane fluidity

(Ca²⁺ + Mg²⁺)-ATPase from sarcoplasmic reticulum has been reconstituted with dipalmitoylphosphatidylcholine, and the activating effect of ATP and Ca²⁺ on this enzyme has been studied at different temperatures. It has been found that two kinetic forms of the enzyme are interconverted at about 31°C, and this is possibly related to a phase change in the phospholipid which is more directly associated with the protein. Above 31°C the enzyme is less dependent on ATP activation at high ATP concentrations but shows positive cooperativity for Ca²⁺ activation. On the other hand, below 31°C, the reconstituted enzyme is more dependent on ATP for activation at high ATP concentrations than the purified ATPase and does not show cooperativity for Ca²⁺ activation.     

Secretion and hydrolysis of ATP by Ehrlich ascites tumor cells during centrifugation. Dependence on calcium and temperature

Precipitation of Ehrlich ascites tumor cells (EATC) by centrifugation causes ATP secretion. ATP secretion is accompanied by an increase of calcium concentration in the cytosol and persists for a long time (minutes) after centrifugation during the storage of cells at a low temperature. During prolonged storage (for more than 1.5 h), the concentration of extracellular ATP decreases to the level of ∼100 nM due to termination of secretion and ATP hydrolysis by surface ATPases. The rate of ATP hydrolysis exponentially falls with the temperature decrease from 36 to 8°C. ARL67156, a selective inhibitor of E-NTPDase-1, effectively suppresses the extracellular ATP hydrolysis. The intensity of ATP secretion does not correlate with the calcium ions concentration in the cytosol and Ca²⁺ mobilization from endoplasmic reticulum but correlates with the intensity of Ca²⁺ influx into the cells. The temperature dependences of ATP secretion intensity and Ca²⁺ entry rate coincide.     

Thermogenic activity of Ca-ATPase from skeletal muscle heavy sarcoplasmic reticulum: The role of ryanodine Ca channel

The sarcoplasmic reticulum Ca²⁺ ATPase 1 (SERCA 1) is able to handle the energy derived from ATP hydrolysis in such a way as to determine the parcel of energy that is used for Ca²⁺ transport and the fraction that is converted into heat. In this work we measured the heat production by SERCA 1 in the two sarcoplasmic reticulum (SR) fractions: the light fraction (LSR), which is enriched in SERCA and the heavy fraction (HSR), which contains both the SERCA and the ryanodine Ca²⁺ channel. We verified that although HSR cleaved ATP at faster rate than LSR, the amount of heat released during ATP hydrolysis by HSR was smaller than that measured by LSR. Consequently, the amount of heat released per mol of ATP cleaved (ΔH^cal) by HSR was lower compared to LSR. In HSR, the addition of 5 mM Mg²⁺ or ruthenium red, conditions that close the ryanodine Ca²⁺ channel, promoted a decrease in the ATPase activity, but the amount of heat released during ATP hydrolysis remained practically the same. In this condition, the ΔH^cal values of ATP hydrolysis increased significantly. Neither Mg²⁺ nor ruthenium red had effect on LSR. Thus, we conclude that heat production by SERCA 1 depends on the region of SR in which the enzyme is inserted and that in HSR, the ΔH^cal of ATP hydrolysis by SERCA 1 depends on whether the ryanodine Ca²⁺ channel is opened or closed.     

Effects of Small Molecule Modulators on ATP Binding to Skeletal Ryanodine Receptor

Calcium release for muscle contraction in skeletal muscle is mediated in part by the ryanodine receptor 1, RyR1, Ca²⁺-channel and is strongly affected by intrinsic modulators like Ca²⁺, Mg²⁺ and ATP. We showed differential effects on ATP binding in the presence of Ca²⁺ or Mg²⁺ ions using ESR spectroscopy and a spin-labeled ATP analog, SL-ATP (Dias et al. Biochemistry 45: 9408–9415, 2006). We here report the effects of RyR1 modulators like ryanodine, caffeine and dantrolene on the ATP binding of RyR1 using the same technique. We present evidence that the exogenous effectors induce changes within RyR1 that lead to different ATP binding characteristics: In the presence of the activating modulator, caffeine, or in the presence of ryanodine, which causes a half-open state of the channel, binding of eight ATP per RyR1 was observed, even in the presence of inhibitory Ca²⁺, suggestive of a stable “open” channel conformation. In the presence of the inhibitory modulator dantrolene, ATP binding affinity decreased in the presence of activating Ca²⁺, while in the presence of inhibitory Ca²⁺, ATP binding affinity increased, but at the same time the number of accessible sites decreased to four, suggestive of a closed conformation of the channel. The results imply that modulation of ATP binding to RyR1 as well as the overall number of accessible ATP binding sites on the channel are crucial for regulation and are in direct correlation with the modified activity of the channel induced by pharmacological agents.     

Temperature dependency of calcium responses in mammary tumour cells

Changes of intracellular calcium concentration ([Ca²⁺]_i) induced by the extracellular application of ATP and bradykinin in mouse mammary tumour cells (MMT060562) were investigated by image analysis of fluo-3 fluorescence at 24°C and 35°C. ATP (0·1–100 μM ) and bradykinin (0·1 nM –1 μM ) induced the increase of [Ca²⁺]_i at both temperatures and Ca²⁺-depletion did not affect these [Ca²⁺]_i responses. Both [Ca²⁺]_i responses became more sensitive at 35°C than at 24°C. A clear latency of [Ca²⁺]_i increased after the application of the agonists was observed, and it changed with the concentration of the agonist. As concentrations of ATP or bradykinin became lower, the latency and rise time became longer. At higher concentrations, the latency and rise time approached a constant value. The latency shortened remarkably at 35°C. These results suggested the involvement of a regenerative or threshold process in the [Ca²⁺]_i responses in mammary tumour cells. © 1997 John Wiley & Sons, Ltd.     

Regulation of ANP-stimulated guanylate cyclase in the presence of Mn2+ in rat lung membranes

The catalytic activity of guanylate cyclase (GCase) coupled to atrial natriuretic peptide (ANP) receptor depends on the metal co-factor, Mn²⁺ or Mg²⁺. ATP synergistically stimulates the ANP-stimulated GCase in the presence of Mg²⁺. We have now shown the ATP regulation of the ANP-stimulated GCase in the presence of Mn²⁺ in rat lung membranes. ANP stimulated the GCase 2.1-fold compared to the control. ATP enhanced both the basal (basal-GCase) and the ANP-stimulated GCase maximally 1.7- and 2.3- fold compared to the control, respectively, at a concentration of 0.1 mM. The stimulation by ATP was smaller in the presence of Mn²⁺ than in the presence of Mg²⁺. The addition of inorganic phosphate to the reaction mixture altered the GCase activities in the presence of Mn²⁺ with or without ANP and/or ATP. In the presence of 10 mM phosphate, ATP dose-dependently stimulated the basal GCase 5-fold compared to the control at a concentration of 1 mM and augmented the ANP-stimulated GCase, which was 4.2-fold compared to the basal-GCase, 5.5-fold compared to the control at a concentration of 0.5 mM. Protein phosphatase inhibitors, okadaic acid (100 nM), H8 (1 M) and staurosporin (1 M), did not alter the activity. Orthovanadate (1 mM), an inorganic phosphate analogue, significantly stimulated both the basal-GCase and the ANP-stimulated GCase, which were inhibited by ATP. It was assumed that phosphate and orthovanadate might interact with the GCase to regulate the activity in the opposite manner. This was the first report that inorganic phosphate and orthovanadate affected the ATP-regulation of the ANP-stimulated GCase in the presence of Mn²⁺.     

Modulation of P2X4 pore closure by magnesium,potassium, and ATP

The P2X4 receptor plays a prominent role in cellular responses to extracellular ATP. Through classical all-atom molecular dynamics (MD) simulations totaling 24 μs we have investigated how metal-complexed ATP stabilizes the channel's open state and prevents its closing. We have identified two metal-binding sites, Mg²⁺ and potassium K⁺, one at the intersection of the three subunits in the ectodomain (MBS1) and the second one near the ATP-binding site (MBS2), similar to those characterized in Gulf Coast P2X. Our data indicate that when Mg²⁺ and K⁺ ions are complexed with ATP, the channel is locked into an open state. Interestingly, irrespective of the number of bound ATP molecules, Mg²⁺ ions bound to the MBS2 impeded the collapse of the open-state protein to a closed state by stabilizing the ATP-protein interactions. However, when Mg²⁺ in the MBS2 was replaced with K⁺ ions, as might be expected when in equilibrium with an extracellular solution, the interactions between the subunits were weakened and the pore collapsed. This collapse was apparent when fewer than two ATPs were bound to MBS2 in the presence of K⁺. Therefore, the different capacities of common cations to stabilize the channel may underlie a mechanism governing P2X4 channel gating in physiological systems. This study therefore provides structural insights into the differential modulation of ATP activation of P2X4 by Mg²⁺ and K⁺.     

Elevating intracellular free Mg2+ preserves sensitivity of Na+−K+ pump to ATP at reduced temperatures in guinea pig red blood cells

Red cells of hibernating species have a higher relative rate of Na⁺–K⁺ pump activity at low temperature than the red cells of a mammal with a typical sensitivity to cold. The kinetics of ATP stimulation of the Na⁺–K⁺ pump were determined in guinea pig and ground squirrel red cells at different temperatures between 5 and 37°C by measuring ouabain-sensitive K⁺ influx at different levels of ATP. In guinea pig cells, elevation of intracellular free Mg²⁺ to 2 mmol·l^-1 by use of the divalent cation ionophore A23187 caused the apparent affinity of the pump for ATP to increase with cooling to 20°C, rather than to decrease, as occurs in cells not loaded with Mg²⁺. In ground squirrel cells raising intracellular free Mg²⁺ had little effect on apparent affinity of the pump for ATP at 20°C. ATP affinity rose slightly with cooling both in Mg²⁺-enriched and in control ground squirrel cells. Increased intracellular free Mg²⁺ in guinea pig cells stimulated Na⁺–K⁺ pump activity so that at 20°C the pump rate was the same in the Mg²⁺-enriched guinea pig and control ground squirrel cells. Pump activity in Mg²⁺-enriched guinea pig cells at 5°C was significantly improved but still lower than pump activity in control cells from ground squirrel. Thus, loss of affinity of the Na⁺–K⁺ pump for ATP that occurs with cooling in cold-sensitive guinea pig red cells can be, at least partially, prevented by elevating cytoplasmic free Mg²⁺. Conversely, in ground squirrel red cells natural rise of free Mg²⁺ may in part account for the preservation of the ATP affinity of their Na⁺–K⁺ pump with cooling.Abbreviations K _m Michaelis-Menten constant for apparent affinity - MOPS 3-(N-morpholino)-propanesulphonic acid - [Mg²⁺]_i intracellular concentration of free Mg²⁺ - OD optical density - RBC red blood cell(s) - T _b body temperature     

High-affinity P2Y2 and low-affinity P2X7 receptor interaction modulates ATP-mediated calcium signaling in murine osteoblasts

The P2 purinergic receptor family implicated in many physiological processes, including neurotransmission, mechanical adaptation and inflammation, consists of ATP-gated non-specific cation channels P2XRs and G-protein coupled receptors P2YRs. Different cells, including bone forming osteoblasts, express multiple P2 receptors; however, how P2X and P2Y receptors interact in generating cellular responses to various doses of [ATP] remains poorly understood. Using primary bone marrow and compact bone derived osteoblasts and BMP2-expressing C2C12 osteoblastic cells, we demonstrated conserved features in the P2-mediated Ca²⁺ responses to ATP, including a transition of Ca²⁺ response signatures from transient at low [ATP] to oscillatory at moderate [ATP], and back to transient at high [ATP], and a non-monotonic changes in the response magnitudes which exhibited two troughs at 10⁻⁴ and 10⁻² M [ATP]. We identified P2Y2 and P2X7 receptors as predominantly contributing to these responses and constructed a mathematical model of P2Y2R-induced inositol trisphosphate (IP₃) mediated Ca²⁺ release coupled to a Markov model of P2X7R dynamics to study this system. Model predictions were validated using parental and CRISPR/Cas9-generated P2Y2 and P2Y7 knockouts in osteoblastic C2C12-BMP cells. Activation of P2Y2 by progressively increasing [ATP] induced a transition from transient to oscillatory to transient Ca²⁺ responses due to the biphasic nature of IP₃Rs and the interaction of SERCA pumps with IP₃Rs. At high [ATP], activation of P2X7R modulated the response magnitudes through an interplay between the biphasic nature of IP₃Rs and the desensitization kinetics of P2X7Rs. Moreover, we found that P2Y2 activity may alter the kinetics of P2X7 towards favouring naïve state activation. Finally, we demonstrated the functional consequences of lacking P2Y2 or P2X7 in osteoblast mechanotransduction. This study thus provides important insights into the biophysical mechanisms underlying ATP-dependent Ca²⁺ response signatures, which are important in mediating bone mechanoadaptation.     

Ultracytochemical detection of guanylate cyclase C activity in alimentary tract and associated glands of the rat. Influence of pH, ATP and the ions Mg2+ and Mn2+

Intestinal guanylate cyclase C is activated by guanylin, an endogenous peptide. This activity seems to be modulated by adenine nucleotides, the ions Mg²⁺ and Mn²⁺, and pH. In this study, we report an ultracytochemical method for the localization of guanylate cyclase C activity at the electron microscope level. We studied the enzymatic activity in the presence or absence of guanylin and/or ATP, in the presence of the ions Mg²⁺ or Mn²⁺, and at different pH levels. The greatest distribution of enzymatic activity was detected in samples incubated at pH 8 and 7.4 in the presence of guanylin, Mg²⁺ and ATP. Guanylate cyclase C activity was detected at the surface epithelium of stomach and intestine, and in liver, exocrine pancreas and parotid gland. In the intestine, enzymatic activity was more widely distributed in the duodenum than in the jejunum–ileum and colon. In the small intestine, activity was more evident in the upper portion than in the basal portion of the villus. In samples incubated at pH 8 and 7.4 in the absence of ATP, enzymatic activity was detected only in small intestine, liver and exocrine pancreas. Enzymatic activity was present in duodenum incubated at pH 8 and 7.4 in the presence of Mn²⁺ and in the presence or absence of ATP. No samples incubated in all these experimental conditions but at pH 5 or samples incubated in the presence of guanylin only or in the absence of guanylin, displayed guanylate cyclase C activity. Our results suggest that a complete ultracytochemical detection of guanylate cyclase C activity requires guanylin as stimulator, and incubation in the presence of Mg²⁺ and ATP atbreak pH 8 and 7.4.